Activation markers of t cells and method for assessing t cell activation

Abstract

The present disclosure relates to methods of assessing T cell activation state within a cell composition which may be used in connection with cell therapy or generating the cell for cell therapy. The cells of the cell composition can express recombinant receptors such as chimeric receptors, e.g., chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). The methods provide markers and how to use them for assessing the activation of T cells within a cell composition.

Description

ACTIVATION MARKERS OF T CELLS AND METHOD FOR ASSESSING T CELL ACTIVATIONCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 468,524, filed May 23, 2023, entitled “ACTIVATION MARKERS OF T CELLS AND METHODS FOR ASSESSING T CELL ACTIVATION” the contents of which are incorporated by reference in their entirety.Reference to An Electronic Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042027540SEQLIST.xml, created on May 21, 2024, which is 99,949 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present invention relates to methods of assessing activation of T cells in a cell composition for use in connection with cell therapy or transduction of cells. The T cells of the cell composition can express recombinant receptor such as chimeric receptors, e.g., chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). The methods provide an assay for assessing activation of T cells using one or more markers and examining surface level expression or the percent of cells positive for the marker to assess the activation.Background

[0004] Various methods for determining a T cell activation state in culture or from a sample exist, often relying on markers present on the cell surface which are upregulated upon activation of the T cell. By examining these defined markers and their surface level expression or the percent of T cells positive for the markers within a cell composition, activation state is assessed. However, markers may only be present on certain subsets of T cells and so combination of markers are used to assess T cell activation, leading to a benefit in having multiple markers to choose from for assessing T cell activation. These methods of assessing T cell activation using markers rely on characterization results demonstrating whichsurface markers expression levels are changed during T cell activation and benefit from larger pools of markers being available. Provided herein are methods that address such needs.Summary

[0005] Provided herein is a method for assessing activation of T cells within a cell composition, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: one or more markers of group (i) are selected from the group consisting of CD36L (SCARB1, SR-BI), CD 120b, CD 107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD 105 (Endoglin), CD262 (DR5, TRAIL-R2), CD 170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CD11c, CD 146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and one or more markers of group (ii) are selected from the group consisting of CD 192 (CCR2), CCRL2, CD96 (TACTILE), CD 195 (CCR5), CD 124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MALA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD 172g (SIRPg), CD 127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR; (b) comparing the level of surface expression or the percent of positive cells in the cell composition to the level of surface expression or the percent of positive cells for each of the one or more markers in a reference, wherein a higher level or higher percent of positivecells for a marker in (i) compared to the reference indicates the T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the reference indicates the T cells are activated.

[0006] In some of any embodiments, the reference is comprised of an unstimulated control cell composition.

[0007] In some of any embodiments, the reference is the level of expression or percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject or donor. In some of any embodiments, the reference is the average level of expression or average percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor. In some of any embodiments, the reference is the median level of expression or median percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.

[0008] Provided herein is a method for assessing T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: one or more markers of group (i) are selected from the group consists of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD 105 (Endoglin), CD262 (DR5, TRAIL-R2), CD 170 (Siglec-5), CD73 (Ecto-5'- nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF- 1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-Rl), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R);; and one or more markers of group (ii) are selected from the group consisting of CD 192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDllb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0009] In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with T cell activation. In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with T cell activation.

[0010] Provided herein is a method of comparing activation of T cells within a donor, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CD11c, CD 146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and group (ii) consists of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195(CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR; (b) comparing the level of surface expression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in an unstimulated cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated cell composition indicates the T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the unstimulated cell composition indicates the T cells are activated.

[0011] In some of any embodiments, the composition comprising T cells has been subjected to incubation with a T cell stimulatory agent under conditions to induce T cell activation prior to the detecting.

[0012] In some of any embodiments, the method comprises incubation of the composition with a T cell stimulatory agent prior to the detecting. In some of any embodiments, the method comprises incubation of the composition with a T cell stimulatory agent following the detecting.

[0013] In some of any embodiments, the incubation with a T cell stimulatory agent is carried out in vivo in a subject. In some of any embodiments, the incubation with a T cell stimulatory agent is carried out in vitro or ex vivo.

[0014] In some of any embodiments, the incubation with a T cell stimulatory agent is for 12-72 hours. In some of any embodiments, the incubation with a T cell stimulatory agent is for about 24 hours.

[0015] In some of any embodiments, the one or more markers of group (i) are selected from CD20, CD 100, CD 123, CD 184 (CXCR4), CD55, TIGIT (VSTM3), CD 105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (LGLR4), CD66a / c / e, and TSLPR (TSLP-R); and the one or more markers of group (ii) areselected from CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD 172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0016] In some of any embodiments, the incubation with a T cell stimulatory agent is for about 48 hours.

[0017] In some of any embodiments, the one or more markers of group (i) are selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CDllc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP (LRRC32); and the one or more markers of group (ii) are selected from CD 192 (CCR2), CD314 (NKG2D), KLRG1 (MALA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDllb, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f.

[0018] In some of any embodiments, the one or more markers of group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and the one or more markers of group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217,CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

[0019] In some of any embodiments, the one or more markers are of (i) and are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56.

[0020] Provided herein is a method of identifying an activated T cell, the method comprising detecting the cell surface expression of one or more of markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and wherein the cells expressing a high level of the one or more markers are activated T cells.

[0021] In some of any embodiments, the one or more markers are selected from group (i) and consist of CD200 (0X2), CD357 (GITR), CD120b, CD155 (PVR), CD107b (LAMP-2).

[0022] In some of any embodiments, the one or more markers are of (ii) and are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

[0023] Provided herein is a method of identifying an activated T cell, the method comprising detecting the cell surface expression of one or more of markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1, and wherein the cells expressing a low level of the one or more markers are activated T cells.

[0024] In some of any embodiments, the detecting is of CD4+ or CD8+ T cells in the composition comprising T cells.

[0025] In some of any embodiments, the one or more markers from group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and the one ormore markers from group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0026] In some of any embodiments, the detecting is of CD4+ T cells in the composition comprising T cells.

[0027] In some of any embodiments, the one or more markers from group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154,and CD165, and the one or more markers from group (ii) are selected from CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MALA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0028] Provided herein is a method for assessing CD4+ T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii) wherein group (i) consist of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MALA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0029] In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with CD4+ T cell activation. In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with CD4+ T cell activation.

[0030] Provided herein is a method for assessing activation of CD4+ T cells, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more marker in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MALA), CD96 (TACTILE), and CD 127 (IL-7Ra); and(b) comparing the level of surfaceexpression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in cells of an unstimulated control cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated control cell composition indicates the CD4+ T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the unstimulated control cell composition indicates the CD4+ T cells are activated.

[0031] In some of any embodiments, the detecting is of CD8+ T cells in the composition comprising T cells.

[0032] In some of any embodiments, the one or more markers selected from group (i) are selected from CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and the one or more markers from group (ii) are selected from CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD 127 (IL-7Ra).

[0033] Provided herein is a method for assessing CD8+ T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii) wherein group (i), consist of CD120b, CD200 (OX2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD 127 (IL-7Ra).

[0034] In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with CD8+ T cell activation. In some of any embodiments, the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with CD8+ T cell activation.

[0035] Provided herein is a method for assessing activation of CD8+ T cells, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consist of CD120b, CD200 (OX2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra); and (b) comparing the level of surface expression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in cells of an unstimulated control cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated control cell composition indicates the CD8+ T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the unstimulated control cell composition indicates the CD8+ T cells are activated.

[0036] In some of any embodiments, the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.

[0037] In some of any embodiments, the one or more markers selected from group (i) consist of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and the one or more markers selected from group (ii) consist of CD49f, CCRL2, CD 124 (IL-4Ra), CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE).

[0038] Provided herein is a method for assessing activation of T cells, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (OX2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and group (ii) consists of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56.

[0039] In some of any embodiments, the one or more markers from group (i) are selected from CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5), and the one or more markers selected from group (ii) are selected from CD49f, CCRL2, CD 124 (IL-4Ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE).

[0040] In some of any embodiments, the detecting is of recombinant receptor-expressing CD4+ T cells in the composition comprising T cells.

[0041] In some of any embodiments, the one or more markers from group (i) are selected from CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), and CD74, and / or the one or more markers selected from group (ii) are selected from CD49f, CCRL2, and CD 124 (IL-4Ra).

[0042] In some of any embodiments, the detecting is of recombinant receptor-expressing CD8+ T cells in the composition comprising T cells.

[0043] In some of any embodiments, the one or more markers from group (i) are selected from CD200 (OX2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56, and / or the one or more markers selected from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE).

[0044] In some of any embodiments, the one or more markers of group (i) are selected from CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (Betos’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and the one or more markers of group (i) are selected from CD96 (TACTILE).

[0045] Provided herein is a method for assessing activation of T cells, the method comprising: (a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in a composition of T cells, which comprises T cells expressing a recombinant receptor, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and group (ii) consists of CD96 (TACTILE).

[0046] In some embodiments, the composition comprising T cells comprises cells that express a recombinant receptor.

[0047] In some of any embodiments, the detecting is of recombinant expressing cells of the composition of T cells

[0048] In some of any embodiments, the surface expression of the one or more markers of group (i) is increased on cells expressing the recombinant receptor compared with cells that are not expressing the recombinant receptor.

[0049] In some of any embodiments, the surface expression of the one or more markers of group (ii) is decreased on cells expressing the recombinant receptor compared with cells that are not expressing the recombinant receptor.

[0050] In some of any embodiments, the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor and wherein the T cell stimulatory agent is a recombinant receptor stimulating agent that induces recombinant receptor-dependent T cell activation.

[0051] In some of any embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0052] In some of any embodiments, the recombinant receptor stimulating agent comprises a recombinant target antigen recognized by the recombinant receptor. In some of any embodiments, the recombinant receptor stimulating agent is an antibody specific to an extracellular antigen binding domain of the recombinant receptor. In some of any embodiments, the recombinant receptor stimulating agent is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor.

[0053] In some of any embodiments, the recombinant receptor stimulating agent is immobilized or attached to a solid support. In some of any embodiments, the solid support is a surface of the vessel, optionally a well of microwell plate or a flask. In some of any embodiments, the solid support is a bead.

[0054] In some of any embodiments, the recombinant receptor stimulating agent is an antigen-expressing cell, optionally wherein the cell is a clone, from a cell line, or a primary cell taken from a subject. In some of any embodiments, the antigen-expressing cell is a cell line. In some of any embodiments, the cell line is a tumor cell line. In some of any embodiments, the antigen-expressing cell is a cell that has been engineered to express the antigen of the recombinant receptor.

[0055] In some of any embodiments, the detecting is of recombinant receptor-expressing T cells in the composition comprising T cells.

[0056] In some of any embodiments, the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (0X40), and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD 195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1.

[0057] In some of any embodiments, the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2,CD165, and CD83, and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

[0058] In some of any embodiments, the detecting is of CD4+ T cells in the composition comprising T cells.

[0059] In some of any embodiments, the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD 165 and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), and CD127 (IL-7Ra).

[0060] In some of any embodiments, the detecting is of CD8+ T cells in the composition comprising T cells.

[0061] In some of any embodiments, the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, CD83, and / or the one or more markers from group (ii) are selected from CDl lb, CX3CR1, and CD127 (IL-7Ra).

[0062] In some of any embodiments, the T cell stimulatory agent is a pan-T cell activation agent. In some of any embodiments, the pan-T cell activation reagent comprises an anti-CD3 antibody and an anti-CD28 antibody, optionally wherein the pan-T cell activation reagent comprises an anti-CD3 Fab and an anti-CD28 Fab. In some of any embodiments, the pan-T cell activation reagent comprises anti-CD3 / anti-CD28 beads. In some of any embodiments, the pan-T cell activation reagent comprises a soluble anti-CD3 / anti-CD28 streptavidin oligomeric reagent.

[0063] In some of any embodiments, prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agents which bind to the one or more markers.

[0064] In some of any embodiments, prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agent comprising a means for binding the one or more markers

[0065] In some of any embodiments, the one or more binding agents are one or more antibodies or antigen-binding fragments. In some of any embodiments, the one or more binding agents are detectably labeled. In some of any embodiments, the one or more binding agents are fluorescently labeled.

[0066] In some of any embodiments, the detecting is by flow cytometry. In some of any embodiments, the detecting of step (a) is carried out in conjunction with CITE-Seq or REAP-seq. In some of any embodiments, the detecting of step (a) is done by immunohistochemistry, optionally immunohistochemistry fluorescence.

[0067] In some of any embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different markers are used for detecting in step (a).

[0068] Provided herein is a kit for determining T cell activation comprising means for detecting the one or more markers in the methods of some of any embodiments.

[0069] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding the one or more markers in the methods of some of any embodiments.

[0070] In some embodiments, the meads for detecting each of the one or more markers is an antibody.

[0071] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R), and group (ii) consists of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282(TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDllb, CX3CR1, NKp80, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0072] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (LGLR4), CD66a / c / e, and TSLPR (TSLP-R) and group (ii) consists of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MALA), CCRL2, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGER.

[0073] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (ILN-g R a chain), CDllc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32) and group (ii) consists of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDl lb, CX3CR1, NKp80, CD127 (IL- 7Ra), and CD49f.

[0074] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

[0075] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0076] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154,and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD 127 (IL-7Ra).

[0077] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (OX2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra).

[0078] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and the one or more markers selected from group (ii) consist of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5), and CD96 (TACTILE).

[0079] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD134 (0X40), and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD 195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1.

[0080] Provided herein is a kit for determining T cell activation comprising a means for detecting one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers from group (ii) are selected from CD96 (TACTILE).

[0081] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (OX2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP- 1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a,CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R), and group (ii) consists of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDllb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0082] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R) and group (ii) consists of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD 172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0083] Provided herein is a kit for determining T cell activation comprising binding agent comprising a a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155(PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32) and group (ii) consists of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDllb, CX3CR1, NKp80, CD 127 (IL-7Ra), and CD49f.

[0084] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDllb, and CX3CR1.

[0085] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0086] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154,and CD165, andgroup (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

[0087] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD 127 (IL-7Ra).

[0088] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD 120b, CD83, CD357 (GITR), CD200 (OX2), CD 134 (0X40), CD 107b (LAMP-2), CD 155 (PVR), CD74, and the one or more markers selected from group (ii) consist of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5), and CD96 (TACTILE).

[0089] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (OX2), and CD 134 (0X40), and / or the one or more markers from group (ii) are selected from KLRG1 (MALA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1.

[0090] Provided herein is a kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (Ecto-5’-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers from group (ii) are selected from CD96 (TACTILE).

[0091] In some of any embodiments, the means for detecting the one or more markers are one or more binding agents which bind to the one or more markers. In some of any embodiments, the one or more binding agents are one or more antibodies or antigen-binding fragments. In some of any embodiments, the one or more binding agents are detectablylabeled. In some of any embodiments, the one or more binding agents are fluorescently labeled.

[0092] Provided herein is a method for isolating activating T cells, the method comprising identifying a population of activated T cells according to the method in some of any embodiment and isolating the population.

[0093] Provided herein is a method for enriching activating T cells, the method comprising identifying a population of activated T cells according to the method in some of any embodiment and selecting the population, thereby obtaining a cell population enriched in activated T cells.

[0094] Provided herein is method of depleting a cell population of activated T cells, the method comprising identifying a population of activated T cells according to the method in some of any embodiment and depleting the population of activated T cells.

[0095] Provided herein is a T cell population produced according to the method in some of any embodiments.Brief Description of the Drawings

[0096] FIG. 1A shows the difference in percent of positive cells for each marker for CD8+ CAR+ T cells, calculated between the TO and T48 hour stimulation timepoint. The top 15 upregulated and bottom 5 downregulated markers are shown here and coded based on whether they are canonical or noncanonical activation markers, based on the literature.

[0097] FIG. IB shows the difference in percent of positive cells for each marker for CD4+ CAR+ T cells, calculated between the TO and T48 hour stimulation timepoint. In this case, the CD4 CAR-T product is shown. The top 15 upregulated and bottom 5 downregulated markers are shown here and coded based on whether they are canonical or noncanonical activation markers, based on the literature.

[0098] FIG. 2A shows the difference in percent of positive cells for each marker for CD8+ PBMC T cells, calculated between the TO and T48 hour stimulation timepoint. The top 15 upregulated and bottom 5 downregulated markers are shown here and coded based on whether they are canonical or noncanonical activation markers, based on the literature.

[0099] FIG. 2B shows the difference in percent of positive cells for each marker for CD4+ PBMC T cells, calculated between the TO and T48 hour stimulation timepoint. The top15 upregulated and bottom 5 downregulated markers are shown here and coded based on whether they are canonical or noncanonical activation markers, based on the literature.

[0100] FIG. 3A shows the same data from the previous four figures, visualized differently with the top 15 upregulated and bottom 5 downregulated markers from each of the previous four figures aggregated together and displayed on the x axis. The CD4 components of both the PBMC and the CAR+ T cells are shown together. If a marker is blank, it does not mean the marker is absent from the CD4 component, but rather that it does not appear in the top 15 and bottom 5 marker list.

[0101] FIG. 3B shows the same data from the previous four figures, visualized differently with the top 15 upregulated and bottom 5 downregulated markers from each of the previous four figures aggregated together and displayed on the x axis. The CD8 components of both the PBMC and the CAR+T cells are shown together. If a marker is blank, it does not mean the marker is absent from the CD8 component, but rather that it does not appear in the top 15 and bottom 5 marker list.

[0102] FIG. 4 shows the abundance of 5 representative non-canonical activation markers within the CAR+T product, depending on whether cells also express at least one canonical activation marker. Cells are split into two bins: cells that express at least one canonical activation marker, and cells that do not express any canonical activation markers. The percentage of cells expressing the five non-canonical activation markers is shown split between the two bins. Cells that express at least one canonical marker are more likely to also express each non-canonical marker.

[0103] FIG. 5A to 5S show pseudocolor plots of the non-canonical markers generated in Figures 1 through 3 that are upregulated, filtered onto CAR+ T cells only, and plotted against the three canonical activation markers.

[0104] FIG. 6A to 6J show pseudocolor plots of the other canonical markers generated in Figures 1 through 3, filtered onto CAR+ T cells only, and plotted against the three canonical activation markers used in FIG. 4.

[0105] FIG. 7 A to 7K show pseudocolor plots of the non-canonical markers generated in Figures 1 through 3 that are downregulated, filtered onto CAR+ T cells only, and plotted against the three canonical activation markers used in FIG. 4.

[0106] FIG. 8 shows a Venn diagram of the number of markers that have a > 10% change, filtered upon all T cells (both CAR-T and PBMC), in each timepoint pair. A few representative markers from each group are shown.

[0107] FIG. 9A to 9N show histograms of expression of 14 different cell surface markers in CAR+ or CAR- cells stimulated for 48 hours (T=48h), 24 hours (T-24h), or unstimulated (T=0h) broken up into CD4+ T cells and CD8+ T cells.

[0108] FIG. 10A to 10B shows the area under the curve (AUC) for different markers for the CAR+ and CAR- cells broken up by each manufacturing process, where the AUC was calculated for each sample by summing the percent positive for each maker across all timepoints.

[0109] FIG. 11A to 11B shows the data and the percent positive for each marker in the CAR+ compartment plotted on the y axis, and time (in hours post-stimulation) plotted on the x axis. Each sample for each marker within each process is represented as a separate line.Detailed Description

[0110] Provided herein are methods for assessing T cell activation in a cell composition, the method including detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers. In some aspects, the provided methods allow for determination of duration or timing of incubations used on the T cells based on their activation. The provided embodiments are based on the identification of T cell markers that are upregulated or downregulated in activated T cells. The T cell markers in many respects include non-canonical markers that have not been previously reported to be associated with T cell activation or not commonly used to assess T cell activation. Among provided embodiments are markers that can be used to assess or determine activation of particular subsets of T cells, such as CD4+ or CD8+ T cells. Also among provided embodiments are markers that can be used to assess or determine activation of T cells engineered with a T cell signaling recombinant receptor, such as a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR). Also provided herein are kits to assess T cell activation in a cell composition containing means for detecting the one or more markers.

[0111] Existing methods for assessing T cell activation, for example during in vitro or ex vivo culture of the T cells, often rely on defined markers or surface proteins, which are expressed on the cell surface upon T cell activation. Existing methods rely on definedcanonical markers for T cell activation. Often, one marker does not perfectly align with whether all T cells present are activated, leading to a benefit in using multiple markers for assessing T cell activation.

[0112] The provided methods allow for assessing T cell activation using markers provided herein. In some aspects, the markers are non-canonical markers. The results provided herein demonstrate that the markers (e.g. non-canonical markers) are upregulated in stimulated cells when compared to unstimulated cells. The results also demonstrate that certain markers are expressed in sub-groups of cells, such as T cells from peripheral blood mononuclear cells (PBMCs) and / or T cells with a chimeric antigen receptor (CAR), indicating that such markers can be used in assessing activation of T cells in such sub-groups. In some embodiments, certain markers disclosed herein are downregulated upon T cell activation. The results provided herein demonstrate that after T cells have been stimulated (e.g., for 72 hours, 48 hours, or 24 hours prior to detecting their activation state by examining surface expression of surface markers on the T cells), that certain markers increase or decrease their expression based on whether the cells are activated, as was verified using canonical activation markers. The results provided herein show that if the cells are separated by whether they express a canonical marker or not, the cells expressing a canonical marker are more likely to be positive for other markers, such as CD200 (0X2), CD357 (GITR), CD120b, CD155 (PVR), and CD107b (LAMP-2). In addition, if markers identified as being differentially expressed on stimulated cell populations are plotted against canonical markers of cell activation, a double positive population of cells appear for non-canonical markers that are increased upon activation, while no double positive cell population appears for the non- canonical markers that decrease upon stimulation. In some embodiments, changes in marker expression are time sensitive and show differences in expression based on the amount of time post cell stimulation. In some embodiments changes in marker expression are cell type specific (e.g., depending on whether the cells are CD4 or CD8 T cells). In some embodiments, changes in marker expression are specific for cell treatment, with changes in cell marker expression happening more drastically in PBMC or CAR+ T cells.

