Materials, methods and systems for cellular redifferentiation and expansion

Abstract

Materials, methods, and systems for the cellular redifferentiation and expansion of γδ T cells from induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) without the use of serum or additional cells is provided.

Description

Attorney Docket No: JBI6895WOPCT1 Materials, Methods and Systems for Cellular Redifferentiation and Expansion CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Applications 63 / 574,573, filed on April 4, 2024 and 63 / 715,885 filed on November 4, 2024, which are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION

[0002] The present invention teaches cell production, including for example materials, methods, and systems for the cellular growth and redifferentiation of T cells, such as γδ T cells, and such as induced pluripotent stem cell (iPSC)-derived γδ T cells (iγδ T cells) from iPSC- derived hematopoietic stem cells (iHSCs). The iγδ T cells produced by these methods can be derived from unedited or gene edited γδ T cell-derived iPSCs and can be expanded in an additional process to produce a cell therapy product. The materials, methods, and systems of the present invention advantageously reduce or eliminate the use of costly materials, energy, carbon feedstock(s), and biomass, etc., such as animal serum, additional cells (e.g., feeder cells or stromal cells), human supervision, and the like, in iγδ T cell manufacture and use for producing a cell therapy product. BACKGROUND

[0003] Cell therapy, such as T cell immunotherapy, including autologous chimeric antigen receptor (CAR) T cell therapies, has been reported to be an efficacious therapy for the treatment of some diseases, such as some cancers, including some hematologic malignancies. However, applications of cell therapy to treat multiple cancer types has met numerous and varied challenges (see, e.g., Advances and challenges of CAR T therapy and suitability of animal models (Review), Authors: Xavier E. Ramos-Cardona Weichuan Luo Sulma I. Mohammed; Published online on: July 12, 2022).Attorney Docket No: JBI6895WOPCT1 SUMMARY OF THE INVENTION

[0004] Advances in T cell immunotherapy have been reported, such as in autologous CAR T cell therapy; however, applications of cell therapy to treat multiple cancer types in a variety of settings has numerous unmet challenges; and the present invention, against this backdrop, meets many of the challenges that impede access to obtaining therapeutic cell products, such as for example prohibitive, high manufacturing costs, undue complexity, lack of consistent and scalable manufacturing processes, automation, avoidance of scarce resources, etc. For example, the inventors of the present invention use iPSCs, which can undergo, inter alia, self-renewal, gene editing, multilineage differentiation, etc., to advance off-the-shelf, allogeneic, T cell therapy platforms with more commercially viable, automated, energy efficient, and resource sparing manufacturing processes. For example, in accordance with the present invention, an iPSC- derived cell therapy platform is based on γδ T cells, which possess intrinsic antitumor activity and whose cellular tumor infiltration is associated with more favorable outcomes as a starting point for reprogramming into iPSCs and ultimately for redifferentiating into iγδ T cells that can be expanded and used as a cell therapy product. There is therefore a need in the art for methods of efficiently redifferentiating iPSC-derived HSCs (iHSCs) into iγδ T cells that can be effectively expanded in an additional process into a cell therapy product with potent antitumor activity. The exemplary methods described herein result in high iγδ T cell purity and fold expansion and and in phenotypically and functionally mature iγδ T cells. The present invention is directed to such improved methods and materials and systems.

[0005] The invention taught herein has multiple aspects. In an aspect, the present invention provides a method of de novo generation of human iPSC-derived γδ (iγδ) T cells under normoxic, serum-free, and feeder-free culture conditions from iPSC-derived hematopoietic stem cells (iHSCs), said method comprising: a) seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand (e.g., Delta-like ligand 4 (DLL4)) and at least one α4β1 ligand (e.g., vascular cell adhesion molecule 1 (VCAM1), and Retronectin); b) culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC-derived cell intermediates; c) replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; d) culturing the iHSC-derived cell intermediates in theAttorney Docket No: JBI6895WOPCT1 first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) harvesting redifferentiated iγδ T cells at about day 14 of culture.

[0006] In an embodiment, the at least one Notch ligand is DLL4. In an embodiment, the at least one α4β1 ligand comprises VCAM1 and RetroNectin. In an embodiment, the coating comprises DLL4, VCAM1, and RetroNectin.

[0007] In an embodiment, step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture.

[0008] In an embodiment, step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture.

[0009] In an embodiment, the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential. In an embodiment, the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13-acetate and ionomycin. In an embodiment, the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining.

[0010] In another aspect, the invention provides a method of de novo generation of human iPSC- derived γδ (iγδ) T cells under normoxic, serum-free and feeder-free culture conditions from iPSC- derived hematopoietic stem cells (iHSCs), said method comprising: a) a step for performing a function of seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; b) a step for performing a function of culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC-derived cell intermediates; c) a step for performing a function of replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with the coating comprising at least one Notch ligand and at least one α4β1 ligand; d) a step for performing a function of culturing the iHSC-derived cell intermediates in the first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) a step for performing a function of harvesting redifferentiated iγδ T cells at about day 14 of culture.