[0113] In some aspects, the provided methods involve assessing T cell activation based on expression or surface levels of the following markers CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2),CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD 107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR. In some embodiments, the provided methods involve assessing T cell activation in engineered T cells that express a recombinant receptor. In some embodiments, the receptor is a chimeric antigen receptor (CAR). In some embodiment, the provided methods involve assessing T cell activation in CD4+ T cells. In some embodiment, the provided methods involve assessing T cell activation in CD8+ T cells. In some embodiment, the provided methods involve assessing T cell activation in CD4+ CAR+ T cells. In some embodiment, the provided methods involve assessing T cell activation in CD8+ CAR+ T cells.

[0114] In some aspects, the provided methods can be used to determine if cells are ready to be transduced. In some aspects, the provided methods further comprise selecting or isolating T cells for engineering the T cells after a determination of activation. In some aspects, the provided methods further comprise engineering the T cells for which activation is determined and / or monitored to produce a cell therapy product. In some embodiments, the provided methods can be used to monitor the dynamics of T cell activation, stimulation or during cultivation such as under conditions for expansion. In some aspects, the provided methods can be used to identify relationships between activation state and outcomes, forinstance how the cells evolve over time. In some aspects, the provided methods allow for determination of when cells can be administered to a patient.

[0115] In some embodiments, the provided methods can be used to predict the quality of T cells subjected to a manufacturing process, e.g., the quality of T cells during or after the manufacturing process. In some aspects, activation status as determined by the provided methods can be used as a readout during manufacturing, e.g., of manufacturing success or of the success of a manufacturing step (e.g., of cell stimulation). In some embodiments, the provided methods can be used to monitor whether T cells are sufficiently activated, such as for expansion of the T cells to a desired threshold number, for instance to numbers needed for clinical doses of the T cells for a T cell therapy.

[0116] In some aspects, the activation of T cells can lead to the differentiation of T cells. Higher proportions of early memory T cells, such as naive-like T cells, in T cell therapies can improve patient outcomes (see, e.g., Jiang et al., Journal of Pharmaceutical Sciences (2021) 110:1871-1876). In some embodiments, the provided methods can be used to monitor the memory status of the T cells, either directly or by monitoring the activation state of the T cells. In some embodiments, the activation state of the T cells is monitored to predict the memory status of the T cells.

[0117] In some aspects, the cell phenotype information obtained by the provided methods can be used during process development to optimize the duration or other conditions of the manufacturing process or steps thereof in order to improve the quality of processed T cells. In some instances, this information can be used to develop process control strategies in which, for example, when a predicted cell phenotype, such as activation state, falls outside a determined range, conditions of one or more (e.g., the current or a subsequent) manufacturing steps can be altered, e.g., the duration of the current or subsequent manufacturing step can be altered, to improve the final quality of the T cells being manufactured. For example, when an activation state falls outside a determined range during cultivation, subsequent cultivation can, in some instances, be performed under perfusion conditions and / or in the presence of small molecules for, e.g., modulating T cell phenotype towards desired profiles.

[0118] In some aspects, information about activation status obtained by the provided methods can be used to assess or reduce batch-to-batch variability of T cells subjected to the manufacturing process. In some aspects, the activation status information can be used to assess or reduce batch-to-batch variability of a drug product produced using themanufacturing process. For instance, by ensuring that T cells across different cell therapy manufacturing runs are at comparable activation states, the differentiation and memory status of the T cells can be kept consistent. This can reduce variability (e.g., patient-to-patient variability) in the resulting T cell therapies (see, e.g., Jiang et al., Journal of Pharmaceutical Sciences (2021) 110:1871-1876).

[0119] In some embodiments, the provided methods can be used to monitor the activation state of T cells prior to or following the engineering of the T cells. In some aspects, transgene expression can be higher in activated vs. non-activated T cells, such as following the viral transduction of the T cells (see, e.g., Ghassemi et al., Nature Biomedical Engineering (2022) 6:118-128). In some aspects, electroporation efficiency for engineering can be higher in activated vs. non-activated T cells (see, e.g., Zhang et al., BMC Biotechnology (2018) 18:4). In some embodiments, the T cells are monitored in accordance with the provided methods prior to engineering, for instance so that engineering can be initiated once the provided methods predict that the T cells are sufficiently activated for improved transgene expression. In some embodiments, the T cells are monitored in accordance with the provided methods following engineering, for instance to determine whether the T cells are or remain sufficiently activated following engineering to improve transgene expression.

[0120] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0121] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS FOR ASSESSING T CELL ACTIVATION

[0122] In some embodiments, the provided methods involve assessing T cells for surface expression of a T cell activation marker in cells of a composition. In some embodiments, the provided methods are for assessing T cell activation within cells of a composition. Exemplary compositions are described in Section II. In some embodiments, the provided methods involve performing any of the cell processing steps described in Section II. In some 1embodiments, the provided methods involve determining T cell activation with the T cells described in Sections II or Sections III.

[0123] In some embodiments, assessing T cells is done by detecting surface expression of a marker. In some embodiments, assessing T cells is done by detecting the expression of one or more markers. In some embodiments, the one or more markers are expressed on the surface of T cells. In some embodiments, the method involves determining the presence or absence of one or more markers on T cells on cells of a composition. In some embodiments, T cell activation is determined by expression of one or more marker. In some embodiment, an increase in the one or more marker indicates T cell activation. In some embodiments, a decrease in the one or more marker indicates T cell activation. In some embodiments, the one or more markers are used to determine the degree to which T cells are activated within cells of a composition. In some embodiments, the one or more markers are used to determine the number or percent of T cells that are activated within cells of a composition.

[0124] In some embodiments, activation of T cells is determined based on expression of a combination of one or more markers in cells of a composition. In some embodiments, T cell activation is determined based on an increase expression of some markers in the one or more markers and a decrease in other markers in the one or more markers on cells of a composition. In some embodiments, activation of T cells is determined based on expression of a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 2 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 3 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 4 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 5 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 6 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 7 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 8 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 9 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 10 markers. In some embodiments, activation of T cells is determined based on expression of a combination of 15 markers. In some embodiments, activation of T cells is determined based on expression of acombination of 20 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 20 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 10 markers. In some embodiments, activation of T cells is determined based on expression of a combination of between 2 and 5 markers.

[0125] In some embodiments, the provided methods involve determining CD4+ T cell activation in cells of a composition. In some embodiments, the provided methods involve determining CD8+ T cell activation in cells of a composition. In some embodiments, the provided methods involve determining activation of T cells containing a recombinant receptor in cells of a composition. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the provided methods involve determining the activation of CD4+ T cells containing a recombinant receptor in cells of a composition. In some embodiments, the provided methods involve determining activation of CD8 + T cells containing a recombinant receptor in cells of a composition.

[0126] In some embodiments, the cells are present in vivo in a subject. In some embodiments, the cells are isolated from a subject and their activation state is assessed ex vivo. In some embodiments, the T cells are stimulated or activated in vitro and assessed for surface expression of the one or more markers in accord with the provided methods. Exemplary stimulatory reagents for activation of cells include any as described in Section III.

[0127] In some embodiments, the stimulatory conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the cells are stimulated and the phenotype is determined by whether or not a soluble factor, e.g., a cytokine or a chemokine, is produced or secreted. In some embodiments, the stimulation is nonspecific, i.e., is not an antigen- specific stimulation. In some embodiments, cells are incubated in the presence of stimulating conditions or a stimulatory agent for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, about 72 hours, or for a duration of time between 1 hour and 4 hours,between 1 hour and 12 hours, between 12 hours and 24 hours, between 12 and 48 hours, or between 12 and 72 hours, each inclusive, or for more than 24 hours.

[0128] In some embodiments, the cells are stimulated with an agent that is an antigen or an epitope thereof that is specific to the recombinant receptor, or is an antibody or fragment thereof that binds to and / or recognizes the recombinant receptor, or a combination thereof. In some embodiments, the recombinant receptor is a CAR, and the agent is an antigen or an epitope thereof that is specific to the CAR, or is an antibody or fragment thereof that binds to and / or recognizes the CAR, or a combination thereof. In particular embodiments, the cells are stimulated by incubating the cells in the presence of target cells with surface expression of the antigen that is recognized by the CAR. In certain embodiments, the recombinant receptor is a CAR, and the agent is an antibody or an active fragment, variant, or portion thereof that binds to the CAR. In certain embodiments, the antibody or the active fragment, variant, or portion thereof that binds to the CAR is an anti-idiotypic (anti-ID) antibody.

[0129] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. In some embodiments, the one or more agents are PM A and ionomycin.A. Markers

[0130] In some embodiments, the provided methods involve assessing T cell activation based on one or more markers present on the T cells. In some embodiments, the one or more markers occur on the surface of T cells and can be used for measuring activation of T cells. In some embodiments, the markers were identified through comparing surface expression of the marker on stimulated T cells compared to unstimulated T cells. In some embodiments, the markers are were identified by comparing the expression of a potential marker to the expression of canonical markers on stimulated and unstimulated T cells to identify markers which correspond to activation. In some embodiments, flow cytometry is used to compare potential markers to canonical markers to identify markers. In some embodiments, infinity flow cytometry is used to perform a screen to identify markers by utilizing canonical markers of T cell activation as a backbone for staining across samples and then potential markers arescreened across wells, each with a unique potential marker. In some embodiments, an infinity flow cytometry screen can be used to correlate potential activation markers with canonical activation to assess which potential activation markers can be utilized as markers for assessing activation of T cells.

[0131] In some embodiments, the one or more markers for assessing activation of T cells are selected from the group consisting of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD 107b (LAMP- 2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0132] In some embodiments, the markers are either markers that (i) are upregulated upon T cell stimulation or activation or (ii) are downregulated upon T cell stimulation or activation. In some embodiments, the one or more markers are selected from group (i) and / or group (ii). In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have notbeen engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0133] In some embodiments, the samples are CD4+ CAR+ T cells, CD8+ CAR+ T cells, CD4+ PBMC T cells, and CD8+ PBMC T cells. In some embodiments, markers of group (i) are selected from the group consisting of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD 107b (LAMP- 2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R). In some embodiments, markers of group (ii) are selected from the group consisting of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MALA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDllb, CX3CR1, NKp80, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0134] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA,CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and / or the one or more markers of group (ii) are selected from the group consisting of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD172g (SIRPg), CD127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR. In some embodiments, the one or more markers are assessed 12-36 hours post stimulation. In some embodiments, the one or more markers are assessed about 24 hours post stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have not been engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0135] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (ILN-g R a chain), CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP(LRRC32); and / or the one or more markers of group (ii) are selected from the group consisting of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDl lb, CX3CR1, NKp80, CD127 (IL-7Ra), and CD49f. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed about 48 hours post stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have not been engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0136] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), GPR56, CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5); and one or more markers of group (ii) are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have not been engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR.

[0137] In some embodiments, the one or more markers are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), GPR56, CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

[0138] In some embodiments, the one or more markers are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

[0139] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56; and / or the one or more markers of group (ii) are selected from the group consisting of CD49f, CD 124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD3+CAR+ T cells. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the T cells are CD8+ CAR+ T cells. In some embodiments, the sample contains CD4+CAR+ T cells and CD8+ CAR T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ and / or CD8+ CAR T cells.

[0140] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56; and / or one or more markers of group (ii) are selected from the group consisting of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD3+CAR+ T cells. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the T cells are CD8+ CAR+ T cells. In some embodiments, the sample contains CD4+CAR+ T cells and CD8+ CAR T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ and / or CD8+ CAR T cells.

[0141] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD 154, and CD 165; and / or one or more markers of group (ii) are selected from the group consisting of CD49f, CD 124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have not been engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD4+ CAR+ T cells.

[0142] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5); and / or one or more markers of group (ii) are selected from the group consisting of CD49f, CCRL2, CD 124 (IL-4Ra), CD217, and CD192 (CCR2). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD4+ T cells engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD4+ CAR+ T cells.

[0143] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2; and / or one or more markers of group (ii) are selected from the group consisting of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers areassessed 48 hours post stimulation. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample. In some embodiments, the T cells are isolated T cells that have not been engineered. In some embodiments, the T cells are engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD8+ CAR+ T cells.

[0144] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), and GPR56; and / or one or more markers of group (ii) are selected from the group consisting of CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE). In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD8+ T cells engineered with a recombinant receptor, such as a CAR. In some embodiments, the T cells are CD8+ CAR+ T cells.

[0145] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD170 (Siglec-5); and one or more markers of group (ii) are selected from the group consisting of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE). In some embodiments, the one or more markers are selective for activation of CAR+ T cells. In some embodiments, the T cells are selective for activation of CAR+ T cells following stimulation with a CAR-dependent agent, such as an anti-idiotypic antibody or by antigen-expressing cells. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD3+CAR+ T cells. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the T cells are CD8+ CAR+ T cells. In some embodiments, the sample contains CD4+CAR+ T cells and CD8+ CAR T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ and / or CD8+ CAR T cells.

[0146] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the groupconsisting of CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), and CD74; and / or one or more markers of group (ii) are selected from the group consisting of CD49f, CCRL2, and CD 124 (IL-4Ra). In some embodiments, the one or more markers are selective for activation of CD4+CAR+ T cells. In some embodiments, the T cells are selective for activation of CD4+CAR+ T cells following stimulation with a CAR-dependent reagent, such as an anti- idiotypic antibody or by antigen-expressing cells. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD4+ CAR+ T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ T cells.

[0147] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56; and one or more markers of group (ii) are selected from the group consisting of CCRL2, CD217, CD96 (TACTILE). In some embodiments, the one or more markers are selective for activation of CD8+CAR+ T cells. In some embodiments, the T cells are selective for activation of CD8+CAR+ T cells following stimulation with a CAR-dependent reagent, such as an anti- idiotypic antibody. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD8+ CAR+ T cells. In some embodiments, the one or more markers are used to assess T cell activation on CD4+ CAR+ T cells.

[0148] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (0X2), and CD134 (0X40); and / or one or more markers of group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In someembodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample.

[0149] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, and CD83); and / or one or more markers of group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD 11b, and CX3CR1. In some embodiments, the T cells are selective for assessment of stimulation of non-engineered T cells. In some embodiments, the T cells are selective for stimulation using a pan-T cell activation such as anti-CD3 / anti-CD28. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells obtained from peripheral blood of a subject, such as from a PBMC sample.

[0150] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD 165; and / or one or more markers of group (ii) are selected from the group consisting of KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), and CD127 (IL-7Ra). In some embodiments, the T cells are selective for assessment of stimulation of nonengineered CD4+ T cells. In some embodiments, the T cells are selective for stimulation of CD4+ T cells using a pan-T cell activation such as anti-CD3 / anti-CD28. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD4+T cells obtained from peripheral blood of a subject, such as from a PBMC sample.

[0151] In some embodiments, the one or more markers are selected from group (i) and / or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, CD83; and / or one or more markers of group (ii) are selected from the group consisting of CD1 lb, CX3CR1, and CD127 (IL-7Ra). In some embodiments, the T cells are selective forassessment of stimulation of non-engineered CD8+ T cells. In some embodiments, the T cells are selective for stimulation of CD8+ T cells using a pan-T cell activation such as anti- CD3 / anti-CD28. In some embodiments, the one or more markers are assessed 24-72 hours post stimulation. In some embodiments, the one or more markers are assessed 48 hours post stimulation. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD8+T cells obtained from peripheral blood of a subject, such as from a PBMC sample.

[0152] In some embodiments, the one or more markers are differentially expressed on cells that express a recombinant receptor. In some embodiments, the one or more markers are differentially expressed on cells that express a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a CAR. In some embodiments, the surface expression of the one or more markers is increased on cells expressing a recombinant receptor than on cells that are not expressing the recombinant receptor. In some embodiments, the surface expression of the one or more markers is decreased on cells expressing a recombinant receptor than on cells that are not expressing the recombinant receptor.

[0153] In some embodiments, the one or more markers are selected from group (i) or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers of group (ii) are selected from CD96 (TACTILE). In some embodiments, the one or more markers are expressed in a composition of T cells which comprise T cells expressing a recombinant receptor. In some embodiments, the surface expression of the one or more markers of group (i) is increased on cells expressing a recombinant receptor than on cells that are not expressing the recombinant receptor. In some embodiments, there surface expression of the one or more markers of group (ii) is decreased on cells expressing a recombinant receptor than on cells that are not expressing the recombinant receptor.

[0154] In some embodiments, the one or more markers are surface proteins. In some embodiments, the one or more markers may be involved with a variety of cellular functions. In some embodiments, the one or more markers may be involved in, inter alia, metabolism, cell proliferation, cell signaling, immune response, apoptosis, etc. In some embodiments, theone or more markers are selected from group (i) or group (ii), wherein the one or more markers of group (i) are selected from the group consisting of CD262 (DR5, Trail-R2), CD105 (Endoglin), CD36L1 (SCARB1, SR-BI), CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers of group (ii) are selected from CD96 (TACTILE). In some embodiments, the one or more markers are markers listed in Table 1. Table 1 lists the markers, alternative names, and a description of the marker and its role.Table 1: Markers and their description

[0155] In some embodiments, the one or more markers are non-canonical markers. In some embodiments, the one or more markers are markers that are not typically used to assess activation of T cells (e.g., non-canonical markers). In some embodiments, the one or more markers are identified by comparing their expression or percent of positive cells to the expression or percent of positive cells expressing a canonical maker of activation, indicating the one or more marker is a non-canonical marker of activation.B. Binding Agents

[0156] In some embodiments, the T cell activation state is determined based on quantity or percent of cells that bind a binding agent. In some embodiments, one or more binding agents bind specifically to the one or more markers. In some embodiments, the one or more binding agents are antibodies. In some embodiments, the one or more antibodies are uniquely labeled. In some embodiments, the one or more antibodies are each uniquely fluorescently labeled. In some embodiments, measuring the unique labels for the antibodies is used to quantify surface expression of the one or more antibodies. In some embodiments, the unique fluorescent labels are used to quantify surface expression of the one or more antibodies. In some embodiments, the one or more antibodies are commercially obtained antibodies. / . Antibodies

[0157] In some embodiments, the markers are detected using any means for detecting a marker selected from the group consisting of CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2,Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD 107b (LAMP- 2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, TSLPR (TSLP-R), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD 127 (IL-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

[0158] In some embodiments, the one or more binding agent is one or more antibodies. As is well known in the art, an “antibody” is an immunoglobulin (Ig) molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site, located in the variable region of the Ig molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof, such as dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, chimeric antibodies, nanobodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0159] In some embodiments, each of the one or more binding agents (e.g., an antibody) comprises a means for binding, such as specifically or preferentially binding, to the respective marker. In some embodiments, binding of the binding agent (e.g, antibody) to the marker can be used for detecting one or more markers on the surface of a T cells, for example by flow cytometry, such as to determine T cell activation. In some embodiments, each of the one or more binding agents (e.g, antibody) comprises a means for detecting the one or more markers for determining T cell activation.

[0160] A binding agent, such as an antibody, that "specifically binds" or "preferentially binds" (used interchangeably herein) to marker is a term well understood in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular marker target than it does with alternative markers. An antibody specifically binds or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. It is also understood by reading this definition that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods to determine such specific or preferential binding are also well known in the art, e.g., an immunoassay.

[0161] In particular embodiments, the antibody is selected from a monoclonal antibody, a humanized antibody, a single chain antibody, an antibody fragment and combinations thereof. In some embodiments, the antibody is a full-length IgG antibody. In some embodiments, the antibody is a Fab antibody fragment. In some embodiments, the one or more antibodies are monoclonal. In some embodiments, the one or more antibodies are polyclonal.

[0162] In some embodiments, the antibody is detectably labeled. In some embodiments, the detectable label is a fluorescent label, a radiolabel or an enzymatic label. In some embodiments, the one or more antibodies are fluorescently labeled with one or more fluorescent moieties.

[0163] Any of a variety of antibody binding reagents are known and available for detecting any one or more of the above markers. Various commercial sources for antibody reagents include, but are not limited to, Thermo Fisher, Biolegend, BD Biosciences, Abeam, Bio X Cell, Invitrogen, Sigma-Aldrich, Miltenyi Biotec, Beckman, LifeSpan BioSciences Inc, Santa Cruz Bioscience, and / or Novus Biologicals.