[0011] In an embodiment, the at least one Notch ligand is DLL4. In an embodiment, the at least one α4β1 ligand comprises VCAM1 and RetroNectin. In an embodiment, the coating comprises DLL4, VCAM1, and RetroNectin.Attorney Docket No: JBI6895WOPCT1

[0012] In an embodiment, step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture.

[0013] In an embodiment, step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture.

[0014] In an embodiment, the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential. In an embodiment, the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13-acetate and ionomycin. In an embodiment, the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Figure 1 depicts an exemplary diagram of the iCD34 to iγδ T cell redifferentiation process outlining exemplary steps.

[0017] Figure 2 depicts an exemplary gating strategy for iCD34 cell samples: All cells^ Singlets^ Live cells^ Lineage marker-negative population ^ CD34+CD43+ cells. The percentage of cells in each gate is shown. Representative plots are shown for iCD34 cells that were generated using the exemplary methods described herein. The Kasumi-1 cell line was used as staining control.

[0018] Figure 3 depicts an exemplary gating strategy to identify hematopoietic lineages: All cells ^ Singlets^ Live cells^ CD19- CD20- ^ CD3- CD11b- ^ CD16- CD14- ^ CD235a-plots are shown for cells that were generated using the exemplary methods described herein. The Kasumi-1 cell line was used for staining control.

[0019] Figure 4 depicts an exemplary gating strategy for iγδ T cell samples: All cells ^ Singlets^ Live cells^ CD3+population ^ TCR Vγ9+Vδ2+cells. The percentage ofinAttorney Docket No: JBI6895WOPCT1 each gate is shown. Representative plots are shown for iγδ T cells that were generated using the exemplary methods described herein.

[0020] Figure 5 depicts an exemplary gating strategy of unstimulated and PMA + Ionomycin stimulated iγδ T cells: TNFα vs IFNγ and CD56 vs Granzyme B on gated Vγ9+Vδ2+T cells (see Figure 4). Data shown are representative plots for iγδ T cells generated using the exemplary methods described herein.

[0021] Figure 6 depicts an exemplary gating strategy for iγδ T cell and primary γδ T cell samples: All Cells ^ Singlets^ Live cells^ CD3+ population ^ Vγ9+Vδ2+T cells. Data shown are plots for iγδ T cells generated using the exemplary methods describedherein.

[0022] Figure 7 demonstrates that iγδ T cells, redifferentiated using the exemplary methods described herein, exhibit high purity and high fold expansion. Data shown are representative plots for iγδ T cells generated using the exemplary methods described herein in which the iHSCs were generated using a two dimensional (2D) culture system or a three dimensional (3D) culture system.

[0023] Figure 8 demonstrates that iγδ T cells redifferentiated using the exemplary methods described herein resemble peripheral γδ T cells in that they are functionally mature (i.e., have the capacity to produce soluble effectors in a short-term assay). Cells were activated with PMA + ionomycin + brefeldin A for 5 hours and then assayed for intracellular IFNγ, TNFα, and Granzyme B expression by flow cytometry. Dot plots show TNFα vs IFNγ and CD56 vs Granzyme B on gated Vγ9+Vδ2+T cells. Data shown are representative plots for iγδ T cells generated using the exemplary methods described herein.

[0024] Figure 9 demonstrates that the exemplary methods of iCD34 to iγδ T cell redifferentiation as described herein provide phenotypically mature iγδ T cells based on the expression results obtained for CD1a, CD11a, HLA-ABC, NKG2D and CD56. CD1a is known to be expressed by immature human γδ thymocytes (Perriman et al. A three-stage developmental pathway for human Vγ9Vδ2 T cells within the postnatal thymus. Sci Immunol. 2023;8:eabo4365.). CD11a and HLA-ABC are known to be expressed by mature human γδ thymocytes (Perriman et al. A three-stage developmental pathway for human Vγ9Vδ2 T cells within the postnatal thymus. Sci Immunol.2023;8:eabo4365.). NKG2D and CD56 are known to be expressed by type 1 cytotoxic effectors (Perriman et al. A three-stage developmental pathwayAttorney Docket No: JBI6895WOPCT1 for human Vγ9Vδ2 T cells within the postnatal thymus. Sci Immunol.2023;8:eabo4365; Sanz et al. Deep characterization of human γδ T cell subsets define shared and lineage-specific traits. Front Immunol.2023;14:1148988.). DETAILED DESCRIPTION

[0025] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0026] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0027] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0028] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0029] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being,Attorney Docket No: JBI6895WOPCT1 or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.

[0030] The term “subject” refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human.