[0164] In some embodiments, the one or more binding agents may include one or more of the following antibodies; anti-human CD107b (LAMP-2) (e.g., clone H4B4, clone 8E2F2, clone 6A10H10, clone 02, or clone AF488) anti-human CD120b (e.g., clone 3G7A02, clone MR2-1, clone 22221, clone utr 1, clone 80M2, clone 7G8B6, or 2H11CR), anti-human CD357 (GITR) (e.g., clone 108-17, clone 2H4, clone 621, clone OTI9G8, clone ANC7D6, clone ANC5E3, AIT 158D, clone 4H2D6, or clone aa26-162), anti-human CD83 (e.g., clone HB15e, clone 3G10-1F4, clone 8A4C11, or clone 6H4G10), anti-human CD200 (0X2) (e.g., clone A18042B, clone OX-104, or clone 6E8B11), anti-human CD134 (0X40) (e.g., clone Ber-ACT35, clone W4-3, clone UMAB276, clone 3G5G7, or clone OTI2F12A2), anti-human CD155 (PVR) (e.g., clone TX24, clone aa314-342, clone aa220-345, clone ANC2B2, or clone ANC6A3), anti-human CD74 (e.g., clone LN2, clone BU45, clone PIN.l, B318, clone CDLA74-1, clone 2D1B11, clone M-B741, or clone 2D1B3), anti-human CD170 (Siglec-5) (e.g., clone 1A5, clone 110, clone 3F5A3, or clone 11), anti-human Notch 1 (e.g., clone MHN1-519, clone OTI3E12, clone A6, clone mNIA, or clone 4G1), anti-human Notch 2 (e.g., clone MHN2-25, clone NOD-15, clone 8A1, cloneOT13E12, cloneA6, cloneOTI2E7, or clone 487CT6.9.2), anti-human CD166 (e.g., clone 3A6, clone 3F8B12, clone 10F1G12, clone 4h9A5, or clone 8E12C7), anti-human CD107a (LAMP-1) (e.g., clone H4A3, clone 6E2, clone 5H6, cloneLylC6, or clone OTI8B1), anti-human CD71 (e.g., clone CY1G4, clone 1E6, clone H68.4, clone 3G291, cloneDF1513, clone 10F11, clone 1A1B2, MEM-189, or clone SOM4D10), anti-human CD245 (p220 / 240) (e.g., clone DY12, or clone OTI3F7), anti-human CD154 (e.g., clone 24-31, clone 2E2, clone 8H10F5, clone 1H4, clone301, or clone5A3A9), anti-human CD165 (e.g., clone SN2, or clone AD2), anti-human CD355 (CRTAM) (e.g., clone Cr24.1, clone 12, clone 08, clone REA1225, or clone 06),. Anti-human GPR56 (e.g., clone CG4, clone REA467, or clone CG4.rMAB), anti-human CD49f (e.g., clone GoH3, clonel29CD49.6.5, clone BQ16, or clone 6B4), anti-human CD124 (IL-4Ra) (e.g., clone G077F6, clone 25463, clone 1D3, clone R401, clone R001, or clone Hil4r-M57), anti-human CCRL2 (e.g., clone K097F7, clone 12K19, or clone 1B2), anti-human CD217 (e.g., clone W15177A, clone 49M4D2, clone 004, clone 6H1B1), anti-human CD192 (CCR2) (e.g., clone K036C2, clone 7A7, clone 3B6B1, clone 4D12, clone 2A9-a, or clone 48607), anti-human CD96 (TACTILE) (e.g., clone NK92.39, clone 6f9, clone 1C8, clone 5E6C12, or clone 8A11F8), anti-human CD127 (IL-7Ra) (e.g., clone A019D5, clone AbD11590, clone HIL-7R-M21, clone IL7R / 2751, clone ANC8F2, or clone 3F5D9), anti-human CDllb (e.g.,clone ICRF44, clone REA713, clone MI / 70.15, clone CBRM1 / 5, clone MI / 70, clone X-5, or clone 3A10H5), and anti-human CX3CR1 (e.g., clone 2A9-1, clone 2B11, or clone REA385).

[0165] In provided embodiments, the binding agent, such as antibody, is conjugated to a fluorescent marker, such as a fluorophore. For instance, the cells may be incubated with one or more fluorescently labeled antibody. In some embodiments, any fluorescent marker or fluorophore suitable for use with flow cytometry analysis can be used. Some non-limiting examples of fluorescent markers include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate) (FITC), Phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF)), nucleic acid colorants (e.g., 4 ', 6-diamidino-2- phenylindole (DAPI), SYT016, propidium iodide (PI), cell membrane stain (e.g., FMI-43), cell functional dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)). Exemplary fluorphores include, but are not limited to, hydroxycoumarin, Cascade Blue, Dylight 405 Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7-Diochlorofluorescien, ATTO 488, Chromeo 488, Dylight 488, HiEyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiEyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700 (AF700), Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, PE-Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800.

[0166] In some embodiments the one or more antibodies are labeled with one or more of the following fluorescent moieties: DyEight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Fluorescein (FITC), DyEight 550, phycoerthrin (PE), Allophycocyanin (APC), Alexa Fluor 647, DyEight 650, Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor 700, StarBright Violet 440, StarBright Violet 515, StarBright Violet 610, StarBright Violet 670, StarBright Violet 700, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, APC-Cy7, cyan fluorescent protein (CFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCHERRY, Cy5, and Cy7. In some embodiments the one or more antibodies are labeled with phycoerthrin (PE).

[0167] In some embodiments, multi-color staining or labeling is carried out using multiple fluorophores in which multiple antibodies against different markers are incubated with cells. In some embodiments, the fluorescent marker, e.g., conjugated to such antibodies, are selected to minimize energy transfer between them, such as to avoid or minimize overlapping emission and absorption spectra. In some embodiments, each fluorescent marker has a different emission spectra. In some embodiments, the multiple fluorescent marker may be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap. In some embodiments, excitation of one or more of the fluorescent markers may be by light at a single or the same wavelength, but whereby different wavelengths of light are emitted therefrom.

[0168] In some embodiments, the one or more antibodies are each labeled with a unique fluorescent moiety. In some embodiments, the one or more fluorescent markers each individually comprise a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.C. Measuring Marker Surface Expression

[0169] In some embodiments, the cells are incubated with the one or more binding agents for staining or detecting cells the level or percent of cells that express the one or more markers. In some embodiments, the cell staining involves incubation with an antibody or binding agent that specifically binds to such markers, which in some embodiments is followed by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of a desired staining reagent and incubated under conditions for staining of the cells. In some embodiments, the staining or labelling is carried out at a temperature between 0°C and 25 °C, such as at or about 4°C. In some embodiments, the staining or labelling is carried out for greater than 5 minutes, typically greater than 15 minutes. In some embodiments, the staining or labelling is carried out for between 15 minutes and 6 hours, such as between 30 minutes and 2 hours. In some embodiments, the staining or labelling is carried out for example, at or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value between any of the foregoing. In some embodiments, the labeling with the one or more staining reagents is carried out simultaneously. In some embodiments, one or more wash steps are carried outprior to measuring or determining the level of the one or more markers or the percent of cells positive for the one or more markers.

[0170] In some embodiments, the one or more markers are measured by quantifying binding agents which bind to the one or more markers. In some embodiments, quantifying the binding agents is done by measuring fluorescent tags on the binding agents. In some embodiments, the binding agents are fluorescently tagged antibodies which selectively bind to the one or more markers. In some embodiments, the T cells within a cell composition are incubated with antibodies. In some embodiments, the T cells are fixed after incubation with the antibodies. In some embodiments, the T cells are alive after incubation with the antibodies. In some embodiments, the T cells are incubated with antibodies which recognize markers in addition to the one or more markers. In some embodiments, the additional markers are canonical markers that help to identify cell populations within the cell composition.

[0171] In some embodiments, the antibodies bound to the one or more markers are measured using flow cytometry. In some embodiments, the antibodies bound to the one or more markers are measured using infinity flow cytometry. In some embodiments, the antibodies are measured through immuno-histochemistry (IHC). In some embodiments, the one or more markers are measured in a tissue. In some embodiments, the cells of the cell composition are fixed and stained with antibodies before being measured with IHC.

[0172] In some embodiments, the measurements of surface markers is analyzed in FloJo. In some embodiments, populations are marked positive or negative based on gating of the cells after quantifying level of expression with flow cytometry. In some embodiments, the cells of the cell composition are determined to be positive if they are in a group with a higher level of expression in a bimodal distribution of the cells. In some embodiments, the cells of the cell composition are determined to be positive if they are in a group with a higher level of expression than other cells in the composition.D. Assessing Activation Status of T Cells

[0173] In some embodiments, assessing activation of T cells within a cell composition is done by comparing the level of surface expression of the one or more activation markers on the T cells to the level of surface expression of the one or more activation markers on an unstimulated control. In some embodiments, the unstimulated control consists of T cells. In some embodiments, the unstimulated control is a composition of cells treated the same way as the cell composition whose T cells are being assessed, except the unstimulated control isnot exposed to any of the stimulating reagents described in Section III. In some embodiments, the unstimulated control may consist of any of the cell compositions as described in Section II but the cell composition is not exposed to any stimulatory reagent, such as those described in Section III. In some embodiments, the unstimulated control comes from a patient or a donor. In some embodiments, the unstimulated control comes from the same patient or donor as the cell composition being assessed.

[0174] In some embodiments, assessing the activation of T cells in cells of a composition is done by comparing the level of surface expression of the one or more markers on the cells of the composition to the surface expression of the one or more markers on an unstimulated control where the surface level of the one or more markers on the cell composition and the unstimulated control were measured at the same time. In some embodiments, assessing the activation of T cells is done by comparing the level of surface expression of the one or more markers on the T cells to the surface expression of the one or more markers on an unstimulated control, where the surface level of the one or more markers on the cell composition and the unstimulated control were measured at different times. In some embodiments, assessing the activation of T cells in cells of a composition is done by determining if the level or percent positive of T cells within the cell composition for each of the one or more markers is higher than the level or percent positive of cells for each of the one or more markers of the unstimulated control. In some embodiments, assessing the activation of T cells in cells of a composition is done by determining if the level or percent positive of T cells within the cell composition for each of the one or more markers is lower than the level or percent positive of cells for each of the one or more markers of the unstimulated control.

[0175] In some embodiments, assessing activation of T cells within a cell composition is done by comparing the level or the percent of surface expression of the one or more activation markers on the T cells within a cell composition to the level or the percent of surface expression of the one or more activation markers in a reference. In some embodiments, assessing activation of T cells within a cell composition is done by comparing the level or the percent of surface expression of the one or more activation markers on the T cells within a cell composition to the level or the percent of surface expression of the one or more activation markers in a reference, wherein the T cells having a higher level or higher percent of the one or more activation markers than the reference indicates activation. In someembodiments, assessing activation of T cells within a cell composition is done by comparing the level or the percent of surface expression of the one or more activation markers on the T cells within a cell composition to the level or the percent of surface expression of the one or more activation markers in a reference, wherein the T cells having a lower level or lower percent of the one or more activation markers than the reference indicates activation.

[0176] In some embodiments, the reference is an unstimulated composition of cells combined from several donors or patients. In some embodiments, the reference is an average level of expression or an average of percent positive cells across a plurality of cell compositions from patients or donors, wherein the cell compositions are unstimulated. In some embodiments, the reference is a median level of expression or a median of percent positive cells across a plurality of cell compositions from patients or donors, wherein the cell compositions are unstimulated. In some embodiments, the reference is a plurality of cell compositions, each cell composition is obtained from a different donor or patient. In some embodiments, the reference is a gating scheme, wherein the gating scheme is determined based off of the level of expression or the amount of positive cells in a plurality of cell compositions, wherein each cell composition is obtain from a different patient or donor.

[0177] In some embodiments, T cell surface expression for the one or more markers is determined to be “low” “lower” or “decreased” if the percent of cells positive for the one or more markers is below the percent of cells positive for the one or more markers in a reference (e.g., an unstimulated control cell composition, the average percent of cells positive in a plurality of cell compositions, the median percent of cells positive in a plurality of cell compositions, the maximum percent of cells positive in a plurality of cell composition, the minimum percent of cells positive in a plurality of cell compositions, ect.). In some embodiments, T cell surface expression for the one or more markers is determined to be “low” “lower” or “decreased” if the surface level expression for the one or more markers is below the surface level expression for the one or more markers in a reference (e.g., the mean or median fluorescence intensity of unstimulated control cell composition, the average mean or median fluorescence intensity across a plurality of cell compositions, the median mean or median fluorescence intensity across a plurality of cell compositions, the maximum mean or median fluorescence intensity across a plurality of cell composition, the minimum mean or median fluorescence intensity across a plurality of cell compositions, ect.).

[0178] In some embodiments, T cell surface expression for the one or more markers is determined to be “high” “higher” or “increased” if the percent of cells positive for the one or more markers is above the percent of cells positive for the one or more markers in a reference (e.g., an unstimulated control cell composition, the average percent of cells positive in a plurality of cell compositions, the median percent of cells positive in a plurality of cell compositions, the maximum percent of cells positive in a plurality of cell composition, the minimum percent of cells positive in a plurality of cell compositions, ect.). In some embodiments, T cell surface expression for the one or more markers is determined to be “high” “higher” or “increased” if the surface level expression for the one or more markers is above the surface level expression for the one or more markers in a reference (e.g., the mean or median fluorescence intensity of unstimulated control cell composition, the average mean or median fluorescence intensity across a plurality of cell compositions, the median mean or median fluorescence intensity across a plurality of cell compositions, the maximum mean or median fluorescence intensity across a plurality of cell composition, the minimum mean or median fluorescence intensity across a plurality of cell compositions, ect.).

[0179] In some embodiment, T cell activation is assessed by T cells that are positive for (marker+ or markerpos) or express high levels (marker111811) of the one or more markers, or that are negative for or express relatively low levels (marker- or marker1168) of the one or more markers. Hence, it is understood that the terms positive, pos or + with reference to a marker of the one or more markers are used interchangeably herein. Likewise, it is understood that the terms negative, neg or - with reference to a marker of the one or more markers are used interchangeably herein. Further, it is understood that reference to cells that are marker1168herein may refer to cells that are negative for the marker as well as cells expressing relatively low levels of the marker, such as a low level that would not be readily detectable compared to control or background levels. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells but are present or expressed at relatively higher levels on certain other populations of lymphocytes (such as NK cells). In some cases, such markers are those that are present or expressed at relatively higher levels on certain populations of T cells but are absent or expressed at relatively low levels on certain other populations of lymphocytes (such as NK cells or subsets thereof).E. Utilization of Activation Status

[0180] In some embodiments, the method comprises incubation of the cell composition with a stimulatory reagent, such as those described in Section III. In some embodiments, the method comprises incubation of the cell composition with a stimulatory reagent before detecting T cell activation using one or more markers. In some embodiments, the method comprises incubation of the cell composition with a stimulatory reagent after detecting T cell activation using one or more markers. In some embodiments, the method comprises determining the time of incubating the cell composition with a stimulatory reagent based on the determination of T cell activation with one or more markers prior to the stimulation. In some embodiments, the method comprises determining the dose of the stimulatory reagent the cell composition is incubated with based off the determination of T cell activation with the one or more markers.

[0181] In some embodiments, the method includes engineering T cells such as described in Section III after determining T cell activation using one or more markers. In some embodiments, the method includes dosing vector or virus based on determination of the activation status of the T cells. In some embodiments, the method consists of incubating the T cells with a vector for a duration determined based off the detection of the one or more markers on the surface of the T cells. In some embodiments, the method consists of incubating the T cells with a vector for a duration determined based off the assessment of T cell activation status.

[0182] In some embodiments, the method comprises cultivating and or expansion of the cell composition after assessing T cell activation. In some embodiments, the method comprises determining the length of time the cell composition is cultivated based on T cell activation assessed using the one or more markers.

[0183] In some embodiments, the T cell population is enriched, such as by isolation or selection, from a sample by the provided methods based on T cell activation assess by the methods disclosed herein. In some embodiments, T cells that are positive for (marker+ or markerpos) or express high levels (marker111811) of the one or more markers, or that are negative for or express relatively low levels (marker- or marker1168) of the one or more markers.

[0184] In some embodiments, any known method for separation based on the one or more markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the expression or expression level of the one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some embodiments, incubation is static (without mixing). In some embodiments, incubation is dynamic (with mixing).II. CELL COMPOSITIONSA. Drug Product

[0185] In some embodiments, the cells of the cell composition are engineered to express a recombinant receptor, such as those listed in Section IV. In some embodiments, the recombinant receptor is a CAR. In some embodiments, the method of assessing T cell activation is used to determine the potency of the cells of the cell composition. In some embodiments, the method of assessing T cell activation is used to assess whether the cells of the cell composition were successfully engineered through any of the methods described in Section III. In some embodiments, the cell composition is a drug product. In some embodiments, the drug product consists of cells engineered using any of the methods listed in Section III. In some embodiments, the drug product is a therapeutic composition, including any that are presented in Section III E. In some embodiments, the drug product is delivered to a patient after the assessment of T cell activation. In some embodiments, the cells of a composition are administered to a patient after assessing T cell activation.B. T Cells being Engineered

[0186] In some embodiments, the cells of the cell composition being engineered to be used as a drug product. In some embodiment, the cells of the cell composition are cells stimulated using any of the methods described in Section III B. In some embodiments, the method for assessing T cell activation is done after the cells of the cell composition are stimulated to assess if the cells are ready to be engineered. In some embodiments, the engineering is done if T cells are determined to be activated by detecting levels of surface markers using any of the methods described in Section III C.

[0187] In some embodiments, the cell composition is assessed for T cell activation prior to transduction or engineering of the T cells. In some embodiments, the cell composition is isolated from a patient to treat a disease. In some embodiments, the cell composition is asample obtained using any of the methods in Section II. In some embodiments, the cell composition is isolated from a patient and assessed for T cell activation, prior to engineering the cell to assess the ability of the cells to be engineered. In some embodiments, the cell composition is a sample obtained using any of the methods in Section II, which were them stimulated using any of the stimulatory reagents and methods in Section II before T cell activation was assessed to prior to engineering of the cell composition.C. Peripheral Blood Mononuclear Cells

[0188] In some embodiments, the cell composition in the method of assessing T cell activation consists of peripheral blood mononuclear cells (PBMCs). In some embodiments, the method for assessing activation of T cells may be utilized in conjunction with the isolation of PBMCs. In some embodiments, the method for assessing activation of T cells is used to assess the activation of T cells within PBMCs. In some embodiments, PBMCs are incubated with stimulatory reagents, such as those listed in Section III B. In some embodiments, after the PBMCs are incubated with the stimulatory reagents, the level of surface expression of one or more markers are detected to quantify the T cell activation within the PBMCs.

[0189] PBMCs used in the methods described herein can be isolated by any standard mean. In some embodiments, PBMCs may be isolated from a patient. In some embodiments, PBMCs may be isolated from a healthy donor. In some embodiments, PBMCs may be isolated from a patient before they are in need of treatment. In some embodiments, PBMCs may be isolated from a patient after they are in need of treatment. In some embodiments, PBMCs may be isolated from a donor and T cell activity is assessed to determine how they will be used.III. METHODS FOR GENERATING ENGINEERED T CELLS

[0190] In some embodiments, the methods of assessing activation of T cells within a cell composition provided herein can be used in connection with generating a therapeutic composition of engineered cells (e.g., output composition), such as engineered CD4+ T cells and / or engineered CD8+ T cells, that express a recombinant protein, e.g., a recombinant receptor such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the methods provided herein are used in connection with manufacturing, generating, or producing a cell therapy, and may be used in connection with additionalprocessing steps, such as steps for the isolation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation of the cells. In some embodiments, the methods of generating or producing engineered cells, e.g., engineered CD4+ T cells and / or engineered CD8+ T cells, include one or more of isolating cells from a subject, preparing, processing, incubating under stimulating conditions, and / or engineering (e.g., transducing) the cells. In some embodiments, the method includes processing steps carried out in an order in which: input cells, e.g., primary cells, are first isolated, such as selected or separated, from a biological sample; input cells are incubated under stimulating conditions, engineered with vector particles, e.g., viral vector particles, to introduce a recombinant polynucleotide into the cells, e.g., by transduction or transfection; cultivating the engineered cells, e.g., transduced cells, such as to expand the cells; and collecting, harvesting, and / or filling a container with all or a portion of the cells for formulating the cells in an output composition. In some embodiments, CD4+ and CD8+ T cells are manufactured independently from one another, e.g., in separate input compositions, but the process for manufacturing includes the same processing steps. In some embodiments, CD4+ and CD8+ T cells are manufactured together, e.g., in the same input composition. In some embodiments, the cells of the generated output composition (e.g., therapeutic cell composition) are re-introduced into the same subject, before or after cryopreservation. In some embodiments, the output compositions of engineered cells (e.g., therapeutic cell composition) are suitable for use in a therapy, e.g., an autologous cell therapy, allogeneic cell therapy. Exemplary manufacturing methods are described in published international patent application, publication no. WO 2019 / 089855, the contents of which are incorporated herein by reference in their entirety.A. Samples and Cell preparations

[0191] In particular embodiments, the provided methods are used in connection with isolating, selecting, and / or enriching cells from a biological sample to generate one or more input compositions of enriched cells, e.g., T cells. In some embodiments, the provided methods include isolation of cells or compositions thereof from biological samples, such as those obtained from or derived from a subject, such as one having a particular disease or condition or in need of a cell therapy or to which cell therapy will be administered. In some aspects, the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated,processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0192] In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0193] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0194] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0195] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, selection and / or enrichment and / orincubation for transduction and engineering, and / or after cultivation and / or harvesting of the engineered cells. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. In some embodiments, the cells are frozen, e.g., cryofrozen or cryopreserved, in media and / or solution with a final concentration of or of about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9. 0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In particular embodiments, the cells are frozen, e.g., cryofrozen or cryopreserved, in media and / or solution with a final concentration of or of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to or to about -80° C. at a rate of or of about 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0196] In some embodiments, isolation of the cells or populations includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0197] In some embodiments, at least a portion of the selection step includes incubation of cells with a selection reagent. The incubation with a selection reagent or reagents, e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins,intracellular markers, or nucleic acid. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0198] In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. The immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. In some embodiments, methods are carried out using particles such as beads, e.g., magnetic beads, that are coated with a selection agent (e.g., antibody) specific to the marker of the cells. The particles (e.g., beads) can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions. In other cases, the methods include selection of cells in which all or a portion of the selection is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with selection reagents, such as immunoaffinity-based selection reagents, is performed in a centrifugal chamber. In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus described in International Patent Application, Publication Number W02009 / 072003, or US 20110003380 Al. In one example, the system is a system as described in International Publication Number W02016 / 073602.