[0031] The term “induced pluripotent stem cell” (hereinafter “iPSC”) means a stem cell that is established by introducing reprogramming factors into a somatic cell (e.g., γδ T cell), that has pluripotency permitting differentiation into many cell types present in living organisms, and that also has self-renewal capacity. It encompasses any cell that can be redifferentiated into a hematopoietic stem cell (HSC) and ultimately into a γδ T cell to be used in the present invention. The iPSC is preferably derived from a primate (e.g., monkey, orangutan, chimpanzee, human), more preferably human. The iPSC is preferably derived from a human γδ T cell. The iPSC may be unedited or may be gene-edited.

[0032] The term “iHSC” refers to hematopoietic stem cells that are derived from iPSCs.

[0033] The term “iγδ T cells” refers to γδ T cells that are derived from iPSCs.

[0034] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. Redifferentiation of the iHSCs into iγδ T Cells

[0035] An exemplary method of redifferentiating human iHSCs into iγδ T cells follows.

[0036] Appropriate planar surfaces, such as tissue or cell culture plates or vessels, can be prepared by coating the surface (e.g., wells) with a coating comprising at least one Notch ligand and at least one α4β1 ligand. A preferred tissue culture vessel includes a 48-well tissue culture plate. In an embodiment, the Notch ligand includes delta like canonical Notch ligand 4 (DLL4) and Jagged 2. In another embodiment, the Notch ligand is DLL4. In an embodiment, the α4β1 ligand includes RetroNectin and / or vascular cell adhesion molecule 1 (VCAM1). RetroNectin is a fragment of recombinant human fibronectin. In an embodiment, the α4β1 ligand includes RetroNectin and VCAM1. In an embodiment, the coating comprises DLL4, VCAM1, and RetroNectin. The coating can be performed by any method known in the art.Attorney Docket No: JBI6895WOPCT1

[0037] Cryopreserved iHSCs can be thawed before beginning the redifferentiation processes described herein. Alternatively, freshly redifferentiated iHSCs can be used. The iHSCs can be counted and their viability determined as discussed herein or as commonly known in the art.

[0038] An exemplary medium for use with the present methods comprises interleukin-7 (IL- 7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L). A non-limiting exemplary medium is StemSpan™ SFEM II (StemCell Technologies) combined with Lymphoid Progenitor Expansion Supplement, also referred to herein as iγδ T cell redifferentiation medium.

[0039] Approximately 1 x 104to 4 x 104live iHSCs, preferably 1.2 x 104live iHSCs, can be seeded in a tissue culture vessel per 250 µL of iγδ T cell redifferentiation medium per well, and the cells can be incubated at 37oC, 5% CO2, 18% O2 (e.g., normoxic conditions) for further culture and differentiation.

[0040] Seeding the cells in the tissue culture vessel with wells coated as described herein initiates and may promote T cell specification and development. In an embodiment, the wells are coated with DLL4, VCAM1, and Retronectin.

[0041] Additional iγδ T cell redifferentiation medium can be added during the culture process as commonly known in the art. The cells can be analyzed during the culture process for viability and / or T cell lineage marker expression by any method known in the art. The iγδ T cell redifferentiation medium can be changed as commonly known in the art. For example, the iγδ T cell redifferentiation medium can be changed every three days, e.g., on Days 3, 7, and 10 of the culture process. The cells can be harvested approximately 14 days after thawing.

[0042] Interleukin 15 (IL-15) can be added to the iγδ T cell redifferentiation medium starting on about Day 7 and continuing for the remainder of the culture process. Addition of IL-15 may promote T cell maturation.

[0043] During the culture process, the cells can be replated into another tissue culture vessel with wells coated as described herein. In an embodiment, the replating occurs on about Day 7 of the culture process. In an embodiment, the wells are coated with a coating comprising DLL4, VCAM1, and Retronectin.Attorney Docket No: JBI6895WOPCT1 EXAMPLES