[0199] In some embodiments, by conducting such selection steps or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of a centrifugal chamber, the user is able to control certain parameters, such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods. For example, the ability to decrease theliquid volume in the cavity during the incubation can increase the concentration of the particles (e.g., bead reagent) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This in turn can enhance the pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, carrying out the incubation step in the chamber, e.g., when associated with the systems, circuitry, and control as described herein, permits the user to effect agitation of the solution at desired time(s) during the incubation, which also can improve the interaction.

[0200] In some embodiments, at least a portion of the selection step is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent. In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinitybased selection reagent that is far less than is normally employed when performing similar selections in a tube or container for selection of the same number of cells and / or volume of cells according to manufacturer’s instructions. In some embodiments, an amount of selection reagent or reagents that is / are no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, no more than 60%, no more than 70% or no more than 80% of the amount of the same selection reagent(s) employed for selection of cells in a tube or container-based incubation for the same number of cells and / or the same volume of cells according to manufacturer’s instructions is employed.

[0201] In some embodiments, for selection, e.g., immunoaffinity-based selection of the cells, the cells are incubated in the cavity of the chamber in a composition that also contains the selection buffer with a selection reagent, such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and / or deplete, but not on other cells in the composition, such as an antibody, which optionally is coupled to a scaffold such as a polymer or surface, e.g., bead, e.g., magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. In some embodiments, as described, the selection reagent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g., is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the selection reagent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation. In some embodiments, the incubation is performed with the addition of a selectionbuffer to the cells and selection reagent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or about at least or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In some embodiments, the selection buffer and selection reagent are pre-mixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are separately added to the cells. In some embodiments, the selection incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall selection reagent while achieving a high selection efficiency.

[0202] In some embodiments, the total duration of the incubation with the selection reagent is from 5 minutes to 6 hours or from about 5 minutes to about 6 hours, such as 30 minutes to 3 hours, for example, at least or about at least 30 minutes, 60 minutes, 120 minutes or 180 minutes.

[0203] In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from 80g to 100g or from about 80g to about 100g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

[0204] In some embodiments, such process is carried out within the entirely closed system to which the chamber is integral. In some embodiments, this process (and in some aspects also one or more additional step, such as a previous wash step washing a sample containing the cells, such as an apheresis sample) is carried out in an automated fashion, such that the cells, reagent, and other components are drawn into and pushed out of the chamber at appropriate times and centrifugation effected, so as to complete the wash and binding step in a single closed system using an automated program.

[0205] In some embodiments, after the incubation and / or mixing of the cells and selection reagent and / or reagents, the incubated cells are subjected to a separation to selectfor cells based on the presence or absence of the particular reagent or reagents. In some embodiments, the separation is performed in the same closed system in which the incubation of cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagents, incubated cells, including cells in which the selection reagent has bound are transferred into a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column.

[0206] Such separation steps can be based on positive selection, in which the cells having bound the reagents, e.g., antibody or binding partner, are retained for further use, and / or negative selection, in which the cells having not bound to the reagent, e.g., antibody or binding partner, are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0207] In some embodiments, the process steps further include negative and / or positive selection of the incubated and cells, such as using a system or apparatus that can perform an affinity-based selection. In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker111811) on the positively or negatively selected cells, respectively. Multiple rounds of the same selection step, e.g., positive or negative selection step, can be performed. In certain embodiments, the positively or negatively selected fraction subjected to the process for selection, such as by repeating a positive or negative selection step. In some embodiments, selection is repeated twice, three times, four times, five times, six times, seven times, eight times, nine times or more than nine times. In certain embodiments, the same selection is performed up to five times. In certain embodiments, the same selection step is performed three times.

[0208] The separation need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers toincreasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0209] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. In certain embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, the positively or negatively selected fraction resulting from a separation step is subjected to the same separation step, such as by repeating the positive or negative selection step. In some embodiments, a single separation step is repeated and / or performed more than once, for example, to increase the yield of positively selected cells, to increase the purity of negatively selected cells, and / or to further remove the positively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed and / or repeated two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times. In certain embodiments, the one or more selection steps are performed and / or repeated between one and ten times, between one and five times, or between three and five times. In certain embodiments, one or more selection steps are repeated three times.

[0210] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibody or binding partner that specifically binds to such markers. In some embodiments, the antibody or binding partner can be conjugated, such as directly or indirectly, to a solid support or matrix to effect selection, such as a magnetic bead or paramagnetic bead. For example, CD3+, CD28+ T cells can be positively selected usingCD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads).

[0211] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0212] In some embodiments, CD8+ T cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al., (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0213] In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0214] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L.

[0215] Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ T cell population or subpopulation, also is used to generate the CD4+ T cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. In some embodiments, the selection for the CD4+ T cell population and the selection for the CD8+ T cell population are carried out simultaneously. In some embodiments, the CD4+ T cell population and the selection for the CD8+ T cell population are carried out sequentially, in either order. In some embodiments, methods for selecting cells can include those as described in published U.S. App. No. US20170037369. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined subsequent to the selecting. In some aspects, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined in a bioreactor bag as described herein. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population are separately processed, whereby the selected CD4+ T cell population is enriched in CD4+ T cells and incubated with a stimulatory reagent (e.g., anti- CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR) and cultivated under conditions to expand T cells and the selected CD8+ T cell population is enriched in CD8+ T cell and incubated with a stimulatory reagent (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR), such as the same recombinant protein as for engineering of the CD4+ T cells from the same donor, and cultivated under conditions to expand T cells, such as in accord with the provided methods.

[0216] In particular embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0217] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ T cells, where both the negative and positive fractions areretained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0218] CD4+ T helper cells may be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ T cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ T cells are CD62L- and CD45RO-.

[0219] In one example, to enrich for CD4+ T cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0220] In some aspects, the incubated sample or composition of cells to be separated is incubated with a selection reagent containing small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0221] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials for use in magnetic separation methods are known, e.g., those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference.Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 also may be used.

[0222] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0223] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0224] In some aspects, separation is achieved in a procedure in which the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0225] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS), e.g., CliniMACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in somemanner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0226] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0227] In some embodiments, the isolation and / or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more separate input composition are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate input compositions are isolated, selected, enriched, and / or obtained from separate biological samples collected, taken, and / or obtained from the same subject.

[0228] In certain embodiments, the one or more input compositions is or includes a composition of enriched T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD3+ T cells. In particular embodiment, the input composition of enriched T cells consists essentially of CD3+ T cells.

[0229] In certain embodiments, the one or more input compositions is or includes a composition of enriched CD4+ T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells. In certain embodiments, the input composition of CD4+ T cells includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells, and / or is free or substantially free of CD8+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD4+ T cells.

[0230] In certain embodiments, the one or more compositions is or includes a composition of CD8+ T cells that is or includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or contains no CD4+ T cells, and / or is free of or substantially free of CD4+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD8+ T cells.

[0231] In some embodiments, the one or more input compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryofrozen, after isolation, selection and / or enrichment. In some embodiments, the one or more input compositions of frozen e.g., cryopreserved and / or cryofrozen, prior to any steps of incubating, activating, stimulating, engineering, transducing, transfecting, cultivating, expanding, harvesting, and / or formulating the composition of cells. In particular embodiments, the one or more cryofrozen input compositions are stored, e.g., at or at about -80°C, for between 12 hours and 7 days, between 24 hours and 120 hours, or between 2 days and 5 days. In particular embodiments, the one or more cryofrozen input compositions are stored at or at about -80°C, for an amount of time of less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the one or more cryofrozen input compositions are stored at or at about -80°C, for or for about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.B. Activation and Stimulation of Cells

[0232] In some embodiments, the provided methods are used in connection with incubating cells under stimulating conditions. In some embodiments, the stimulating conditions include conditions that activate or stimulate, and / or are capable of activating or stimulating a signal in the cell, e.g., a CD4+ T cell or CD8+ T cell, such as a signal generated from a TCR and / or a coreceptor. In some embodiments, the stimulating conditions include one or more steps of culturing, cultivating, incubating, activating, propagating the cells with and / or in the presence of a stimulatory reagent, e.g., a reagent that activates or stimulates, and / or is capable of activating or stimulating a signal in the cell. In some embodiments, the stimulatory reagent stimulates and / or activates a TCR and / or a coreceptor. In particular embodiments, the stimulatory reagent is a reagent described in Section II-B-1.

[0233] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions prior to genetically engineering the cells, e.g., transfecting and / or transducing the cell such as by a technique provided in Section II-C. Inparticular embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions after the one or more compositions have been isolated, selected, enriched, or obtained from a biological sample. In particular embodiments, the one or more compositions are input compositions. In particular embodiments, the one or more input compositions have been previously cryofrozen and stored, and are thawed prior to the incubation.

[0234] In certain embodiments, the one or more compositions of enriched T cells are or include two separate compositions, e.g., separate input compositions, of enriched T cells. In particular embodiments, two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are separately incubated under stimulating conditions. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells. In particular embodiments, the two separate compositions include a composition of enriched CD8+ T cells. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are separately incubated under stimulating conditions.

[0235] In some embodiments, a single composition of enriched T cells is incubated under stimulating conditions. In certain embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that have been combined from separate compositions prior to the incubation.

[0236] In some embodiments, the composition of enriched CD4+ T cells that is incubated under stimulating conditions includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells. In certain embodiments, the composition of enriched CD4+ T cells that is incubated under stimulating conditions includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0237] In some embodiments, the composition of enriched CD8+ T cells that is incubated under stimulating conditions includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, thecomposition of enriched CD8+ T cells that is incubated under stimulating conditions includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or contains no CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0238] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and are incubated under stimulating conditions. In certain embodiments, separate stimulated compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after the incubation has been performed and / or completed. In some embodiments, separate stimulated compositions of stimulated CD4+ and stimulated CD8+ T cells are separately processed after the incubation has been performed and / or completed, whereby the stimulated CD4+ T cell population (e.g., incubated with stimulatory an anti-CD3 / anti-CD28 magnetic bead stimulatory reagent) is transduced with a viral vector encoding a recombinant protein (e.g., CAR) and cultivated under conditions to expand T cells and the stimulated CD8+ T cell population (e.g., incubated with stimulatory an anti-CD3 / anti-CD28 magnetic bead stimulatory reagent) is transduced with a viral vector encoding a recombinant protein (e.g., CAR), such as the same recombinant protein as for engineering of the CD4+ T cells from the same donor, and cultivated under conditions to expand T cells, such as in accord with the provided methods.

[0239] In some embodiments, the incubation under stimulating conditions can include culture, cultivation, stimulation, activation, propagation, including by incubation in the presence of stimulating conditions, for example, conditions designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. In particular embodiments, the stimulating conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0240] In some aspects, the stimulation and / or incubation under stimulating conditions is carried out in accordance with techniques such as those described in US Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0241] In some embodiments, the cells, e.g., T cells, compositions of cells, and / or cell populations, such as CD4+and CD8+T cells or compositions, populations, or subpopulations thereof, are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0242] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. In some embodiments, a temperature shift is effected during culture, such as from 37 degrees Celsius to 35 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0243] In embodiments, populations of CD4+and CD8+that are antigen specific can be obtained by stimulating naive or antigen specific T lymphocytes with antigen. Lor example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen. Naive T cells may also be used.

[0244] In particular embodiments, the stimulating conditions include incubating, culturing, and / or cultivating the cells with a stimulatory reagent. In particular embodiments, the stimulatory reagent is a reagent described in Section ILB-l. In certain embodiments, the stimulatory reagent contains or includes a bead. An exemplary stimulatory reagent is or includes anti-CD3 / anti-CD28 magnetic beads. In certain embodiments, the start and / or initiation of the incubation, culturing, and / or cultivating cells under stimulating conditions occurs when the cells come into contact with and / or are incubated with the stimulatoryreagent. In particular embodiments, the cells are incubated prior to, during, and / or subsequent to genetically engineering the cells, e.g., introducing a recombinant polynucleotide into the cell such as by transduction or transfection.

[0245] In some embodiments, the composition of enriched T cells are incubated at a ratio of stimulatory reagent and / or beads, e.g., anti-CD3 / anti-CD28 magnetic beads, to cells at or at about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In particular embodiments, the ratio of stimulatory reagent and / or beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1. In particular embodiments, the ratio of stimulatory reagent to cells is about 1:1 or is 1:1. In some embodiments, the ratio is determined after assessing T cell activation using the methods provided herein.

[0246] In particular embodiments, incubating the cells at a ratio of less than 3: 1 or less than 3 stimulatory reagents, e.g., anti-CD3 / anti-CD28 magnetic beads per cell, such as a ratio of 1:1, reduces the amount of cell death that occurs during the incubation, e.g., such as by activation-induced cell death. In some embodiments, the cells are incubated with the stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of beads to cells of less than 3 (or 3:1 or less than 3 beads per cell). In particular embodiments, incubating the cells at a ratio of less than 3:1 or less than 3 beads per cell, such as a ratio of 1:1, reduces the amount of cell death that occurs during the incubation, e.g., such as by activation-induced cell death.

[0247] In particular embodiments, the composition of enriched T cells is incubated with the stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of less than 3:1 stimulatory reagents and / or beads per cell, such as a ratio of 1:1, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the T cells survive, e.g., are viable and / or do not undergo necrosis, programed cell death, or apoptosis, during or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after the incubation is complete. In particular embodiments, the composition of enriched T cells is incubated with the stimulatory reagent at a ratio of less than 3:1 stimulatory reagents and / or beads per cell, e.g., a ratio of 1:1, and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1% or less than 0.01% of the cells undergo activation induced cell death during the incubation.

[0248] In certain embodiments, the composition of enriched T cells is incubated with the stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, at a ratio of less than 3:1 beads per cell, e.g., a ratio of 1:1, and the cells of the composition have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100- fold greater survival as compared to cells undergoing an exemplary and / or alternative process where the composition of enriched T cells in incubated with the stimulatory reagent at a ratio of 3:1 or greater.

[0249] In some embodiments, the composition of enriched T cells incubated with the stimulatory reagent comprises from 1.0 x 105cells / mL to 1.0 x 108cells / mL or from about 1.0 x 105cells / mL to about 1.0 x 108cells / mL, such as at least or about at least or about 1.0 x 105cells / mL, 5 x 105cells / mL, 1 x 106cells / mL, 5 x 106cells / mL, 1 x 107cells / mL, 5 x 107cells / mL or 1 x 108cells / mL. In some embodiments, the composition of enriched T cells incubated with the stimulatory reagent comprises about 0.5 x 106cells / mL, 1 x 106cells / mL,1.5 x 106cells / mL, 2 x 106cells / mL, 2.5 x 106cells / mL, 3 x 106cells / mL, 3.5 x 106cells / mL, 4 x 106cells / mL, 4.5 x 106cells / mL, 5 x 106cells / mL, 5.5 x 106cells / mL, 6 x 106cells / mL,6.5 x 106cells / mL, 7 x 106cells / mL, 7.5 x 106cells / mL, 8 x 106cells / mL, 8.5 x 106cells / mL, 9 x 106cells / mL, 9.5 x 106cells / mL, or 10 x 106cells / mL, such as about 2.4 x 106cells / mL.

[0250] In some embodiments, the composition of enriched T cells is incubated with the stimulatory reagent at a temperature from about 25 to about 38°C, such as from about 30 to about 37°C, for example at or about 37 °C ± 2 °C. In some embodiments, the composition of enriched T cells is incubated with the stimulatory reagent at a CO2 level from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example at or about 5% ± 0.5%. In some embodiments, the composition of enriched T cells is incubated with the stimulatory reagent at a temperature of or about 37 °C and / or at a CO2 level of or about 5%.

[0251] In particular embodiments, the stimulating conditions include incubating, culturing, and / or cultivating a composition of enriched T cells with and / or in the presence of one or more cytokines, In particular embodiments, the one or more cytokines are recombinant cytokines, In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and / or are capable of binding to receptors that are expressed by and / or are endogenous to T cells. Inparticular embodiments, the one or more cytokines is or includes a member of the 4-alpha- helix bundle family of cytokines. In some embodiments, members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL- 15), granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colonystimulating factor (GM-CSF). In some embodiments, the one or more cytokines is or includes IL- 15. In particular embodiments, the one or more cytokines is or includes IL-7. In particular embodiments, the one or more cytokines is or includes IL-2. In some embodiments, the stimulating conditions include incubating composition of enriched T cells, such as enriched CD4+ T cells or enriched CD8+ T cells, in the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, as described and in the presence or one or more recombinant cytokines.

[0252] In particular embodiments, the composition of enriched CD4+ T cells are incubated with IL-2, e.g., recombinant IL-2. Without wishing to be bound by theory, particular embodiments contemplate that CD4+ T cells that are obtained from some subjects do not produce, or do not sufficiently produce, IL-2 in amounts that allow for growth, division, and expansion throughout the process for generating a composition of output cells, e.g., engineered cells suitable for use in cell therapy. In some embodiments, incubating a composition of enriched CD4+ T cells under stimulating conditions in the presence of recombinant IL-2 increases the probability or likelihood that the CD4+ T cells of the composition will continue to survive, grow, expand, and / or activate during the incubation step and throughout the process. In some embodiments, incubating the composition of enriched CD4+ T cells in the presence of recombinant IL-2 increases the probability and / or likelihood that an output composition of enriched CD4+ T cells, e.g., engineered CD4+ T cells suitable for cell therapy, will be produced from the composition of enriched CD4+ T cells by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold as compared to an alternative and / orexemplary method that does not incubate the composition of enriched CD4+ T cells in the presence of recombinant IL-2.

[0253] In certain embodiments, the amount or concentration of the one or more cytokines are measured and / or quantified with International Units (IU). International units may be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, IU are or include units of measure of the potency of biological preparations by comparison to an international reference standard of a specific weight and strength e.g., WHO 1st International Standard for Human IL-2, 86 / 504. International Units are the only recognized and standardized method to report biological activity units that are published and are derived from an international collaborative research effort. In particular embodiments, the IU for composition, sample, or source of a cytokine may be obtained through product comparison testing with an analogous WHO standard product. For example, in some embodiments, the lU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to the WHO standard IL-2 product (NIBSC code: 86 / 500), the WHO standard IL- 17 product (NIBSC code: 90 / 530) and the WHO standard IL- 15 product (NIBSC code: 95 / 554), respectively.

[0254] In some embodiments, the biological activity in lU / mg is equivalent to (ED50 in ng / ml)'1xlO6. In particular embodiments, the ED50 of recombinant human IL-2 or IL- 15 is equivalent to the concentration required for the half-maximal stimulation of cell proliferation (XTT cleavage) with CTLL-2 cells. In certain embodiments, the ED50 of recombinant human IL-7 is equivalent to the concentration required for the half-maximal stimulation for proliferation of PHA-activated human peripheral blood lymphocytes. Details relating to assays and calculations of IU for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114 (1-2): 3-9; details relating to assays and calculations of IU for IL-15 are discussed in Soman et al. Journal of Immunological Methods (2009) 348 (1- 2): 83-94; hereby incorporated by reference in their entirety.

[0255] In particular embodiments, a composition of enriched CD8+ T cells is incubated under stimulating conditions in the presence of IL-2 and / or IL- 15. In certain embodiments, a composition of enriched CD4+ T cells is incubated under stimulating conditions in the presence of IL-2, IL-7, and / or IL- 15. In some embodiments, the IL-2, IL-7, and / or IL- 15 are recombinant. In certain embodiments, the IL-2, IL-7, and / or IL-15 are human. In particularembodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and / or IL- 15. In some aspects, the incubation of the enriched T cell composition also includes the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads.

[0256] In some embodiments, the cells are incubated with a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 lU / ml and 1,000 lU / ml, between 10 lU / ml and 50 lU / ml, between 50 lU / ml and 100 lU / ml, between 100 lU / ml and 200 lU / ml, between 100 lU / ml and 500 lU / ml, between 250 lU / ml and 500 lU / ml, or between 500 lU / ml and 1,000 lU / ml. In some embodiments, the lU / mL of the cytokine the cells are incubated with is based on assessing T cell activation with any of the methods provided herein.

[0257] In some embodiments, a composition of enriched T cells is incubated with IL-2, e.g., human recombinant IL-2, at a concentration between 1 lU / ml and 200 lU / ml, between 10 lU / ml and 200 lU / ml, between 10 lU / ml and 100 lU / ml, between 50 lU / ml and 150 lU / ml, between 80 lU / ml and 120 lU / ml, between 60 lU / ml and 90 lU / ml, or between 70 lU / ml and 90 lU / ml. In particular embodiments, the composition of enriched T cells is incubated with recombinant IL-2 at a concentration at or at about 50 lU / ml, 55 lU / ml, 60 lU / ml, 65 lU / ml, 70 lU / ml, 75 lU / ml, 80 lU / ml, 85 lU / ml, 90 lU / ml, 95 lU / ml, 100 lU / ml, 110 lU / ml, 120 lU / ml, 130 lU / ml, 140 lU / ml, or 150 lU / ml. In some embodiments, the composition of enriched T cells is incubated in the presence of or of about 85 lU / ml recombinant IL-2. In some embodiments, the composition incubated with recombinant IL-2 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the population of T cells is a population of CD4+ T cells. In some embodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD8+ T cells, where CD4+ T cells are not enriched for and / or where CD4+ T cells are negatively selected for or depleted from the composition. In some embodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition. In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-7 and / or recombinant IL- 15, such as in amounts described. In some embodiments, an enriched CD8+ T cell composition incubated withrecombinant IL-2 may also be incubated with recombinant IL- 15, such as in amounts described.