[0044] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. Serum-free, feeder-free, two-dimensional redifferentiation of iCD34 cells into iγδ T cells Table 1.1: Abbreviations used in Example 1 Abbreviation Definition 2D Two-dimensional 3D Three-dimensional CAR Chimeric antigen receptor Cat. # Catalog number DLL4 Delta-like Canonical Notch Ligand 4 DMSO Dimethyl Sulfoxide DPBS Dulbecco’s Phosphate-buffered Saline γδ T-iPSC γδ T cell-derived iPSC iγδ T cell iPSC-derived γδ T cell IMDM Iscove's Modified Dulbecco's Medium (wash medium) iCD34 iPSC-derived hemopoietic progenitor cells IFNγ Interferon γ iPSC induced pluripotent stem cell IL-15 Interleukin-15 ICS Intracellular staining N / A Not applicable PMA Phorbol 12-myristate 13-acetate rh Recombinant human RN RetroNectin RT Room temperature SFFF Serum-free, feeder-free TC Tissue culture TCR T cell receptor TNFα Tumor necrosis factor α VCAM-1 Vascular Cell Adhesion Molecule-1 Vγ9 Variable region domain γ9 Vδ1 Variable region domain δ1 Vδ2 Variable region domain δ2 2.1 Materials 1.3.1. iCD34 cells derived from unedited or gene-edited, research-grade or GMP-grade, γδ T- iPSC clones, which were redifferentiated in 2D or 3D culture formats (eg, 2D adherent or non-adherent cell fractions and 3D single cell or cluster cell fractions). 1.3.2. Reagents necessary to perform cell culture.Attorney Docket No: JBI6895WOPCT1 Table 1.2: Reagents for iγδ T cell redifferentiation Reagent Vendor, Product No. Storage condition StemCell Technologies, -20 °C, upon thaw use StemSpan™ SFEM II cat.# 09655 within two weeks and store at 4°C Lymphoid Progenitor Expansion StemCell Technologies, -20 °C, upon thaw use Supplement (10X) cat.# 09915 within two weeks and store at 4°Crh VCAM-1-Fc chimera proteinR&D, cat.# 862-VC-100 -20 °Crh DLL4-Fc chimera protein R&D, cat.# 10185-D4-050 -20 °Crh RetroNectin, GMP-gradeTakara Bio, cat.# T201 -80 °Crh IL-15PeproTech, cat.# 200-15 -20 °CThermoFisher Scientific, 4 °C IMDM cat.# 12440053 Table 1.3: Supplies and consumables needed for cell culture Item Product No. Corning™ Costar™ 48-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile Corning, cat.# 3548 96-well Conical (V)-Bottom Plate, Non-Treated Surface Thermo Scientific, cat.# 277143 96-well Round (U)-Bottom Plate, TC Surface Thermo Scientific, cat.# 163320 Thermo Scientific, Fisherbrand™ Sterile Polystyrene Disposable Serological cat.# 13-676-10H, Pipets with Magnifier Stripe (various sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11FPipette Tips RT LTS 20 µL F 960A / 10 (P20)Rainin, cat.# 30389225Pipette Tips RT LTS 200 µL F 960A / 10 (P200)Rainin, cat.# 30389239Pipette Tips RT LTS 1000 µL F 768A / 8 (P1000)Rainin, cat.# 3038921250 mL conical tubesCorning, cat.# 352098Falcon® 15 mL Polystyrene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile Corning, cat.# 3520951.8 mL cryovialsThermo Scientific, cat.# 3754181.5 mL Eppendorf tubesGreiner-bio-one cat.# 616261Cell liftersTMCorning, cat.# 3008Corning CoolCell Freezing containerCorning, cat.# 432006Moxi GO Cassette Type S+ Orflo, cat.# MXC030 Orflo Moxi Cyte Viability Reagent Orflo, cat.# MX055 Kasumi-1 (myeloblast cell line used as positive control for ATCC, cat.# CRL-2724 iCD34 flow panel) Table 1.4: Reagents for flow cytometry Reagent Vendor Product No. Dilution Storage Flow staining ReagentsStain BufferBD Biosciences 554656 1X 4 °CBrilliant Stain BufferBD Biosciences 361651 1:10 4 °CAttorney Docket No: JBI6895WOPCT1 Reagent Vendor Product No. Dilution Storage Stabilizing Fixative 3X Concentrate BD Biosciences 338036 1:3 4 °C Human TruStain FcX™ (Fc Receptor Blocking BioLegend 422302 1:20 4 °C Solution) ICS Reagents BD Cytofix / Cytoperm kit 1:10 for Perm / Wash Buffer (Perm / Wash buffer and 1X for Fixation / Permeabilization BD Biosciences 554714 Fix / Perm 4 °C solution) solution Cell Activation Reagents Cell Activation Cocktail with brefeldin ABioLegend 423304 1:100 4 °CBrefeldin A Solution -20°C, aliquot (1000X) BD Biosciences 420601 1:1000 to avoid freeze / thaw Staining