[0258] In some embodiments, a composition of enriched T cells is incubated with recombinant IL-7, e.g., human recombinant IL-7, at a concentration between 100 lU / ml and 2,000 lU / ml, between 500 lU / ml and 1,000 lU / ml, between 100 lU / ml and 500 lU / ml, between 500 lU / ml and 750 lU / ml, between 750 lU / ml and 1,000 lU / ml, or between 550 lU / ml and 650 lU / ml. In particular embodiments, the composition of enriched T cells is incubated with recombinant IL-7 at a concentration at or at about 50 IU / ml,100 lU / ml, 150 lU / ml, 200 lU / ml, 250 lU / ml, 300 lU / ml, 350 lU / ml, 400 lU / ml, 450 lU / ml, 500 lU / ml, 550 lU / ml, 600 lU / ml, 650 lU / ml, 700 lU / ml, 750 lU / ml, 800 lU / ml, 750 lU / ml, 750 lU / ml, 750 lU / ml, or 1,000 lU / ml. In particular embodiments, the composition of enriched T cells is incubated in the presence of or of about 600 lU / ml of recombinant IL-7. In some embodiments, the composition incubated with recombinant IL-7 is enriched for a population of T cells, e.g., CD4+ T cells. In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL-7 may also be incubated with recombinant IL-2 and / or recombinant IL- 15, such as in amounts described. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition. In some embodiments, an enriched CD8+ T cell composition is not incubated with recombinant IL-7.

[0259] In some embodiments, a composition of enriched T cells is incubated with recombinant IL- 15, e.g., human recombinant IL- 15, at a concentration between 0.1 lU / ml and 100 lU / ml, between 1 lU / ml and 100 lU / ml, between 1 lU / ml and 50 lU / ml, between 5 lU / ml and 25 lU / ml, between 25 lU / ml and 50 lU / ml, between 5 lU / ml and 15 lU / ml, or between 10 lU / ml and 100 lU / ml. In particular embodiments, the composition of enriched T cells is incubated with recombinant IL- 15 at a concentration at or at about 1 lU / ml, 2 lU / ml, 3 lU / ml, 4 lU / ml, 5 lU / ml, 6 lU / ml, 7 lU / ml, 8 lU / ml, 9 lU / ml, 10 lU / ml, 11 lU / ml, 12 lU / ml, 13 lU / ml, 14 lU / ml, 15 lU / ml, 20 lU / ml, 25 lU / ml, 30 lU / ml, 40 lU / ml, or 50 lU / ml. In some embodiments, the composition of enriched T cells is incubated in or in about 10 lU / ml of recombinant IL- 15. In some embodiments, the composition incubated with recombinant IL- 15 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the population of T cells is a population of CD4+ T cells. In someembodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD8+ T cells, where CD4+ T cells are not enriched for and / or where CD4+ T cells are negatively selected for or depleted from the composition. In some embodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition. In some embodiments, an enriched CD4+ T cell composition incubated with recombinant IL- 15 may also be incubated with recombinant IL-7 and / or recombinant IL-2, such as in amounts described. In some embodiments, an enriched CD8+ T cell composition incubated with recombinant IL- 15 may also be incubated with recombinant IL-2, such as in amounts described.

[0260] In particular embodiments, the cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated with the stimulatory reagent in the presence of one or more antioxidants. In some embodiments, antioxidants include, but are not limited to, one or more antioxidants comprise a tocopherol, a tocotrienol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alphatocopherolquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), a flavonoids, an isoflavone, lycopene, beta-carotene, selenium, ubiquinone, luetin, S-adenosylmethionine, glutathione, taurine, N-acetyl cysteine (NAC), citric acid, L-camitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, gluthatione, cystamine and cysstathionine, and / or glycine-glycine-histidine. In some aspects, the incubation of the enriched T cell composition, such as enriched CD4+ T cells and / or enriched CD8+ T cells, with an antioxidant also includes the presence of a stimulatory reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, and one or more recombinant cytokines, such as described.

[0261] In some embodiments, the one or more antioxidants is or includes a sulfur containing oxidant. In certain embodiments, a sulfur containing antioxidant may include thiol-containing antioxidants and / or antioxidants which exhibit one or more sulfur moieties, e.g., within a ring structure. In some embodiments, the sulfur containing antioxidants may include, for example, N- acetylcysteine (NAC) and 2,3- dimercaptopropanol (DMP) , L-2-oxo-4-thiazolidinecarboxylate (OTC) and lipoic acid. In particular embodiments, the sulfur containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule which may be modified in one or more steps within a cell to derived glutathione. In particular embodiments, a glutathione precursor may include, but is not limited to N-acetyl cysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (Procysteine), lipoic acid, S-allyl cysteine, or methylmethionine sulfonium chloride.

[0262] In some embodiments, incubating the cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, under stimulating conditions includes incubating the cells in the presence of one or more antioxidants. In particular embodiments, the cells are stimulated with the stimulatory reagent in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and Ipg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 1 500 pg / ml and 2 mg / ml, 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of the one or more antioxidants. In some embodiments, the cells are incubated in the presence of or of about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of the one or more antioxidant. In some embodiments, the one or more antioxidants is or includes a sulfur containing antioxidant. In particular embodiments, the one or more antioxidants is or includes a glutathione precursor.

[0263] In some embodiments, the one or more antioxidants is or includes N-acetyl cysteine (NAC). In some embodiments, incubating the cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, under stimulating conditions includes incubating the cells in the presence of NAC. In particular embodiments, the cells are stimulated with the stimulatory reagent in the presence of NAC. In some embodiments, the cells are incubated in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and Ipg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 1-500 pg / ml and 2 mg / ml, 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of NAC. In some embodiments, the cells are incubated in the presence of or of about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of NAC. In some embodiments, the cells are incubated with or with about 0.8 mg / ml. In particular embodiments, incubating the composition of enriched T cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, in the presence of one or more antioxidants, e.g., NAC, reduces the activation in the cells as compared to cells that are incubated in alternative and / or exemplary processes without the presence of antioxidants.

[0264] In some embodiments, the compositions or cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated in the presence of stimulating conditions or a stimulatory agent, such as described. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. Exemplary stimulatory reagents, such as anti-CD3 / anti-CD28 magnetic beads, are described below. The incubation with the stimulatory reagent may also be carried out in the presence of one or more stimulatory cytokine, such as in the presence of one or more of recombinant IL-2, recombinant IL-7 and / or recombinant IL- 15 and / or in the presence of at least one antioxidant such as NAC, such as described above. In some embodiments, a composition of enriched CD4+ T cells are incubated under stimulatory conditions with a stimulatory agent, recombinant IL-2, recombinant IL-7, recombinant IL- 15 and NAC, such as in amounts as described. In some embodiments, a composition of enriched CD8+ T cells are incubated under stimulatory conditions with a stimulatory agent, recombinant IL-2, recombinant IL- 15 and NAC, such as in amounts as described.

[0265] In some embodiments, the conditions for stimulation and / or activation can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0266] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0267] In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or a stimulatory agents is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation, such as described in International Publication Number WO2016 / 073602. In some embodiments, at least a portion of the incubation performed in a centrifugal chamber includes mixing with a reagent or reagents to induce stimulation and / or activation. In some embodiments, cells, such as selected cells, are mixed with a stimulating condition or stimulatory agent in the centrifugal chamber. In some aspects of such processes, a volume of cells is mixed with an amount of one or more stimulating conditions or agents that is far less than is normally employed when performing similar stimulations in a cell culture plate or other system.

[0268] In some embodiments, the stimulating agent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g., is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the stimulating agent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed without mixing in a centrifugal chamber, e.g., in a tube or bag with periodic shaking or rotation. In some embodiments, the incubation is performed with the addition of an incubation buffer to the cells and stimulating agent to achieve a target volume with incubation of the reagent of, for example, about 10 mL to about 200 mL, or about 20 mL to about 125 mL, such as at least or about at least or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 105 mL, 110 mL, 115 mL, 120 mL, 125 mL, 130 mL, 135 mL, 140 mL, 145 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, or 200 mL. In some embodiments, the incubation buffer and stimulating agent are pre-mixed before addition to the cells. In some embodiments, the incubation buffer and stimulating agent are separately added to the cells. In some embodiments, the stimulating incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall stimulating agent while achieving stimulating and activation of cells.

[0269] In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from 80g to 100g or from about 80g to about 100g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and / or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

[0270] In some embodiments, the total duration of the incubation, e.g., with the stimulating agent, is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, 18 hours and 30 hours, or 12 hours and 24 hours, such as at least or about at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours. In some embodiments, the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.

[0271] In some embodiments, the cells are cultured, cultivated, and / or incubated under stimulating conditions prior to and / or during a step for introducing a polynucleotide, e.g., a polynucleotide encoding a recombinant receptor, to the cells, e.g., by transduction and / or transfection, such as described by Section II-C. In certain embodiments the cells are cultured, cultivated, and / or incubated under stimulating conditions for an amount of time between 30 minutes and 2 hours, between 1 hour and 8 hours, between 1 hour and 6 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 16 hours and 24 hours, between 12 hours and 36 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours between 96 hours and 120 hours, between 90 hours and between 1 days and 7 days, between 3 days and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days prior to the genetic engineering. In some embodiments, the cells are incubated for or for about 2 days prior to the engineering.

[0272] In certain embodiments, the cells are incubated with and / or in the presence of the stimulatory reagent prior to and / or during genetically engineering the cells. In certain embodiments the cells are incubated with and / or in the presence of the stimulatory reagent for an amount of time between 12 hours and 36 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours between 96 hours and 120 hours, between 90 hours and between 2 days and 7 days, between3 days and 8 days, between 1 day and 8 days, between 4 days and 6 days, or between 4 days and 5 days. In particular embodiments, the cells are cultured, cultivated, and / or incubated under stimulating conditions prior to and / or during genetically engineering the cells for an amount of time of less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, or for an amount of time less than 168 hours, 162 hours, 156 hours, 144 hours, 138 hours, 132 hours, 120 hours, 114 hours, 108 hours, 102 hours, or 96 hours. In particular embodiments, the cells are incubated with and / or in the presence of the stimulatory reagent for or for about4 days, 5 days, 6 days, or 7 days. In some embodiments, the cells are incubated with and / or in the presence of the stimulatory reagent for or for about 4 days. In particular embodiments, the cells are incubated with and / or in the presence of the stimulatory reagent for or for about5 days. In certain embodiments, the cells are incubated with and / or in the presence of the stimulatory reagent for less than 7 days.

[0273] In some embodiments, incubating the cells under stimulating conditions includes incubating the cells with a stimulatory reagent that is described in Section II-B-1. In some embodiments, the stimulatory reagent contains or includes a bead, such as a paramagnetic bead, and the cells are incubated with the stimulatory reagent at a ratio of less than 3:1 (beads :cells), such as a ratio of 1:1. In particular embodiments, the cells are incubated with the stimulatory reagent in the presence of one or more cytokines and / or one or more antioxidants. In some embodiments, a composition of enriched CD4+ T cells is incubated with the stimulatory reagent at a ratio of 1:1 (beads :cells) in the presence of recombinant IL- 2, IL-7, IL- 15, and NAC. In certain embodiments, a composition of enriched CD8+ T cells is incubated with the stimulatory reagent at a ratio of 1:1 (beads :cells) in the presence of recombinant IL-2, IL-15, and NAC. In some embodiments, the stimulatory reagent is removed and / or separated from the cells at, within, or within about 6 days, 5 days, or 4 days from the start or initiation of the incubation, e.g., from the time the stimulatory reagent is added to or contacted with the cells. / . Stimulatory S 'eagents

[0274] In some embodiments, incubating a composition of enriched cells under stimulating conditions is or includes incubating and / or contacting the composition of enriched cells with a stimulatory reagent that is capable of activating and / or expanding T cells. In some embodiments, the stimulatory reagent is capable of stimulating and / or activating one or more signals in the cells. In some embodiments, the one or more signals aremediated by a receptor. In particular embodiments, the one or more signals are or are associated with a change in signal transduction and / or a level or amount of secondary messengers, e.g., cAMP and / or intracellular calcium, a change in the amount, cellular localization, confirmation, phosphorylation, ubiquitination, and / or truncation of one or more cellular proteins, and / or a change in a cellular activity, e.g., transcription, translation, protein degradation, cellular morphology, activation state, and / or cell division. In particular embodiments, the stimulatory reagent activates and / or is capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules.

[0275] In certain embodiments, the stimulatory reagent contains a particle, e.g., a bead, that is conjugated or linked to one or more agents, e.g., biomolecules, that are capable of activating and / or expanding cells, e.g., T cells. In some embodiments, the one or more agents are bound to a bead. In some embodiments, the bead is biocompatible, i.e., composed of a material that is suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells, e.g., cultured T cells. In some embodiments, the beads may be any particles which are capable of attaching agents in a manner that permits an interaction between the agent and a cell.

[0276] In some embodiments, a stimulatory reagent contains one or more agents that are capable of activating and / or expanding T cells, e.g., a pan-T cell activation reagent. In some embodiments, the pan-T cell activation reagent comprises anti-CD3 / antiCD28 beads. In some embodiments, the pan-T cell activation reagent comprises anti-CD3 / antiCD28 streptavidin oligomeric reagents.

[0277] In some embodiments, a stimulatory reagent contains one or more agents that are capable of activating and / or expanding cells, e.g., T cells, that are bound to or otherwise attached to a bead, for example to the surface of the bead. In certain embodiments, the bead is a non-cell particle. In particular embodiments, the bead may include a colloidal particle, a microsphere, nanoparticle, a magnetic bead, or the like. In some embodiments the beads are agarose beads. In certain embodiments, the beads are sepharose beads.

[0278] In particular embodiments, the stimulatory reagent contains beads that are monodisperse. In certain embodiments, beads that are monodisperse comprise size dispersions having a diameter standard deviation of less than 5% from each other.

[0279] In some embodiments, the bead contains one or more agents, such as an agent that is coupled, conjugated, or linked (directly or indirectly) to the surface of the bead. In some embodiments, an agent as contemplated herein can include, but is not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids lectins, or any other biomolecule with an affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiment, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137 (4-1- BB), 0X40, or ICOS. The one or more agents may be attached directly or indirectly to the bead by a variety of methods known and available in the art. The attachment may be covalent, noncovalent, electrostatic, or hydrophobic and may be accomplished by a variety of attachment means, including for example, a chemical means, a mechanical means, or an enzymatic means. In some embodiments, a biomolecule (e.g., a biotinylated anti-CD3 antibody) may be attached indirectly to the bead via another biomolecule (e.g., anti-biotin antibody) that is directly attached to the bead.

[0280] In some embodiments, the stimulatory reagent contains a bead and one or more agents that directly interact with a macromolecule on the surface of a cell. In certain embodiments, the bead (e.g., a paramagnetic bead) interacts with a cell via one or more agents (e.g., an antibody) specific for one or more macromolecules on the cell (e.g., one or more cell surface proteins). In certain embodiments, the bead (e.g., a paramagnetic bead) is labeled with a first agent described herein, such as a primary antibody (e.g., an anti-biotin antibody) or other biomolecule, and then a second agent, such as a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other second biomolecule (e.g., streptavidin), is added, whereby the secondary antibody or other second biomolecule specifically binds to such primary antibodies or other biomolecule on the particle.

[0281] In some embodiments, the stimulatory reagent contains one or more agents (e.g., antibody) that is attached to a bead (e.g., a paramagnetic bead) and specifically binds to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHCI, MHCII, CTLA-4, ICOS, PD-1, 0X40, CD27L (CD70), 4-1BB (CD137), 4- 1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gammaR, TNF-alphaR, IL-4R, IL- 10R, CD18 / CD1 la (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligand(e.g., Delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3 or fragment thereof including the corresponding ligands to these macromolecules or fragments thereof. In some embodiments, an agent (e.g., antibody) attached to the bead specifically binds to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD 127, CD3, CD4, CD8, CD45RA, and / or CD45RO.In some embodiments, one or more of the agents attached to the bead is an antibody. The antibody can include a polyclonal antibody, monoclonal antibody (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulatory reagent is an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. It will be appreciated that constant regions of any isotype can be used for the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species (e.g., murine species).

[0282] In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of a T cell receptor. In particular embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulatory reagent comprises an anti-CD28 antibody. In particular embodiments, the stimulatory agent contains an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the antibody is a Fab. In some embodiments, the stimulatory agent contains an anti-CD3 Fab and an anti-CD28 Fab.

[0283] In some embodiments, the stimulating agent is an anti-CD3 / anti-CD28 streptavidin oligomeric reagent, such as described in PCT publication No. WO / 2015 / 158868 or WO2019 / 197949. In some embodiments, the streptavidin is a a recombinant Streptactin, which is a mutant streptavidin. In some embodiments, Streptactin is a mutant streptavidin that contains mutations Val-Thr-Ala-Arg or Ile-Gly-Ala-Arg at positions 44-47 of a wild-type streptavidin thus replacing wild-type amino acids Glu-Ser-Ala-Val. In some embodiments, the stimulating agent is soluble. In some embodiments, the reagent is a soluble reagent of anti-CD3 / CD28 Fab fragments linked to a recombinant Streptactin backbone. In some embodiments, the stimulating agent is an Expamer™.

[0284] In some embodiments, the simulating agents are anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads).

[0285] In some embodiments, the bead has a diameter of greater than about 0.001 pm, greater than about 0.01 pm, greater than about 0.1 pm, greater than about 1.0 pm, greater than about 10 pm, greater than about 50 pm, greater than about 100 pm or greater than about 1000 pm and no more than about 1500 pm. In some embodiments, the bead has a diameter of about 1.0 pm to about 500 pm, about 1.0 pm to about 150 pm, about 1.0 pm to about 30 pm, about 1.0 pm to about 10 pm, about 1.0 pm to about 5.0 pm, about 2.0 pm to about 5.0 pm, or about 3.0 pm to about 5.0 pm. In some embodiments, the bead has a diameter of about 3 pm to about 5 pm. In some embodiments, the bead has a diameter of at least or at least about or about 0.001 pm, 0.01 pm, 0.1 pm, 0.5 pm, 1.0 pm, 1.5 pm, 2.0 pm, 2.5 pm, 3.0 pm, 3.5 pm, 4.0 pm, 4.5 pm, 5.0 pm, 5.5 pm, 6.0 pm, 6.5 pm, 7.0 pm, 7.5 pm, 8.0 pm, 8.5 pm, 9.0 pm, 9.5 pm, 10 pm, 12 pm, 14 pm, 16 pm, 18 pm or 20 pm. In certain embodiments, the bead has a diameter of or about 4.5 pm. In certain embodiments, the bead has a diameter of or about 2.8 pm.

[0286] In some embodiments, the beads have a density of greater than 0.001 g / cm3, greater than 0.01 g / cm3, greater than 0.05 g / cm3, greater than 0.1 g / cm3, greater than 0.5 g / cm3, greater than 0.6 g / cm3, greater than 0.7 g / cm3, greater than 0.8 g / cm3, greater than 0.9 g / cm3, greater than 1 g / cm3, greater than 1.1 g / cm3, greater than 1.2 g / cm3, greater than 1.3 g / cm3, greater than 1.4 g / cm3, greater than 1.5 g / cm3, greater than 2 g / cm3, greater than 3 g / cm3, greater than 4 g / cm3, or greater than 5g / cm3. In some embodiments, the beads have a density of between about 0.001 g / cm3and about 100 g / cm3, about 0.01 g / cm3and about 50 g / cm3, about 0.1 g / cm3and about 10 g / cm3, about 0.1 g / cm3and about .5 g / cm3, about 0.5 g / cm3and about 1 g / cm3, about 0.5 g / cm3and about 1.5 g / cm3, about 1 g / cm3and about 1.5 g / cm3, about 1 g / cm3and about 2 g / cm3, or about 1 g / cm3and about 5 g / cm3. In some embodiments, the beads have a density of about 0.5 g / cm3, about 0.5 g / cm3, about 0.6 g / cm3, about 0.7 g / cm3, about 0.8 g / cm3, about 0.9 g / cm3, about 1.0 g / cm3, about 1.1 g / cm3, about 1.2 g / cm3, about 1.3 g / cm3, about 1.4 g / cm3, about 1.5 g / cm3, about 1.6 g / cm3, about 1.7 g / cm3, about 1.8 g / cm3, about 1.9 g / cm3, or about 2.0 g / cm3. In certain embodiments, the beads have a density of about 1.6 g / cm3. In particular embodiments, the beads or particles have a density of about 1.5 g / cm3. In certain embodiments, the particles have a density of about 1.3 g / cm3.

[0287] In certain embodiments, a plurality of the beads has a uniform density. In certain embodiments, a uniform density comprises a density standard deviation of less than 10%, less than 5%, or less than 1% of the mean bead density.

[0288] In some embodiments, the beads have a surface area of between about 0.001 m2per each gram of particles (m2 / g) to about 1,000 m2 / g, about .010 m2 / g to about 100 m2 / g, about 0.1 m2 / g to about 10 m2 / g, about 0.1 m2 / g to about 1 m2 / g, about 1 m2 / g to about 10 m2 / g, about 10 m2 / g to about 100 m2 / g, about 0.5 m2 / g to about 20 m2 / g, about 0.5 m2 / g to about 5 m2 / g, or about 1 m2 / g to about 4 m2 / g. In some embodiments, the particles or beads have a surface area of about 1 m2 / g to about 4 m2 / g.

[0289] In some embodiments, the bead reacts in a magnetic field. In some embodiments, the bead is a magnetic bead. In some embodiments, the magnetic bead is paramagnetic. In particular embodiments, the magnetic bead is superparamagnetic. In certain embodiments, the beads do not display any magnetic properties unless they are exposed to a magnetic field.

[0290] In particular embodiments, the bead comprises a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal can be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium or any combinations thereof. In certain embodiments, the magnetic core comprises metal oxides (e.g., iron oxides), ferrites (e.g., manganese ferrites, cobalt ferrites, nickel ferrites, etc.), hematite and metal alloys (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, an iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), maghemite (yFe2O3), or greigite (Fe3S4). In some embodiments, the inner core comprises an iron oxide (e.g., FeaC ).