Control Kasumi-1 (myeloblast cell line used as positive control for iCD34 flow ATCC CRL-2724 N / A panel) Table 1.5: Antibodies for flow panels Fluorochrome Marker Clone Vendor Product No. iCD34 Panel (without CAR detection) PE CD34 581 BioLegend 343506 APC CD43 1G10 BD 560198 BUV395 CD45 HI30 BD 563792 BV421 CD38 HIT2 BioLegend 303526 CD3 HIT3a BioLegend 300306 CD235a HI264 BioLegend 349104 CD11c Bu15 BioLegend 337214 CD11b ICRF44 BioLegend 301330 FITC CD14 HCD14 BioLegend 325604 Dump Channel CD56 HCD56 BioLegend 318304 CD19 HIB19 BioLegend 302206 CD20 2H7 BioLegend 302304 CD16 3G8 BioLegend 302006 CD2 RPA-2.10 BioLegend 300206 iCD34 Panel (with CAR detection) BV421 CD34 581 BD 562577Attorney Docket No: JBI6895WOPCT1 Fluorochrome Marker Clone Vendor Product No. BV711 CD43 1G10 BD 743614 AF488 CAR detection reagent PerCP Cy5.5 CD38 HIT2 BioLegend 303522 CD3 HIT3a BioLegend 300324 CD235a HI264 BioLegend 349122 CD11c Bu15 BioLegend 337220 CD11b ICRF44 BioLegend 301356 AF700 CD14 HCD14 BioLegend 325614 Dump Channel CD56 HCD56 BioLegend 318316 CD19 HIB19 BioLegend 302226 CD20 2H7 BioLegend 302322 CD16 3G8 BioLegend 302026 CD2 RPA-2.10 BioLegend 300238 22-color Multilineage Panel BUV395 CD2RPA-2.10BD 563819BUV496 CD11bICRF44BD 741138BUV563 Notch-1MHN1-519BD 748756BUV615 CD14M5E2BD 751150BUV661 CD202H7BD 749952BUV737 CD45HI30BD 748719BUV805 CD3SK7BD 612893BV421 CD41HIP2BD 563312BV480 CD56NCAM16.2BD 566124BV570 Live / dead BD 565694BV605 SSEA4MC813-70BD 563119BV650 CD11cB-ly6BD 563404BV711 CD163G8BioLegend 302044BV750 CD33WM53BD 747086BV786 CD7M-T701BD 740964BB515 SSEA3MC-631BioLegend 330306BB700 CD19SJ25C1BD 566396PE-Cy7 CD38HIT2BioLegend 908312APC CD43CD43-10G7BioLegend 343206Alexa 700 CD235a-R718GA-R2 (HIR2)BD 751931APC / Fire™ 750 CD3269C4BioLegend 324234iγδ T cell redifferentiation panel (without CAR detection) BUV395 CD3 HIT3a BD 740283 BUV496 CD25 M-A251 BD 741144Attorney Docket No: JBI6895WOPCT1 Fluorochrome Marker Clone Vendor Product No. BUV563 CD45RA HI100 BD 612926 BUV615 CD2 S5.2 BD 751450 BUV661 CD277 (BTN3) 232-5 BD 750227 BUV737 CD56 NCAM16.2 BD 612766 BUV805 CD11a HI111 BD 748572 BV421 CD197 (CCR7) G043H7 BioLegend 353208 BV480 CD95 DX2 BD 746675 BV605 CD5 L17F12 BioLegend 364020 BV650 CD16 3G8 BioLegend 302042 BV711 HLA-ABC G46-2.6 BD 565333 BV786 CD122 Mik-β3 BD 743118 FITC Vγ9 TCR B3 BioLegend 331306 PE Vδ1 TCR TS8.2 Thermo Fisher 12-5679-42 PE-CF594 NKG2D 1D11 BD 562498 PE-Cy5 CD1a HI149 BioLegend 300108 PE-Cy7 CD27 356412 BioLegend 356412 APC Vδ2 TCR 123R3 Miltenyi Biotec 130-121-339 APC-R700 CD127 HIL-7R-M21 BD 565185 APC-Cy7 Live / dead Thermo Fisher L10119 iγδ T cell redifferentiation panel (with CAR detection) BUV395 CD3 HIT3a BD 740283 BUV496 CD25 M-A251 BD 741144 BUV563 CD45RA HI100 BD 612926 BUV615 CD2 S5.2 BD 751450 BUV661 CD4 SK3 BD 612962 BUV737 CD56 NCAM16.2 BD 612766 BUV805 CD11a HI111 BD 748572 BV421 CD161 DX12 BD 562615 BV480 CD95 DX2 BD 746675 BV605 CD5 L17F12 BioLegend 364020Attorney Docket No: JBI6895WOPCT1 Fluorochrome Marker Clone Vendor Product No. BV650 CD16 3G8 BioLegend 302042 BV711 HLA-ABC G46-2.6 BD 565333 BV750 CD8 OKT8 BD 753750 BV786 CD122 Mik-β3 BD 743118 Alexa 488 CAR detection reagent BB700 LDLR C7 BD 745794 PE-CF594 NKG2D NKG2D 1D11 BD PE-Cy5 CD1a HI149 BioLegend 300108 PE-Cy7 CD27 356412 BioLegend 356412 APC Vδ2 TCR 123R3 Miltenyi Biotec 130-121-339 Alexa700 CD7 M-T701 BD 561603 APC-Cy7 Live / dead Thermo Fisher L10119 iγδ T cell functional potential panel (without CAR detection) BUV395 CD3 HIT3a BD 740283 BUB563 CD7 M-T701 BD 741355 BUV496 CD8β 2ST8.5H7 BD 749837 BUV615 CD161 DX12 BD 751151 BV421 IFNγ 4S.B3 BioLegend 502532 BV570 CD45 HI30 BioLegend 304034 BV650 CD122 Mik-β3 BD 743117 BV711 CD4 OKT4 BioLegend 317440 BV750 CD16 3G8 BioLegend 302082 BB700 CD8α RPA-T8 BD 566452 PE TNFα MAb11 BioLegend 502909 PE-Cy5 CD5 L17F12 BioLegend 364032 PE-Cy7 Granzyme B QA16A02 BioLegend 372214 APC Vδ2 TCR 123R3 Miltenyi Biotec 130-121-339 AF700 Vγ9 TCR B3 BioLegend 331318 APC-Cy7 Live / dead Near IR Thermo Fisher L10119Attorney