[0291] In certain embodiments, the bead contains a magnetic, paramagnetic, and / or superparamagnetic core that is covered by a surface functionalized coat or coating. In some embodiments, the coat can contain a material that can include, but is not limited to, a polymer, a polysaccharide, a silica, a fatty acid, a protein, a carbon, agarose, sepharose, or a combination thereof. In some embodiments, the polymer can be a polyethylene glycol, poly (lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or a polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In particular embodiments, the outer coating is surface functionalized.

[0292] In some embodiments, the stimulatory reagent comprises a bead that contains a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coat comprises at least one polysaccharide (e.g., amino dextran), at least one polymer (e.g., polyurethane) and silica. In some embodiments the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents include an antibody or antigen-binding fragment thereof. In particular embodiments, the one or more agents include an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragments thereof. In some embodiments, the stimulatory reagent comprises an anti-CD3 antibody, anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulatory reagent comprises an anti-biotin antibody. In some embodiments, the bead has a diameter of about 3 pm to about 10 pm. In some embodiments, the bead has a diameter of about 3 pm to about 5 pm. In certain embodiments, the bead has a diameter of about 3.5 pm.

[0293] In some embodiments, the stimulatory reagent comprises one or more agents that are attached to a bead comprising a metal oxide core (e.g., an iron oxide inner core) and a coat (e.g., a protective coat), wherein the coat comprises polystyrene. In certain embodiments, the beads are monodisperse, paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core, e.g., a core comprising magnetite ( FC3O4) and / or maghemite (yFc2O3) c and a polystyrene coat or coating. In some embodiments, the bead is non-porous. In some embodiments, the beads contain a functionalized surface to which the one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads at the surface. In some embodiments, the one or more agents include an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents include an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent is or comprises anti- CD3 / anti-CD28 magnetic beads. In some embodiments, the one or more agents include an anti-CD3 antibody and / or an anti-CD28 antibody, and an antibody or antigen fragment thereof capable of binding to a labeled antibody (e.g., biotinylated antibody), such as a labeled anti-CD3 or anti-CD28 antibody. In certain embodiments, the beads have a density of about 1.5 g / cm3and a surface area of about 1 m2 / g to about 4 m2 / g. In particular embodiments; the beads are monodisperse superparamagnetic beads that have a diameter of about 4.5 pm and a density of about 1.5 g / cm3. In some embodiments, the beads the beadsare monodisperse superparamagnetic beads that have a mean diameter of about 2.8 pm and a density of about 1.3 g / cm3.

[0294] In some embodiments, the composition of enriched T cells is incubated with stimulatory reagent a ratio of beads to cells at or at about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In particular embodiments, the ratio of beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1. In particular embodiments, the ratio of stimulatory reagent to cells is about 1:1 or is 1:1.C. Engineering Cells

[0295] In some embodiments, the provided methods involve engineering cells with a recombinant antigen receptor. Various methods for the introduction of genetically engineered components, e.g., recombinant receptors, e.g., CARs or TCRs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[0296] Among the cells expressing the receptors and administered by the provided methods are engineered cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into a composition containing the cells, such as by retroviral transduction, transfection, or transformation.

[0297] In some embodiments, the methods provided herein are used in association with engineering one or more compositions of enriched T cells. In certain embodiments, the engineering is or includes the introduction of a polynucleotide, e.g., a recombinant polynucleotide encoding a recombinant protein. In particular embodiments, the recombinant proteins are recombinant receptors, such as any described in Section II. Introduction of the nucleic acid molecules encoding the recombinant protein, such as recombinant receptor, in the cell may be carried out using any of a number of known vectors. Such vectors include viral and non- viral systems, including lentiviral and gammaretroviral systems, as well as transposon-based systems such as PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation. In some embodiments, the engineering produces one or more engineered compositions of enriched T cells.

[0298] In certain embodiments, one or more compositions of enriched T cells are engineered, e.g., transduced or transfected, prior to cultivating the cells, e.g., under conditions that promote proliferation and / or expansion, such as by a method provided in Section III-D. In particular embodiments, one or more compositions of enriched T cells are engineered after the one or more compositions have been stimulated, activated, and / or incubated under stimulating conditions, such as described in methods provided in Section III-B. In particular embodiments, the one or more compositions are stimulated compositions. In particular embodiments, the one or more stimulated compositions have been previously cryofrozen and stored, and are thawed prior to engineering.

[0299] In certain embodiments, the one or more compositions of stimulated T cells are or include two separate stimulated compositions of enriched T cells. In particular embodiments, two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells that have been selected, isolated, and / or enriched from the same biological sample, are separately engineered. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells. In particular embodiments, the two separate compositions include a composition of enriched CD8+ T cells. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells, such as following incubation under stimulating conditions as described above, are genetically engineered separately. In some embodiments, a single composition of enriched T cells is genetically engineered. In certain embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that have been combined from separate compositions prior to the engineering.

[0300] In some embodiments, the composition of enriched CD4+ T cells, such as stimulated CD4+ T cells, that is engineered, e.g., transduced or transfected, includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells. In certain embodiments, the composition of enriched CD4+ T cells, such as stimulated CD4+ T cells, that is engineered includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0301] In some embodiments, the composition of enriched CD8+ T cells, such as stimulated CD8+ T cells, that is engineered, e.g., transduced or transfected, includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, the composition of enriched CD8+ T cells that, such as stimulated CD8+ T cells, that is engineered includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or contains no CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0302] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and are genetically engineered, e.g., transduced or transfected. In certain embodiments, separate engineered compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after the genetic engineering has been performed and / or completed. In particular embodiments, separate compositions of enriched CD4+ and CD8+ T cells, such as separate compositions of stimulated CD4+ and CD8+ T cells are separately engineered and are separately processed for cultivation and / or expansion of T cells after the genetic engineering and been performed and / or completed.

[0303] In some embodiments, the introduction of a polynucleotide, e.g., a recombinant polynucleotide encoding a recombinant protein, is carried out by contacting enriched CD4+ or CD8+ T cells, such as stimulated CD4+ or CD8+ T cells, with a viral particles containing the polynucleotide. In some embodiments, contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing cells, viral particles and reagent can be rotated, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, the rotation is carried at a force, e.g., a relative centrifugal force, of from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), such as at or about 693 g, as measured for example at an internal or external wall of the chamber or cavity. The term “relative centrifugal force” or RCF is generally understood to be the effective force imparted on an object or substance(such as a cell, sample, or pellet and / or a point in the chamber or other container being rotated), relative to the earth’s gravitational force, at a particular point in space as compared to the axis of rotation. The value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured). In some embodiments, at least a portion of the contacting, incubating, and / or engineering of the cells, e.g., cells from an stimulated composition of enriched CD4+ T cell or enriched CD8+ T cells, with the virus is performed with a rotation of between about 100 g and 3200 g, 1000 g and 2000 g, 1000 g and 3200 g, 500 g and 1000 g, 400 g and 1200 g, 600g and 800 g, 600 and 700g, or 500 g and 700 g. In some embodiments, the rotation is between 600 g and 700 g, e.g., at or about 693 g.

[0304] In certain embodiments, at least a portion of the engineering, transduction, and / or transfection is performed with rotation, e.g., spinoculation and / or centrifugation. In some embodiments, the rotation is performed for, for about, or for at least or about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or for at least 7 days. In some embodiments, the rotation is performed for or for about 60 minutes. In certain embodiments, the rotation is performed for about 30 minutes. In some embodiments, the rotation performed for about 30 minutes at between 600 g and 700 g, e.g., at or about 693 g.

[0305] In certain embodiments, the number of viable cells to be engineered, transduced, and / or transfected ranges from about 5 x 106cells to about 100 x 107cells, such as from about 10 x 106cells to about 100 x 106cells, from about 100 x 106cells to about 200 x 106cells, from about 200 x 106cells to about 300 x 106cells, from about 300 x 106cells to about 400 x 106cells, from about 400 x 106cells to about 500 x 106cells, or from about 500 x 106cells to about 100 x 107cells. In particular examples, the number of viable cells to be engineered, transduced, and / or transfected is about or less than about 300 x 106cells.

[0306] In certain embodiments, at least a portion of the engineering, transduction, and / or transfection is conducted at a volume e.g., the spinoculation volume) from about 5 mL to about 100 mL, such as from about 10 mL to about 50 mL, from about 15 mL to about 45 mL, from about 20 mL to about 40 mL, from about 25 mL to about 35 mL, or at or at about 30 mL. In certain embodiments, the cell pellet volume after spinoculation ranges from about 1mL to about 25 mL, such as from about 5 mL to about 20 mL, from about 5 mL to about 15 mL, from about 5 mL to about 10 mL, or at or at about 10 mL.

[0307] In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications. In certain embodiments, the gene transfer is accomplished by first incubating the cells under stimulating conditions, such as by any of the methods described in Section LB.

[0308] In some embodiments, methods for genetic engineering are carried out by contacting one or more cells of a composition with a nucleic acid molecule encoding the recombinant protein, e.g., recombinant receptor. In some embodiments, the contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation). Such methods include any of those as described in International Publication Number WO20 16 / 073602. Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A- 200 / F and A-200 centrifugal chambers and various kits for use with such systems.Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008 / 0171951, and published international patent application, publication no. WO 00 / 38762, the contents of each of which are incorporated herein by reference in their entirety. Exemplary kits for use with such systems include, but are not limited to, single-use kits sold by BioSafe SA under product names CS-430.1, CS- 490.1, CS-600.1 or CS-900.2.

[0309] In some embodiments, the system is included with and / or placed into association with other instrumentation, including instrumentation to operate, automate, control and / or monitor aspects of the transduction step and one or more various other processing steps performed in the system, e.g., one or more processing steps that can be carried out with or in connection with the centrifugal chamber system as described herein or in International Publication Number W02016 / 073602. This instrumentation in some embodiments is contained within a cabinet. In some embodiments, the instrumentation includes a cabinet, which includes a housing containing control circuitry, a centrifuge, a cover, motors, pumps,sensors, displays, and a user interface. An exemplary device is described in US Patent No. 6,123,655, US Patent No. 6,733,433 and US 2008 / 0171951.

[0310] In some embodiments, the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber. In some embodiments, the containers, such as bags, include one or more containers, such as bags, containing the cells to be transduced and the viral vector particles, in the same container or separate containers, such as the same bag or separate bags. In some embodiments, the system further includes one or more containers, such as bags, containing medium, such as diluent and / or wash solution, which is pulled into the chamber and / or other components to dilute, resuspend, and / or wash components and / or compositions during the methods. The containers can be connected at one or more positions in the system, such as at a position corresponding to an input line, diluent line, wash line, waste line and / or output line.

[0311] In some embodiments, the chamber is associated with a centrifuge, which is capable of effecting rotation of the chamber, such as around its axis of rotation. Rotation may occur before, during, and / or after the incubation in connection with transduction of the cells and / or in one or more of the other processing steps. Thus, in some embodiments, one or more of the various processing steps is carried out under rotation, e.g., at a particular force. The chamber is typically capable of vertical or generally vertical rotation, such that the chamber sits vertically during centrifugation and the side wall and axis are vertical or generally vertical, with the end wall(s) horizontal or generally horizontal.

[0312] In some embodiments, the composition containing cells and composition containing viral vector particles, and optionally air, can be combined or mixed prior to providing the compositions to the cavity. In some embodiments, the composition containing cells and composition containing viral vector particles, and optionally air, are provided separately and combined and mixed in the cavity. In some embodiments, a composition containing cells, a composition containing viral vector particles, and optionally air, can be provided to the internal cavity in any order. In any of such some embodiments, a composition containing cells and viral vector particles is the input composition once combined or mixed together, whether such is combined or mixed inside or outside the centrifugal chamber and / or whether cells and viral vector particles are provided to the centrifugal chamber together or separately, such as simultaneously or sequentially.

[0313] In some embodiments, intake of a volume of gas, such as air, occurs prior to the incubating the cells and viral vector particles, such as rotation, in the transduction method. In some embodiments, intake of the volume of gas, such as air, occurs during the incubation of the cells and viral vector particles, such as rotation, in the transduction method.

[0314] In some embodiments, the liquid volume of the cells or viral vector particles that make up the transduction composition, and optionally the volume of air, can be a predetermined volume. The volume can be a volume that is programmed into and / or controlled by circuitry associated with the system.

[0315] In some embodiments, intake of the transduction composition, and optionally gas, such as air, is controlled manually, semi-automatically and / or automatically until a desired or predetermined volume has been taken into the internal cavity of the chamber. In some embodiments, a sensor associated with the system can detect liquid and / or gas flowing to and from the centrifuge chamber, such as via its color, flow rate and / or density, and can communicate with associated circuitry to stop or continue the intake as necessary until intake of such desired or predetermined volume has been achieved. In some aspects, a sensor that is programmed or able only to detect liquid in the system, but not gas (e.g., air), can be made able to permit passage of gas, such as air, into the system without stopping intake. In some such embodiments, a non-clear piece of tubing can be placed in the line near the sensor while intake of gas, such as air, is desired. In some embodiments, intake of gas, such as air, can be controlled manually.

[0316] In aspects of the provided methods, the internal cavity of the centrifuge chamber is subjected to high speed rotation. In some embodiments, rotation is effected prior to, simultaneously, subsequently or intermittently with intake of the liquid input composition, and optionally air. In some embodiments, rotation is effected subsequent to intake of the liquid input composition, and optionally air. In some embodiments, rotation is by centrifugation of the centrifugal chamber at a relative centrifugal force at the inner surface of side wall of the internal cavity and / or at a surface layer of the cells of at or about or at least at or about 800 g, 1000 g, 1100 g, 1500, 1600 g, 1800 g, 2000 g, 2200 g, 2500 g, 3000 g, 3500 g or 4000 g. In some embodiments, rotation is by centrifugation at a force that is greater than or about 1100 g, such as by greater than or about 1200 g, greater than or about 1400 g, greater than or about 1600 g, greater than or about 1800 g, greater than or about 2000 g, greater than or about 2400 g, greater than or about 2800 g, greater than or about 3000 g or greater than orabout 3200 g. In some embodiments, rotation is by centrifugation at a force that is or is about 1600 g.

[0317] In some embodiments, the method of transduction includes rotation or centrifugation of the transduction composition, and optionally air, in the centrifugal chamber for greater than or about 5 minutes, such as greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes or greater than or about 120 minutes. In some embodiments, the transduction composition, and optionally air, is rotated or centrifuged in the centrifugal chamber for greater than 5 minutes, but for no more than 60 minutes, no more than 45 minutes, no more than 30 minutes or no more than 15 minutes. In particular embodiments, the transduction includes rotation or centrifugation for or for about 60 minutes.

[0318] In some embodiments, the method of transduction includes rotation or centrifugation of the transduction composition, and optionally air, in the centrifugal chamber for between or between about 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive, and at a force at the internal surface of the side wall of the internal cavity and / or at a surface layer of the cells of at least or greater than or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g. In particular embodiments, the method of transduction includes rotation or centrifugation of the transduction composition, e.g., the cells and the viral vector particles, at or at about 1600 g for or for about 60 minutes.

[0319] In some embodiments, the gas, such as air, in the cavity of the chamber is expelled from the chamber. In some embodiments, the gas, such as air, is expelled to a container that is operably linked as part of the closed system with the centrifugal chamber. In some embodiments, the container is a free or empty container. In some embodiments, the air, such as gas, in the cavity of the chamber is expelled through a filter that is operably connected to the internal cavity of the chamber via a sterile tubing line. In some embodiments, the air is expelled using manual, semi-automatic or automatic processes. In some embodiments, air is expelled from the chamber prior to, simultaneously, intermittently or subsequently with expressing the output composition containing incubated cells and viral vector particles, suchas cells in which transduction has been initiated or cells have been transduced with a viral vector, from the cavity of the chamber.

[0320] In some embodiments, the transduction and / or other incubation is performed as or as part of a continuous or semi-continuous process. In some embodiments, a continuous process involves the continuous intake of the cells and viral vector particles, e.g., the transduction composition (either as a single pre-existing composition or by continuously pulling into the same vessel, e.g., cavity, and thereby mixing, its parts), and / or the continuous expression or expulsion of liquid, and optionally expelling of gas (e.g., air), from the vessel, during at least a portion of the incubation, e.g., while centrifuging. In some embodiments, the continuous intake and continuous expression are carried out at least in part simultaneously. In some embodiments, the continuous intake occurs during part of the incubation, e.g., during part of the centrifugation, and the continuous expression occurs during a separate part of the incubation. The two may alternate. Thus, the continuous intake and expression, while carrying out the incubation, can allow for a greater overall volume of sample to be processed, e.g., transduced.

[0321] In some embodiments, the incubation is part of a continuous process, the method including, during at least a portion of the incubation, effecting continuous intake of said transduction composition into the cavity during rotation of the chamber and during a portion of the incubation, effecting continuous expression of liquid and, optionally expelling of gas (e.g., air), from the cavity through the at least one opening during rotation of the chamber.

[0322] In some embodiments, the semi-continuous incubation is carried out by alternating between effecting intake of the composition into the cavity, incubation, expression of liquid from the cavity and, optionally expelling of gas (e.g., air) from the cavity, such as to an output container, and then intake of a subsequent (e.g., second, third, etc.) composition containing more cells and other reagents for processing, e.g., viral vector particles, and repeating the process. For example, in some embodiments, the incubation is part of a semi- continuous process, the method including, prior to the incubation, effecting intake of the transduction composition into the cavity through said at least one opening, and subsequent to the incubation, effecting expression of fluid from the cavity; effecting intake of another transduction composition comprising cells and the viral vector particles into said internal cavity; and incubating the another transduction composition in said internal cavity under conditions whereby said cells in said another transduction composition are transduced withsaid vector. The process may be continued in an iterative fashion for a number of additional rounds. In this respect, the semi-continuous or continuous methods may permit production of even greater volume and / or number of cells.

[0323] In some embodiments, a portion of the transduction incubation is performed in the centrifugal chamber, which is performed under conditions that include rotation or centrifugation.

[0324] In some embodiments, the method includes an incubation in which a further portion of the incubation of the cells and viral vector particles is carried out without rotation or centrifugation, which generally is carried out subsequent to the at least portion of the incubation that includes rotation or centrifugation of the chamber. In certain embodiments, the incubation of the cells and viral vector particles is carried out without rotation or centrifugation for at least 1 hour, 6 hours, 12 hours, 24 hours, 32 hours, 48 hours, 60 hours, 72 hours, 90 hours, 96 hours, 3 days, 4 days, 5 days, or greater than 5 days. In certain embodiments, the incubation is carried out for or for about 72 hours.

[0325] In some such embodiments, the further incubation is effected under conditions to result in integration of the viral vector into a host genome of one or more of the cells. It is within the level of a skilled artisan to assess or determine if the incubation has resulted in integration of viral vector particles into a host genome, and hence to empirically determine the conditions for a further incubation. In some embodiments, integration of a viral vector into a host genome can be assessed by measuring the level of expression of a recombinant protein, such as a heterologous protein, encoded by a nucleic acid contained in the genome of the viral vector particle following incubation. A number of well-known methods for assessing expression level of recombinant molecules may be used, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins, such as by flow cytometry. In some examples, the expression is measured by detection of a transduction marker and / or reporter construct. In some embodiments, nucleic acid encoding a truncated surface protein is included within the vector and used as a marker of expression and / or enhancement thereof.

[0326] In some embodiments, the composition containing cells, the vector, e.g., viral particles, and reagent can be rotated, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700rpm). In some embodiments, the rotation is carried at a force, e.g., a relative centrifugal force, of from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), as measured for example at an internal or external wall of the chamber or cavity. The term “relative centrifugal force” or RCF is generally understood to be the effective force imparted on an object or substance (such as a cell, sample, or pellet and / or a point in the chamber or other container being rotated), relative to the earth’s gravitational force, at a particular point in space as compared to the axis of rotation. The value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured).

[0327] In some embodiments, during at least a part of the genetic engineering, e.g., transduction, and / or subsequent to the genetic engineering the cells are transferred to the bioreactor bag assembly for culture of the genetically engineered cells, such as for cultivation or expansion of the cells, as described above.

[0328] In certain embodiments, a composition of enriched T cells in engineered, e.g., transduced or transfected, in the presence of a transduction adjuvant. In some embodiments, a composition of enriched T cells is engineered in the presence of one or more polycations. In some embodiments, a composition of enriched T cells is transduced, e.g., incubated with a viral vector particle, in the presence of one or more transduction adjuvants. In particular embodiments, a composition of enriched T cells is transfected, e.g., incubated with a non- viral vector, in the presence of one or more transduction adjuvants. In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of gene delivery, such as by increasing the amount, portion, and / or percentage of cells of the composition that are engineered (e.g., transduced or transfected). In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of transfection. In certain embodiments, the presence of one or more transduction adjuvants increases the efficiency of transduction. In particular embodiments, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells that are engineered in the presence of a polycation contain or express the recombinant polynucleotide. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold more cells of a composition are engineered to contain or express the recombinant transduction adjuvants in the presence of a polycation as compared to an alternative and / or exemplary method of engineering cells without the presence of a transduction adjuvant.

[0329] In some embodiments, the composition of enriched cells are engineered in the presence of less than 100 pg / ml, less than 90 pg / ml, less than 80 pg / ml, less than 75 pg / ml, less than 70 pg / ml, less than 60 pg / ml, less than 50 pg / ml, less than 40 pg / ml, less than 30 pg / ml, less than 25 pg / ml, less than 20 pg / ml, or less than pg / ml, less than 10 pg / ml of a transduction adjuvant. In certain embodiments, transduction adjuvants suitable for use with the provided methods include, but are not limited to polycations, fibronectin or fibronectinderived fragments or variants, RetroNectin, and combinations thereof.

[0330] In some embodiments, the cells are engineered in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 lU / ml and 1,000 lU / ml, between 10 lU / ml and 50 lU / ml, between 50 lU / ml and 100 lU / ml, between 100 lU / ml and 200 lU / ml, between 100 lU / ml and 500 lU / ml, between 250 lU / ml and 500 lU / ml, or between 500 lU / ml and 1,000 lU / ml.

[0331] In some embodiments, a composition of enriched T cells is engineered in the presence of IL-2, e.g., human recombinant IL-2, at a concentration between 1 lU / ml and 200 lU / ml, between 10 lU / ml and 100 lU / ml, between 50 lU / ml and 150 lU / ml, between 80 lU / ml and 120 lU / ml, between 60 lU / ml and 90 lU / ml, or between 70 lU / ml and 90 lU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of recombinant IL-2 at a concentration at or at about 50 lU / ml, 55 lU / ml, 60 lU / ml, 65 lU / ml, 70 lU / ml, 75 lU / ml, 80 lU / ml, 85 lU / ml, 90 lU / ml, 95 lU / ml, 100 lU / ml, 110 lU / ml, 120 lU / ml, 130 lU / ml, 140 lU / ml, or 150 lU / ml. In some embodiments, the composition of enriched T cells is engineered in the presence of or of about 85 lU / ml. In some embodiments, the population of T cells is a population of CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition. In particular embodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition ofenriched T cells is enriched for CD8+ T cells, where CD4+ T cells are not enriched for and / or where CD4+ T cells are negatively selected for or depleted from the composition.

[0332] In some embodiments, a composition of enriched T cells is engineered in the presence of recombinant IL-7, e.g., human recombinant IL-7, at a concentration between 100 lU / ml and 2,000 lU / ml, between 500 lU / ml and 1,000 lU / ml, between 100 lU / ml and 500 lU / ml, between 500 lU / ml and 750 lU / ml, between 750 lU / ml and 1,000 lU / ml, or between 550 lU / ml and 650 lU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of IL-7 at a concentration at or at about 50 lU / ml, 100 lU / ml, 150 lU / ml, 200 lU / ml, 250 lU / ml, 300 lU / ml, 350 lU / ml, 400 lU / ml, 450 lU / ml, 500 lU / ml, 550 lU / ml, 600 lU / ml, 650 lU / ml, 700 lU / ml, 750 lU / ml, 800 lU / ml, 750 lU / ml, 750 lU / ml, 750 lU / ml, or 1,000 lU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of or of about 600 lU / ml of IL-7. In some embodiments, the composition engineered in the presence of recombinant IL-7 is enriched for a population of T cells, e.g., CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition.

[0333] In some embodiments, a composition of enriched T cells is engineered in the presence of recombinant IL-15, e.g., human recombinant IL-15, at a concentration between 0.1 lU / ml and 100 lU / ml, between 1 lU / ml and 50 lU / ml, between 5 lU / ml and 25 lU / ml, between 25 lU / ml and 50 lU / ml, between 5 lU / ml and 15 lU / ml, or between 10 lU / ml and 100 lU / ml. In particular embodiments, the composition of enriched T cells is engineered in the presence of IL- 15 at a concentration at or at about 1 lU / ml, 2 lU / ml, 3 lU / ml, 4 lU / ml, 5 lU / ml, 6 lU / ml, 7 lU / ml, 8 lU / ml, 9 lU / ml, 10 lU / ml, 11 lU / ml, 12 lU / ml, 13 lU / ml, 14 lU / ml, 15 lU / ml, 20 lU / ml, 25 lU / ml, 30 lU / ml, 40 lU / ml, or 50 lU / ml. In some embodiments, the composition of enriched T cells is engineered in or in about 10 lU / ml of IL-15. In some embodiments, the composition of enriched T cells is incubated in or in about 10 lU / ml of recombinant IL-15. In some embodiments, the composition engineered in the presence of recombinant IL-15 is enriched for a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the composition of enriched T cells is a composition of enriched CD8+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD8+ T cells, where CD4+ T cells are not enriched for and / or where CD4+ T cells are negatively selected for or depleted from the composition. In someembodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells. In particular embodiments, the composition of enriched T cells is enriched for CD4+ T cells, where CD8+ T cells are not enriched for and / or where CD8+ T cells are negatively selected for or depleted from the composition.

[0334] In particular embodiments, a composition of enriched CD8+ T cells is engineered in the presence of IL-2 and / or IL-15. In certain embodiments, a composition of enriched CD4+ T cells is engineered in the presence of IL-2, IL-7, and / or IL- 15. In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant. In certain embodiments, the IL- 2, IL-7, and / or IL-15 are human. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and / or IL- 15.

[0335] In particular embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, antioxidants include, but are not limited to, one or more antioxidants comprise a tocopherol, a tocotrienol, alpha-tocopherol, beta-tocopherol, gammatocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, alpha-tocopherolquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), a flavonoids, an isoflavone, lycopene, betacarotene, selenium, ubiquinone, luetin, S-adenosylmethionine, glutathione, taurine, N-acetyl cysteine (NAC), citric acid, L-camitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, gluthatione, cystamine and cystathionine, and / or glycine-glycine-histidine .

[0336] In some embodiments, the one or more antioxidants is or includes a sulfur containing oxidant. In certain embodiments, a sulfur containing antioxidant may include thiol-containing antioxidants and / or antioxidants which exhibit one or more sulfur moieties, e.g., within a ring structure. In some embodiments, the sulfur containing antioxidants may include, for example, N- acetylcysteine (NAC) and 2,3- dimercaptopropanol (DMP) , L-2- oxo-4-thiazolidinecarboxylate (OTC) and lipoic acid. In particular embodiments, the sulfur containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule which may be modified in one or more steps within a cell to derived glutathione. In particular embodiments, a glutathione precursor may include, but is not limited to N-acetyl cysteine (NAC), L-2-oxothiazolidine-4-carboxylic acid (Procysteine), lipoic acid, S-allyl cysteine, or methylmethionine sulfonium chloride.

[0337] In some embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, the cells are engineered in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 pg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 500 pg / ml and 2 mg / ml, 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of the one or more antioxidants. In some embodiments, the cells are engineered in the presence of or of about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of the one or more antioxidant. In some embodiments, the one or more antioxidants is or includes a sulfur containing antioxidant. In particular embodiments, the one or more antioxidants is or includes a glutathione precursor.

[0338] In some embodiments, the cells are engineered in the presence of NAC. In some embodiments, the cells are engineered in the presence of between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 pg / ml, between 100 ng / ml and 10 pg / ml, between 1 pg / ml and 100 pg / ml, between 10 pg / ml and 1 mg / ml, between 100 pg / ml and 1 mg / ml, between 1,500 pg / ml and 2 mg / ml, 500 pg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml of NAC. In some embodiments, the cells are engineered in the presence of or of about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml of NAC. In some embodiments, the cells are engineered with or with about 0.8 mg / ml.

[0339] In some embodiments, a composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of one or more polycations. In some embodiments, a composition of enriched T cells, , such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is transduced, e.g., incubated with a viral vector particle, in the presence of one or more polycations. In particular embodiments, a composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is transfected, e.g., incubated with a non- viral vector, in the presence of one or more polycations. In certainembodiments, the presence of one or more polycations increases the efficiency of gene delivery, such as by increasing the amount, portion, and / or percentage of cells of the composition that are engineered (e.g., transduced or transfected). In certain embodiments, the presence of one or more polycations increases the efficiency of transfection. In certain embodiments, the presence of one or more polycations increases the efficiency of transduction. In particular embodiments, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells that are engineered in the presence of a polycation contain or express the recombinant polynucleotide. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold more cells of a composition are engineered to contain or express the recombinant polynucleotide in the presence of a polycation as compared to an alternative and / or exemplary method of engineering cells without the presence of a polycation.

[0340] In certain embodiments, the composition of enriched cells, e.g., the composition of enriched CD4+ T cells or enriched CD8+ T cells, such as stimulated T cells thereof, is engineered in the presence of a low concentration or amount of a polycation, e.g., relative to an exemplary and / or alternative method of engineering cells in the presence of a polycation. In certain embodiments, the composition of enriched cells, , such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of less than 90%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, of less than 0.01% of the amount and / or concentration of the polycation of an exemplary and / or alternative process for engineering cells. In some embodiments, the composition of enriched cells, , such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, are engineered in the presence of less than 100 pg / ml, less than 90 pg / ml, less than 80 pg / ml, less than 75 pg / ml, less than 70 pg / ml, less than 60 pg / ml, less than 50 pg / ml, less than 40 pg / ml, less than 30 pg / ml, less than 25 pg / ml, less than 20 pg / ml, or less than pg / ml, less than 10 pg / ml of the polycation. In particular embodiments, the composition of enriched cells, , such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of or of about 1 pg / ml, 5 pg / ml, 10pg / ml, 15 pg / ml, 20 pg / ml, 25 pg / ml, 30 pg / ml, 35 pg / ml, 40 pg / ml, 45 pg / ml, or 50 pg / ml, of the polycation.

[0341] In particular embodiments, engineering the composition of enriched cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, in the presence of a polycation reduces the amount of cell death, e.g., by necrosis, programed cell death, or apoptosis. In some embodiments, the composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of a low amount of a polycation, e.g., less than 100 pg / ml, 50 pg / ml, or 10 pg / ml, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the cells survive, e.g., do not undergo necrosis, programed cell death, or apoptosis, during or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days after the engineering step is complete. In some embodiments, the composition is engineered in the presence of a low concentration or amount of polycation as compared to the alternative and / or exemplary method of engineering cells in the presence of higher amount or concentration of polycation, e.g., more than 50 pg / ml, 100 pg / ml, 500 pg / ml, or 1,000 pg / ml, and the cells of the composition have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater survival as compared to cells undergoing the exemplary and / or alternative process.

[0342] In some embodiments, the polycation is positively-charged. In certain embodiments, the polycation reduces repulsion forces between cells and vectors, e.g., viral or non-viral vectors, and mediates contact and / or binding of the vector to the cell surface. In some embodiments, the polycation is polybrene, DEAE-dextran, protamine sulfate, poly-L- lysine, or cationic liposomes.

[0343] In particular embodiments, the polycation is protamine sulfate. In some embodiments, the composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, are engineered in the presence of less than or about 500 pg / ml, less than or about 400 pg / ml, less than or about 300 pg / ml, less than or about 200 pg / ml, less than or about 150 pg / ml, less than or about 100 pg / ml, less than or about 90 pg / ml, less than or about 80 pg / ml, less than or about 75 pg / ml, less than or about70 pg / ml, less than or about 60 |Jg / ml, less than or about 50 |Jg / ml, less than or about 40 |jg / ml, less than or about 30 |Jg / ml, less than or about 25 |Jg / ml, less than or about 20 |Jg / ml, or less than or about 15 |Jg / ml, or less than or about 10 |Jg / ml of protamine sulfate. In particular embodiments, the composition of enriched cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is engineered in the presence of or of about 1 pg / ml, 5 pg / ml, 10 pg / ml, 15 pg / ml, 20 pg / ml, 25 pg / ml, 30 pg / ml, 35 pg / ml, 40 pg / ml, 45 pg / ml, 50 pg / ml, 55 pg / ml, 60 pg / ml, 75 pg / ml, 80 pg / ml, 85 pg / ml, 90 pg / ml, 95 pg / ml, 100 pg / ml, 105 pg / ml, 110 pg / ml, 115 pg / ml, 120 pg / ml, 125 pg / ml, 130 pg / ml, 135 pg / ml, 140 pg / ml, 145 pg / ml, or 150 pg / ml of protamine sulfate.

[0344] In some embodiments, the engineered composition of enriched CD4+ T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells, includes at least 40, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells. In certain embodiments, the composition of enriched CD4+ T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells, that is engineered includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells, and / or is free or substantially free of CD8+ T cells.

[0345] In some embodiments, the composition of enriched CD8+ T cells, such as stimulated T cells, e.g., stimulated CD8+ T cells, that is engineered includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, the composition of enriched CD8+ T cells, such as stimulated T cells, e.g., stimulated CD8+ T cells, that is engineered includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or contains no CD4+ T cells, and / or is free or substantially free of CD4+ T cells.

[0346] In some embodiments, engineering the cells includes a culturing, contacting, or incubation with the vector, e.g., the viral vector of the non- viral vector. In certain embodiments, the engineering includes culturing, contacting, and / or incubating the cells with the vector is performed for, for about, or for at least 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 hours, 54 hours, 60 hours, 72hours, 84 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or more than 7 days. In particular embodiments, the engineering includes culturing, contacting, and / or incubating the cells with the vector for or for about 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 84 hours, or for or for about 2 days, 3 days, 4 days, or 5 days. In some embodiments, the engineering step is performed for or for about 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 84 hours. In certain embodiments, the engineering is performed for about 60 hours or about 84 hours, for or for about 72 hours, or for or for about 2 days.

[0347] In some embodiments, the engineering is performed at a temperature from about 25 to about 38°C, such as from about 30 to about 37°C, from about 36 to about 38°C, or at or about 37 °C ± 2 °C. In some embodiments, the composition of enriched T cells is engineered at a CO2 level from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example at or about 5% ± 0.5%. In some embodiments, the composition of enriched T cells is engineered at a temperature of or about 37 °C and / or at a CO2 level of or about 5%.

[0348] In some embodiments, the cells, e.g., the CD4+ and / or the CD8+ T cells, are cultivated, after one or more steps are performed for genetic engineering, e.g., transducing or transfection the cells to contain a polynucleotide encoding a recombinant receptor. In some embodiments, the cultivation may include culture, incubation, stimulation, activation, expansion, and / or propagation. In some such embodiments, the further cultivation is effected under conditions to result in integration of the viral vector into a host genome of one or more of the cells. The incubation and / or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0349] In some embodiments, the further incubation is carried out at temperatures greater than room temperature, such as greater than or greater than about 25 °C, such as generally greater than or greater than about 32 °C, 35 °C or 37 °C. In some embodiments, the further incubation is effected at a temperature of at or about 37 °C ± 2 °C, such as at a temperature of at or about 37 °C.

[0350] In some embodiments, the further incubation is performed under conditions for stimulation and / or activation of cells, which conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0351] In some embodiments, the stimulating conditions or agents include one or more agent (e.g., stimulatory and / or accessory agents), e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell, such as agents suitable to deliver a primary signal, e.g., to initiate activation of an ITAM-induced signal, such as those specific for a TCR component, and / or an agent that promotes a costimulatory signal, such as one specific for a T cell costimulatory receptor, e.g., anti-CD3, anti-CD28, or anti-41-BB, for example, optionally bound to solid support such as a bead, and / or one or more cytokines. Among the stimulating agents are anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads). Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti-CD28 antibody to the culture medium. In some embodiments, the stimulating agents include IL-2 and / or IL- 15, for example, an IL-2 concentration of at least about 10 units / mL.

[0352] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2 and / or IL- 15, for example, an IL-2 concentration of at least about 10 units / mL, at least about 50 units / mL, at least about 100 units / mL or at least about 200 units / mL.

[0353] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions,and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0354] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,1 77 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0355] In some embodiments, the further incubation is carried out in the same container or apparatus in which the contacting occurred. In some embodiments, the further incubation is carried out without rotation or centrifugation, which generally is carried out subsequent to the at least portion of the incubation done under rotation, e.g., in connection with centrifugation or spinoculation. In some embodiments, the further incubation is carried out outside of a stationary phase, such as outside of a chromatography matrix, for example, in solution.

[0356] In some embodiments, the further incubation is carried out in a different container or apparatus from that in which the contacting occurred, such as by transfer, e.g., automatic transfer, of the cell composition into a different container or apparatus subsequent to contacting with the viral particles and reagent.

[0357] In some embodiments, the further culturing or incubation, e.g., to facilitate ex vivo expansion, is carried out of for greater than or greater than about 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In some embodiments, the further culturing or incubation is carried out for no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days or no more than 24 hours.

[0358] In some embodiments, the total duration of the incubation, e.g., with the stimulating agent, is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, such as at least or about at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours. In some embodiments, the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.

[0359] In some embodiments, the methods provided herein do not include further culturing or incubation, e.g., do not include ex vivo expansion step, or include a substantially shorter ex vivo expansion step.

[0360] In some embodiments, the stimulatory reagent is removed and / or separated from the cells prior to the engineering. In particular embodiments, the stimulatory reagent is removed and / or separated from the cells after the engineering. In certain embodiments, the stimulatory agent is removed and / or separated from the cells subsequent to the engineering and prior to cultivating the engineered cells, .e.g., under conditions that promote proliferation and / or expansion. In certain embodiments, the stimulatory reagent is a stimulatory reagent that is described in Section I-B-l. In particular embodiments, the stimulatory reagent is removed and / or separated from the cells as described in Section I-B-2. / . Vectors and Methods

[0361] In some embodiments, the cells, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is carried out by introducing one or more polynucleotide(s) that encode the recombinant receptor or portions or components thereof. Also provided are polynucleotides encoding a recombinant receptor, and vectors or constructs containing such nucleic acids and / or polynucleotides.

[0362] In particular embodiments, the vector is a viral vector a non-viral vector. In some cases, the vector is a viral vector, such as a retroviral vector, e.g., a lentiviral vector or a gammaretroviral vector.

[0363] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter that is operatively linked to control expression of the recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control expression of the recombinant receptor. In some embodiments, polynucleotide can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the polynucleotide is to be introduced, as appropriate and taking into consideration whether the polynucleotide is DNA- or RNA-based. In some embodiments, the polynucleotide can contain regulatory / control elements, such as a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor. In some embodiments, the polynucleotide can contain a nonnative promoter operably linked to theI llnucleotide sequence encoding the recombinant receptor and / or one or more additional polypeptide(s). In some embodiments, the promoter is selected from among an RNA pol I, pol II or pol III promoter. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV, SV40 early region or adenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., a U6 or Hl promoter). In some embodiments, the promoter can be a non- viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other known promoters also are contemplated.

[0364] In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, e.g., simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EFla), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken P- Actin promoter coupled with CMV early enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters can include, but are not limited to, human elongation factor 1 alpha (EFla) promoter or a modified form thereof or the MND promoter.

[0365] In another embodiment, the promoter is a regulated promoter (e.g., inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog thereof or is capable of being bound by or recognized by a Lac repressor or a tetracycline repressor, or an analog thereof. In some embodiments, the polynucleotide does not include a regulatory element, e.g., promoter.

[0366] In some cases, the nucleic acid sequence encoding the recombinant receptor, e.g., chimeric antigen receptor (CAR) contains a signal sequence that encodes a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFRalpha chain signal peptide set forth in SEQ ID NO: 10 and encoded by the nucleotide sequence set forth in SEQ ID NO: 9, the CD8 alpha signal peptide set forth in SEQ ID NO: 11, or the CD33 signal peptide set forth in SEQ ID NO: 12.

[0367] In some embodiments, the polynucleotide contains a nucleic acid sequence encoding one or more additional polypeptides, e.g., one or more marker(s) and / or one or more effector molecules. In some embodiments, the one or more marker(s) includes a transduction marker, a surrogate marker and / or a resistance marker or selection marker. Among additional nucleic acid sequences introduced, e.g., encoding for one or more additional polypeptide(s), include nucleic acid sequences that can improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; nucleic acid sequences to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; nucleic acid sequences to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also WO 1992008796 and WO 1994028143 describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker, and US Patent No. 6,040,177.

[0368] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells that have been introduced with the polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker can indicate or confirm modification of a cell. In some embodiments, the surrogate marker is a protein that is made to be coexpressed on the cell surface with the recombinant receptor, e.g., CAR. In particular embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence. Extrinsic marker genes may in some cases be utilized in connection with engineered cell to permit detection or selection of cells and, in some cases, also to promote cell elimination and / or cell suicide.

[0369] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and to not transduce or are not capable of transducing a signal or a signal ordinarily transduced by the full-length form of the cell surface polypeptide, and / or do not or are not capable of internalizing. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO: 2 or 3) or a prostate-specific membrane antigen (PSMA) or modified form thereof, such as a truncated PSMA (tPSMA). In some aspects, tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and an encoded exogenous protein, and / or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g., surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, a CD 19 or a truncated CD19, e.g., a truncated non-human CD19. An exemplary polypeptide for a truncated EGFR (e.g., tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 2 or 3 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2 or 3.

[0370] In some embodiments, the marker is or comprises a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, codon-optimized, stabilized and / or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), P-galactosidase, chloramphenicol acetyltransferase (CAT), P-glucuronidase (GUS) or variants thereof. In some aspects, expression of the enzyme can be detected byaddition of a substrate that can be detected upon the expression and functional activity of the enzyme.

[0371] In some embodiments, the marker is a resistance maker or selection marker. In some embodiments, the resistance maker or selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the resistance marker or selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

[0372] Any of the recombinant receptors and / or the additional polypeptide(s) described herein can be encoded by one or more polynucleotides containing one or more nucleic acid sequences encoding recombinant receptors, in any combinations, orientation or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different polypeptides, e.g., recombinant receptors or portions or components thereof, and / or one or more additional polypeptide(s), e.g., a marker and / or an effector molecule. In some embodiments, one polynucleotide contains a nucleic acid sequence encoding a recombinant receptor, e.g., CAR, or portion or components thereof, and a nucleic acid sequence encoding one or more additional polypeptide(s). In some embodiments, one vector or construct contains a nucleic acid sequence encoding a recombinant receptor, e.g., CAR, or portion or components thereof, and a separate vector or construct contains a nucleic acid sequence encoding one or more additional polypeptide(s). In some embodiments, the nucleic acid sequence encoding the recombinant receptor and the nucleic acid sequence encoding the one or more additional polypeptide(s) are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is present upstream of the nucleic acid encoding the one or more additional polypeptide(s). In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding one or more additional polypeptide(s).