Docket No: JBI6895WOPCT1 Fluorochrome Marker Clone Vendor Product No. iγδ T cell functional potential panel (with CAR detection) BUV395 CD3 HIT3a BD 740283 BUB563 CD7 M-T701 BD 741355 BUV496 CD8β 2ST8.5H7 BD 749837 BUV615 CD161 DX12 BD 751151 BV421 IFNγ 4S.B3 BioLegend 502532 BV570 CD45 HI30 BioLegend 304034 BV650 CD122 Mik-β3 BD 743117 BV711 CD4 OKT4 BioLegend 317440 BV750 CD16 3G8 BioLegend 302082 Alexa 488 CAR detection reagent BB700 CD8α RPA-T8 BD 566452 PE TNFα MAb11 BioLegend 502909 PE-Cy5 CD5 L17F12 BioLegend 364032 PE-Cy7 Granzyme B QA16A02 BioLegend 372214 APC Vδ2 TCR 123R3 Miltenyi Biotec 130-121-339 AF700 Vγ9 TCR B3 BioLegend 331318 APC-Cy7 Live / dead Near IR Thermo Fisher L10119 .2. Equipment .2.1. Moxi GO II Cell Counter .2.2. EVOS M7000 imager .2.3. CO2 Incubator [37 °C, 5% CO2, 18% O2 (normoxic)] .2.4. 4 °C Refrigerator .2.5. -20 °C Freezer .2.6. -80 °C Freezer .2.7. Centrifuge .2.8. Configuration for BD LSRFortessaTMcytometer Table 1.6: Configuration for BD LSRFortessaTMcytometer Lasers Filter SetFluorochrome379 / 28 BUV395 354 (UV) 690LP, 740 / 35 BUV737Attorney Docket No: JBI6895WOPCT1Lasers Filter SetFluorochrome450LP, 515 / 30 Indo-1, BUV496 410LP, 431 / 28 BV421 505LP, 525 / 50 BV510 600LP, 610 / 20 BV605 405 (violet) 635LP, 670 / 30 BV650 690LP, 710 / 50 BV711 750LP, 780 / 60 BV786 505LP, 530 / 30 FITC, Alexa 488, BB515 488 (blue) 690P, 710 / 50 PerCP-Cy5.5 Additional FSC-A, H, W and SSC-A, H, W 586 / 15 PE 600LP, 610 / 20 PE-Texas Red 561 (green) 650LP, 670 / 30 PE-Cy5 750LP, 780 / 60 PE-Cy7 650LP, 670 / 30 APC, Alexa 647 640 (red) 690LP, 730 / 45 Alexa 700 750LP, 780 / 60 APC-Cy7 Configuration for FACSymphonyTMA5 cytometer Table 1.7: Configuration for FACSymphonyTMA5 cytometer Lasers Filter Set Fluorochrome 379 / 28 BUV395 450LP, 515 / 30 BUV496 550LP, 586 / 15 BUV563 354 (UV) 100 mW 600LP, 610 / 20 BUV615 635LP, 670 / 30 BUV661 690LP, 740 / 35 BUV737 770LP, 820 / 60 BUV805 410LP, 431 / 28 BV421 450LP, 470 / 14 BV480 550LP, 586 / 15 BV570 600LP, 610 / 20 BV605 405 (violet) 200 mW 630LP, 670 / 30 BV650 685LP, 710 / 50 BV711 710LP, 740 / 35 BV750 750LP, 780 / 60 BV786Attorney Docket No: JBI6895WOPCT1 505LP, 515 / 20 BB515 600LP, 610 / 20 BB630 635LP, 670 / 30 BB660 8 (blue) 100 mW 690LP, 710 / 50 PerCP-Cy5.5, BB700 750LP, 780 / 60 BB790 Additional FSC-A, H, W and SSC-A, H, W 586 / 15 PE 600LP, 610 / 20 PE-Texas Red 1 (green) 150 mW 635LP, 670 / 30 PE-Cy5 690LP, 710 / 50 PE-Cy5.5 750LP, 780 / 60 PE-Cy7 670 / 30 APC, Alexa 647 0 (red) 140 mW 690LP, 710 / 50 Alexa 700 750LP, 780 / 60 APC-Cy7 Exemplary Procedure Overview of iCD34 to iγδ T cell redifferentiation process. a. The redifferentiation of iCD34 into iγδ T cells consists of two major steps (Figure 1). i. Promoting T cell specification and commitment by culturing iCD34 cells on immobilized (i.e., plate-bound) DLL4 (Notch ligand), VCAM-1 (α4β1 integrin ligand), and RetroNectin (RN) (another α4β1 integrin ligand that is a fragment of recombinant human fibronectin) for the entire 14-day redifferentiation process. ii. Promoting iγδ T cell maturation on Day 7 by replating the redifferentiating cells onto freshly coated DLL4 / VCAM-1 / RN plates in the presence of IL-15 until harvest on Day 14. b. Prior to setting up the iγδ T cell redifferentiation process, flow cytometry was used to determine iCD34 cell purity and phenotype following Step 4.1.a. Exemplary purity of iCD34 cells [defined as Lineage marker-negative (Lin‒) CD34+] was >70% and fold expansion per input iPSC was >100-fold for 2D format and >0.5-fold for 3D format. c. Small scale SFFF 2D redifferentiation of iCD34s into iγδ T cells was performed as described in 1.3.2.Attorney Docket No: JBI6895WOPCT1 d. Large scale SFFF 2D redifferentiation of iCD34s into iγδ T cells was performed as described in 1.3.3. iγδ T cells generated using the large-scale process can be used in a subsequent expansion step. Small scale SFFF 2D culture method to redifferentiate iCD34 cells into iγδ T cells a. DLL4 / VCAM-1 / RN-coated 48-well plates were prepared following directions outlined in Table 1.8. Table 1.8: Reagents for coating the 48-well plate with DLL4, VCAM-1, and RN. Stock Reagent Concentration Number of wells Volume for a 48- Final Concentration (µg / mL) of a 48-well Plate* well Plate (µL) (µg / mL) DLL4 250 2 8 10 VCAM-1 100 2 10 5 RN 1000 2 2 10 1X DPBS N / A 2 255 N / A *An extra volume was prepared to account for pipetting errors. For example, if coating 2 wells of the 48-well plate, the coating reagents were calculated based on 3 wells. b. Lyophilized DLL4 and VCAM-1 were reconstituted in 1X DPBS to prepare stock solutions. i. DLL4 stock solution was prepared by adding 200 µL 1X DPBS to the vial containing 50 µg of lyophilized DLL4 for 250 µg / mL stock solution. The lyophilized powder was allowed to completely dissolve and then 100 µL aliquots were prepared. ii. VCAM-1 stock solution was prepared by adding 1 mL 1X DPBS to the vial containing 100 µg of lyophilized VCAM-1 for 100 µg / mL stock solution. The lyophilized powder was allowed to completely dissolve and then 100 µL aliquots were prepared. iii. The aliquots of DLL4 and VCAM-1 stock solutions were stored in -20 °C freezer for up to 6 months and freeze / thaw cycles were avoided. c. RN aliquots were prepared by thawing the RN vial on ice or at 4 °C. i. 100 μL aliquots were prepared. ii. The aliquots were stored at -80 °C for up to 6 months and freeze / thaw cycles were avoided.Attorney Docket No: JBI6895WOPCT1 d. DLL4 (10 μg / mL), VCAM-1 (5 μg / mL), and RN (10 μg / mL) coating solution was prepared following directions outlined in Table 1.8. i. For each source of iCD34 cells to be redifferentiated, two wells of a 48-well tissue culture-treated plate were coated with 100 μL / well of the DLL4 / VCAM-1 / RN coating solution and the plate was manually rocked to ensure the solution covered the surface of the well. ii. For uncoated wells, 100 μL / well of 1X DPBS was added. iii. The plate was incubated at 37 °C for one hour. a) Note: the plate can be stored overnight at 4 °C. Prior to use, the plate was incubated at 37 °C for one hour. e. Day 0: iCD34 cells for redifferentiation were prepared: i. StemSpanTMSFEM II medium and Lymphoid Progenitor Expansion Supplement (10X) were thawed overnight at 4 °C. ii. 5 mL of iγδ T cell redifferentiation medium was prepared. The medium was brought to room temperature (RT, 15-25 °C) prior to use. The differentiation medium should not be warmed up using a water bath. Table 1.9: iγδ T redifferentiation medium components for small research-scale redifferentiation. Reagent Stock Concentration Final Concentration Volume for 48-well plate StemSpan™ SFEM II1X 1X 4.5 mLLymphoid Progenitor Expansion Supplement (10X) 10X 1X 0.5 mL iii. For freshly redifferentiated iCD34 cells, the purity of the Lin‒ CD34+ cell population was calculated as in Step 1.4.1 before determining the fold expansion of the Lin‒ CD34+ cell population. After determining the purity and fold expansion of the iCD34 cells was satisfactory, cell counting was performed as in Step 1.3.2.e.iv. iii. For cryopreserved iCD34 cells, cells were thawed as outlined in Step 4.1.a.i.b. iv. The cells were counted using Moxi GO II. a) 10 μL of cell suspension was mixed with 90 μL of Orflo Moxi Cyte Viability Reagent (this was considered a dilution factor of 10).Attorney Docket No: JBI6895WOPCT1 b) The cells were counted on Moxi GO II following the program “Cell Count and Viability.” c) The prompts were followed within the program and 60 μL of diluted cell reagent was loaded. d) Calcuate the number of total live cells. e) The cell diameter gates were adjusted to permit a cell size between 6 to 16 μm and the cell viability gate was adjusted based on live / dead population separation. f) The below formula was used to calculate the final cell count: [(Live cell density (cell no. / mL)) x (Dilution factor of 10) x (Volume of cell resuspension (mL))] v. The volume of iγδ T cell redifferentiation medium was adjusted such that the iCD34 cells were at a final cell