[0373] In certain cases, one polynucleotide contains nucleic acid sequences encode two or more different polypeptide chains, e.g., a recombinant receptor and one or more additional polypeptide(s), e.g., a marker and / or an effector molecule. In some embodiments, the nucleicacid sequences encoding two or more different polypeptide chains, e.g., a recombinant receptor and one or more additional polypeptide(s), are present in two separate polynucleotides. For exa...

Claims

ClaimsWHAT IS CLAIMED:

1. A method for assessing activation of T cells within a cell composition, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: one or more markers of group (i) are selected from the group consisting of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (0X2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD 170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM- 1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD 123, CD 184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and one or more markers of group (ii) are selected from the group consisting of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD 172g (SIRPg), CD 127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR;(b) comparing the level of surface expression or the percent of positive cells in the cell composition to the level of surface expression or the percent of positive cells for eachof the one or more markers in a reference, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the reference indicates the T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the reference indicates the T cells are activated.

2. The method of claim 1, wherein the reference is comprised of an unstimulated control cell composition.

3. The method of claim 1, wherein the reference is the level of expression or percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.

4. The method of claim 1, wherein the reference is the average level of expression or average percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.

5. The method of claim 1, wherein the reference is the median level of expression or median percent of positive cells across a plurality of cell compositions, wherein each cell composition is from a different patient, subject, or donor.

6. A method for assessing T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein: one or more markers of group (i) are selected from the group consists of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (0X2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD 170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7,Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CDl lc, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM- 1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD 123, CD 184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin-1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and one or more markers of group (ii) are selected from the group consisting of CD 192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

7. The method of claim 6, wherein the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with T cell activation.

8. The method of claim 6 or 7, wherein the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with T cell activation.

9. A method of comparing activation of T cells within a donor, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD 120b, CD 107b (LAMP-2), CD200 (0X2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD 105 (Endoglin), CD262 (DR5, TRAIL-R2), CD 170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317(BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CD11c, CD 146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGF-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and group (ii) consists of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MAFA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD13, CDl lb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR;(b) comparing the level of surface expression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in an unstimulated cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated cell composition indicates the T cells are activated and a lower level or lower percent of positive cells for a marker in (ii) compared to the unstimulated cell composition indicates the T cells are activated.

10. The method of any one of claims 1-9, wherein the composition comprising T cells has been subjected to incubation with a T cell stimulatory agent under conditions to induce T cell activation prior to the detecting.

11. The method of any one of claims 1-9, wherein the method comprises incubation of the composition with a T cell stimulatory agent prior to the detecting.

12. The method of any one of claims 1-9, wherein the method comprises incubation of the composition with a T cell stimulatory agent following the detecting.

13. The method of any one of claims 10-12, wherein the incubation with a T cell stimulatory agent is carried out in vivo in a subject.

14. The method of any one of claims 10-12, wherein the incubation with a T cell stimulatory agent is carried out in vitro or ex vivo.

15. The method of any one of claims 10-14, wherein the incubation with a T cell stimulatory agent is for 12-72 hours.

16. The method of any one of claims 10-15, wherein the incubation with a T cell stimulatory agent is for about 24 hours.

17. The method of any of claims 1-16, wherein the one or more markers of group (i) are selected from CD20, CD 100, CD 123, CD 184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (OX2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R); and the one or more markers of group (ii) are selected from CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRE2, CD172g (SIRPg), CD127 (IE-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

18. The method of any one of claims 10-15, wherein the incubation with a T cell stimulatory agent is for about 48 hours.

19. The method of any of claims 1-15 and 18, wherein the one or more markers of group (i) are selected from CD20, CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b,CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL- 18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD 164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGE- 1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), and GARP (LRRC32) and the one or more markers of group (ii) are selected from CD192 (CCR2), CD314 (NKG2D), KLRG1 (MALA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDl lb, CX3CR1, NKp80, CD 127 (IL-7Ra), and CD49f.

20. The method of any of claims 1-19, wherein the one or more markers of group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and the one or more markers of group (ii) are selected from CD49f, CD 124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MALA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

21. The method of any of claims 1-20, wherein the one or more markers are of (i) and are selected from the group consisting of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56.

22. A method of identifying an activated T cell, the method comprising detecting the cell surface expression of one or more of markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of CD 107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (OX2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and wherein the cells expressing a high level of the one or more markers are activated T cells.

23. The method of any of claims 1-22, wherein the one or more markers are selected from group (i) and consist of CD200 (0X2), CD357 (GITR), CD120b, CD155 (PVR), CD 107b (LAMP-2).

24. The method of any of claims 1-20, wherein the one or more markers are of (ii) and are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CD 11b, and CX3CRl.

25. A method of identifying an activated T cell, the method comprising detecting the cell surface expression of one or more of markers in cells of a composition comprising T cells, wherein the one or more markers are selected from the group consisting of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1, and wherein the cells expressing a low level of the one or more markers are activated T cells.

26. The method of any of claims 1-25, wherein the detecting is of CD4+ or CD 8+ T cells in the composition comprising T cells.

27. The method of any of claims 1-26, wherein the one or more markers from group (i) are selected from CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), andGPR56, and the one or more markers from group (ii) are selected from CD49f, CD 124 (IL- 4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

28. The method of any of claims 1-25, wherein the detecting is of CD4+ T cells in the composition comprising T cells.

29. The method of claim 28, wherein the one or more markers from group (i) are selected from CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154,and CD165, and the one or more markers from group (ii) are selected from CD49f, CD 124 (IL- 4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD 127 (IL-7Ra).

30. A method for assessing CD4+ T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii) wherein group (i) consist of CD 107b (LAMP-2), CD 120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

31. The method of claim 30, wherein the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with CD4+ T cell activation.

32. The method of claim 30 or 31, wherein the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with CD4+ T cell activation.

33. A method for assessing activation of CD4+ T cells, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more marker in CD4+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group(i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, and CD165, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra); and(b) comparing the level of surface expression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in cells of an unstimulated control cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated control cell composition indicates the CD4+ T cells are activated and a lower level or lower percent of positive cells for a marker in(ii) compared to the unstimulated control cell composition indicates the CD4+ T cells are activated.

34. The method of any of claims 1-25, wherein the detecting is of CD8+ T cells in the composition comprising T cells.

35. The method of claim 34, wherein the one or more markers selected from group (i) are selected from CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and the one or more markers from group (ii) are selected from CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra).

36. A method for assessing CD8+ T cells for surface expression of a T cell activation marker, the method comprising detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii) wherein group (i), consist of CD 120b, CD200 (0X2), CD 134 (0X40), CD 107b (LAMP-2), CD357 (GITR), CD 155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra).

37. The method of claim 36, wherein the level of surface expression or percent of positive cells of the one or more markers in (i) positively correlates with CD8+ T cell activation.

38. The method of claim 37, wherein the level of surface expression or percent of positive cells of the one or more markers in (ii) negatively correlates with CD8+ T cell activation.

39. A method for assessing activation of CD8+ T cells, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consist of CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra); and(b) comparing the level of surface expression or the percent of positive cells to the level or the percent of positive cells for each of the one or more marker in cells of an unstimulated control cell composition, wherein a higher level or higher percent of positive cells for a marker in (i) compared to the unstimulated control cell composition indicates the CD8+ T cells are activated and a lower level or lower percent of positive cells for a marker in(ii) compared to the unstimulated control cell composition indicates the CD8+ T cells are activated.

40. The method of any of claims 1-39, wherein the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor.

41. The method of claim 27, wherein the one or more markers selected from group (i) consist of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and the one or more markers selected from group (ii) consist of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD195 (CCR5), and CD96 (TACTILE).

42. A method for assessing activation of T cells, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in CD8+ T cells of a composition comprising T cells, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56, and group (ii) consists of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD355 (CTRAM), CD155 (PVR), CD74, CD170 (Siglec-5), and GPR56.

43. The method of claim 41 or claim 42, wherein the one or more markers from group (i) are selected from CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and CD 170 (Siglec-5), and the one or more markers selected from group (ii) are selected from CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD355 (CRTAM), GPR56, and CD96 (TACTILE).

44. The method of any of claims 41-43, wherein the detecting is of recombinant receptor-expressing CD4+ T cells in the composition comprising T cells.

45. The method of claim 44, wherein the one or more markers from group (i) are selected from CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), and CD74, and / or the one or more markers selected from group (ii) are selected from CD49f, CCRL2, and CD 124 (IL-4Ra).

46. The method of any of claims 41-43, wherein the detecting is of recombinant receptor-expressing CD8+ T cells in the composition comprising T cells.

47. The method of claim 46, wherein the one or more markers from group (i) are selected from CD200 (0X2), CD107b (LAMP-2), CD155 (PVR), CD355 (CRTAM), and GPR56, and / or the one or more markers selected from group (ii) are selected from CCRL2, CD217, CD96 (TACTILE).

48. The method of any of claims 1-16, wherein the one or more markers of group (i) are selected from CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and the one or more markers of group (i) are selected from CD96 (TACTILE).

49. A method for assessing activation of T cells, the method comprising:(a) detecting the level of surface expression of one or more markers or the percent of cells positive for one or more markers in a composition of T cells, which comprises T cells expressing a recombinant receptor, wherein the one or more markers are selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin), CD73 (Ecto-5’-nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and group (ii) consists of CD96 (TACTILE).

50. The method of claim 48, wherein the composition comprising T cells comprises cells that express a recombinant receptor.

51. The method of claim 49 or claim 50, wherein the detecting is of recombinant receptor expressing cells of the composition of T cells52. The method of any of claims 49-51, wherein the surface expression of the one or more markers of group (i) is increased on cells expressing the recombinant receptor compared with cells that are not expressing the recombinant receptor.

53. The method of any of claims 49-52, wherein the surface expression of the one or more markers of group (ii) is decreased on cells expressing the recombinant receptor compared with cells that are not expressing the recombinant receptor.

54. The method of any one of claims 3-47, wherein the composition comprising T cells comprises T cells genetically engineered to express a recombinant receptor and wherein the T cell stimulatory agent is a recombinant receptor stimulating agent that induces recombinant receptor-dependent T cell activation.

55. The method of any one of claims 27-54, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

56. The method of claim 54 or claim 55, wherein the recombinant receptor stimulating agent comprises a recombinant target antigen recognized by the recombinant receptor.

57. The method of claim 54 or claim 55, wherein the recombinant receptor stimulating agent is an antibody specific to an extracellular antigen binding domain of the recombinant receptor.

58. The method of claim 54 or claim 55, wherein the recombinant receptor stimulating agent is an anti-idiotypic antibody specific to an extracellular antigen binding domain of the recombinant receptor.

59. The method of any one of claims 54-58, wherein the recombinant receptor stimulating agent is immobilized or attached to a solid support.

60. The method of claim 59, wherein the solid support is a surface of the vessel, optionally a well of microwell plate or a flask.

61. The method of claim 59, wherein the solid support is a bead.

62. The method of claim 54 or claim 55, wherein the recombinant receptor stimulating agent is an antigen-expressing cell, optionally wherein the cell is a clone, from a cell line, or a primary cell taken from a subject.

63. The method of claim 62, wherein the antigen-expressing cell is a cell line.

64. The method of claim 63, wherein the cell line is a tumor cell line.

65. The method of claim 63, wherein the antigen-expressing cell is a cell that has been engineered to express the antigen of the recombinant receptor.

66. The method of any one of claims 27-65, wherein the detecting is of recombinant receptor-expressing T cells in the composition comprising T cells.

67. The method of any one of claims 1-26, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (0X2), and CD134 (0X40), and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1.

68. The method of any one of claims 1-26, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240),CD154, Notch 2, CD165, and CD83, and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CDl lb, and CX3CR1.

69. The method of claim 67 or 68, wherein the detecting is of CD4+ T cells in the composition comprising T cells.

70. The method of claim 69, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, and CD 165 and / or the one or more markers from group (ii) are selected from KLRG1 (MAFA), CD195 (CCR5), CD96 (TACTILE), and CD127 (IL-7Ra).

71. The method of claim 67 or 68, wherein the detecting is of CD8+ T cells in the composition comprising T cells.

72. The method of claim 71, wherein the one or more markers from group (i) are selected from CD71, Notch 1, CD107a (LAMP-1), CD166, Notch 2, CD165, CD83, and / or the one or more markers from group (ii) are selected from CD1 lb, CX3CR1, and CD127 (IL-7Ra).

73. The method of any one of claims 67-72, wherein the T cell stimulatory agent is a pan-T cell activation agent.

74. The method of claim 73, wherein the pan-T cell activation reagent comprises an anti-CD3 antibody and an anti-CD28 antibody, optionally wherein the pan-T cell activation reagent comprises an anti-CD3 Fab and an anti-CD28 Fab.

75. The method of claim 73 or claim 74, wherein the pan-T cell activation reagent comprises anti-CD3 / anti-CD28 beads.

76. The method of claim 73 or claim 74, wherein the pan-T cell activation reagent comprises a soluble anti-CD3 / anti-CD28 streptavidin oligomeric reagent.

77. The method of any one of claims 1-76, wherein prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agents which bind to the one or more markers.

78. The method of any one of claims 1-76, wherein prior to the detecting of step (a), the method comprises contacting cells of the composition comprising T cells with one or more binding agent comprising a means for binding the one or more markers.

79. The method of claim 77 or claim 78, wherein the one or more binding agents are one or more antibodies or antigen-binding fragments.

80. The method of any of claims 77-79, wherein the one or more binding agents are detectably labeled.

81. The method of claim 80, wherein the one or more binding agents are fluorescently labeled.

82. The method of any of one claims 1-81, wherein the detecting is by flow cytometry.

83. The method of any of one claims 1-81, wherein the detecting of step (a) is carried out in conjunction with CITE-Seq or REAP-seq.

84. The method of any of one claims 1-81, wherein the detecting of step (a) is done by immunohistochemistry, optionally immunohistochemistry fluorescence.

85. The method of any one of claims 1-84, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different markers are used for detecting in step (a).

86. A kit for determining T cell activation comprising a binding agent comprising a means for binding the one or more markers in the methods of any of claims 1-85.

87. The kit of claim 86, wherein the means for detecting each of the one or more markers is an antibody.

88. A kit for determining T cell activation comprising a binding agent comprising a means for bindingone or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD120b, CD107b (LAMP-2), CD200 (0X2), CD357 (GITR), CD134 (0X40), CD83, CD155 (PVR), CD74, GPR56, Notch 1, CD119 (IFN-g R a chain), CD105 (Endoglin), CD262 (DR5, TRAIL-R2), CD170 (Siglec-5), CD73 (Ecto-5'-nucleotidase), CD360 (IL-21R ), CD20, CD107a (LAMP-1), CD109, CD132 (common g chain), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD97, Integrin b7, Mouse IgG3 k, Notch 2, CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, Ig light chain k, Ig light chain 1, IgM, CD11c, CD 146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22, CD221 (IGE-1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD266 (Enl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD 122 (IL-2Rb), CD 100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD196 (CCR6), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32), CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R), and group (ii) consists of CD192 (CCR2), CCRL2, CD96 (TACTILE), CD195 (CCR5), CD124 (IL-4Ra), CD49f, CD314 (NKG2D), KLRG1 (MALA), CD96 (TACTILE), HLA-E, CD195 (CCR5), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337(NKp30), CD13, CDllb, CX3CR1, NKp80, CD172g (SIRPg), CD127 (IL-7Ra), a / b T Cell Receptor, CD229 (Ly-9), CD84, and EGFR.

89. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD105 (Endoglin), CD107a (LAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL-18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD116, CD334 (FGFR4), CD66a / c / e, and TSLPR (TSLP-R) and group (ii) consists of CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRE2, CD 172g (SIRPg), CD 127 (IE-7Ra), CD49f, a / b T Cell Receptor, CD229 (Ey-9), CD84, and EGFR.

90. A kit for determining T cell activation comprising binding agent comprising a a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD20, CD105 (Endoglin), CD107a (EAMP-1), CD109, CD120b, CD132 (common g chain), CD134 (0X40), CD148, CD150 (SLAM), CD151 (PETA-3), CD154, CD165, CD166, CD170 (Siglec-5), CD200 (0X2), CD200 R, CD217, CD218a (IL- 18Ra), CD227 (MUC-1), CD245 (p220 / 240), CD30, CD317 (BST2, Tetherin), CD323 (JAM3), CD357 (GITR), CD45RA, CD49c (integrin a3), CD54, CD63, CD71, CD8, CD83, CD97, Integrin b7, Mouse IgG3 k, Notch 1, Notch 2, CD107b (LAMP-2), CD155 (PVR), CD 164, CD319 (CRACC), Cd355 (CRTAM), CD4, CD49a, CD49b, CD74, GPR56, Ig light chain k, Ig light chain 1, IgM, CD119 (IFN-g R a chain), CD11c, CD146, CD183 (CXCR3), CD185 (CXCR5), CD19, CD194 (CCR4), CD21, CD210 (IL-10 R), CD22 ,CD221 (IGF- 1R), CD226 (DNAM-1), CD23, CD258 (LIGHT), CD26, CD262 (DR5, TRAIL-R2), CD266 (Fnl4, TWEAK R), CD276 (B7-H3), CD326 (Ep-CAM), CD36L1 (SCARB1, SR-BI),CD49e, CD87, CD8a, CD9, DcRl (TRAIL-R3, CD263), Ganglioside GD2, MERTK, TMEM8A, CD122 (IL-2Rb), CD360 (IL-21R), CD100, CD123, CD184 (CXCR4), CD55, TIGIT (VSTM3), CD230 (Prion), CD235ab, CD261 (DR4, TRAIL-R1), CD304 (Neuropilin- 1), CD49d, CD73 (Ecto-5'-nucleotidase), DR3 (TRAMP), CD186 (CXCR6), GARP (LRRC32) and group (ii) consists of CD192 (CCR2), CD314 (NKG2D), KLRG1 (MAFA), CCRL2, CD96 (TACTILE), HLA-E, CD195 (CCR5), CD124 (IL-4Ra), CD198 (CCR8), CD282 (TLR2), CD294 (CRTH2), CD337 (NKp30), CDllb, CX3CR1, NKp80, CD127 (IL- 7Ra), and CD49f.

91. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154, CD165, CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), CD127 (IL-7Ra), CDllb, and CX3CR1.

92. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), CD355 (CRTAM), and GPR56, and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD96 (TACTILE), and CD195 (CCR5).

93. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD107b (LAMP-2), CD120b, CD357 (GITR), CD83, CD200 (0X2), CD134 (0X40), CD155 (PVR), CD74, CD170 (Siglec-5), Notch 1, Notch 2, CD166, CD107a (LAMP-1), CD71, CD245 (p220 / 240), CD154,and CD165,and group (ii) consists of CD49f, CD124 (IL-4Ra), CCRL2, CD217, CD192 (CCR2), CD195 (CCR5), KLRG1 (MAFA), CD96 (TACTILE), and CD127 (IL-7Ra).

94. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD357 (GITR), CD155 (PVR), CD355 (CRTAM), GPR56, CD71, CD107a (LAMP1), Notch 1, CD166, CD165, CD83, and Notch 2, and group (ii) consists of CD96 (TACTILE), CD 195 (CCR5), CD217, CCRL2, CD192 (CCR2), CDl lb, CX3CR1, and CD127 (IL-7Ra).

95. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD120b, CD83, CD357 (GITR), CD200 (0X2), CD134 (0X40), CD107b (LAMP-2), CD155 (PVR), CD74, and the one or more markers selected from group (ii) consist of CD49f, CCRL2, CD124 (IL-4Ra), CD217, CD192 (CCR2), CD355 (CRTAM), GPR56, CD195 (CCR5), and CD96 (TACTILE).

96. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD71, Notch 1, CD107a (LAMP-1), CD166, CD245 (p220 / 240), CD154, Notch 2, CD165, CD120b, CD83, CD357 (GITR), CD200 (0X2), and CD134 (0X40), and / or the one or more markers from group (ii) are selected from KLRG1 (MALA), CD195 (CCR5), CD96 (TACTILE), CD127 (IL-7Ra), CD192 (CCR2), CDl lb, and CX3CR1.

97. A kit for determining T cell activation comprising a binding agent comprising a means for binding one or more markers selected from group (i) and / or group (ii), wherein group (i) consists of CD36L (SCARB1, SR-BI), CD262 (DR5, Trail-R2), CD105 (Endoglin),CD73 (Ecto-5’ -nucleotidase), CD83, CD119 (IFN-g R a chain), CD154, CD170 (Siglec-5), Notch 1, and CD360 (IL-21R), and / or the one or more markers from group (ii) are selected from CD96 (TACTILE).

98. The kit of any of claims 86-97, wherein the binding agent is one or more antibodies or antigen-binding fragments.

99. The kit of any of claims 86-98, wherein the binding agent is detectably labeled.

100. The kit of claim 99, wherein the binding agent is fluorescently labeled.

101. A method for isolating activating T cells, the method comprising identifying a population of activated T cells according to the method of any of claims 1-85 and isolating the population.

102. A method for enriching activating T cells, the method comprising identifying a population of activated T cells according to the method of any of claims 1-85 and selecting the population, thereby obtaining a cell population enriched in activated T cells.

103. A method of depleting a cell population of activated T cells, the method comprising identifying a population of activated T cells according to the method of any of claims 1-85 and depleting the population of activated T cells.

104. A T cell population produced according to the method of any of claims 101-103.

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