concentration of 4.8 x104cells / mL before plating. Any remaining iγδ T cell redifferentiation medium was stored at 4 °C for up to 3 days. vi. iCD34 cells were seeded onto a 48-well plate: a) VCAM-1 / DLL4 / RN coating solution was removed from each coated well and the wells were washed once with 0.5 mL 1X DPBS / well. b) With a P1000, 250 µL of iCD34 cell suspension was added to each of the two wells to seed 1.2x104cells / well onto the pre-coated 48-well plate. c) The plate was incubated at 37 °C, 5% CO2, 18% O2 to initiate redifferentiation. f. Day 3: Cells were fed by adding iγδ T cell redifferentiation medium (Table 1.9) to the wells. i. The iγδ T cell differentiation medium was brought to RT. ii. 250 µL of medium was gently added to each well containing cells. iii. The plate was incubated at 37 °C, 5% CO2, 18% O2. g. Day 7:Each well of redifferentiating cells was replated into a freshly coated DLL4 / VCAM-1 / RN wells in RT iγδ T cell redifferentiation medium + IL-15. i. A 48-well plate that was freshly coated with DLL4 / VCAM-1 / RN coating solution following Steps 1.3.2.d.i-iii. was prepared.Attorney Docket No: JBI6895WOPCT1 ii. The iγδ T cell redifferentiation medium was prepared with exogenous IL-15 shown in Table 1.10. a) To prepare the IL-15 stock solution, 1 mL of 1X DPBS was added to a vial of 100 µg lyophilized IL-15. The IL-15 solution was mixed well by vortexing. The vial was centrifuged at 500 x g for 3 minutes. The solution was aliquoted into Eppendorf tubes with 50 µL / vial and stored at -20 °C. Table 1.10: iγδ T cell redifferentiation medium + IL-15 for small scale feeding on Day 7. Reagent Stock Final Concentration Volume for 48-well Concentration plate StemSpan™ SFEM II1X 1X 4.5 mLLymphoid Progenitor Expansion Supplement (10X) 10X 1X 0.5 mL rhIL-15 100 µg / mL 10 ng / mL 0.5 µL iii. Using a P1000, cells were gently pipetted up and down (4-6 times) and then the iγδ T cells were collected in a 15 mL conical tube. iv. The wells were rinsed with 250 µL of IMDM wash medium 3 times and the cell suspensions were collected in the 15 mL conical tube. v. The tube containing the cell suspensions was centrifuged at 300 x g for 5 minutes at RT. vi. The resulting cell pellet was resuspended in 1 mL of iγδ T cell redifferentiation medium + IL-15 (Table 1.10). vii. From the new pre-coated plate, VCAM-1 / DLL4 / RN coating solution was removed and the new pre-coated plate washed once with 0.5 mL 1X DPBS / well. viii. 500 µL / well of resuspended cells was pipetted onto the side of each well. The plate was incubated at 37 °C, 5% CO2, 18% O2. h. Day 10: 50% medium exchange with iγδ T cell redifferentiation medium+ IL-15 (Table 1.10). i. The iγδ T cell redifferentiation medium was exchanged by gently removing 250 µL of culture medium and adding 250 µL fresh room temperature iγδ TAttorney Docket No: JBI6895WOPCT1 cell redifferentiation medium + IL-15 (Table 1.10) to each well of the 48-well plate. i. Day 14: iγδ T cells were harvested and cell purity, phenotype, and maturity were evaluated by flow cytometry. i. The cells were collected in a 50 mL conical tube by using a P1000, and pipetted up and down 4-6 times (gently to avoid creating bubbles). ii. The wells were rinsed with IMDM wash medium and the resulting rinsate was pipetted into the 50 mL conical tube. iii. The cells were centrifuged at 300 x g for 5 minutes at RT. iv. The supernatant was removed and the cell pellets were resuspended to 1-1.5 mL of culture depending on the size of the cell pellet. v. The cells were counted using Moxi GO II as in Step 1.3.2.e.iv. vi. 0.5x106-1.0x106cells were aliquoted in 100 µL / well of a 96-well V-bottom plate for flow cytometric analysis to assess the purity and phenotype of iγδ T cells according to Step 1.4.1.b. vii. iγδ T cell redifferentiation flow panel is shown in Table 1.17 and Table 1.18. viii. Exemplary success criteria for iγδ T cell redifferentiation step were cell purity (defined by CD3+ Vγ9+ Vδ2+ population) >60 % and cell fold expansion per input iCD34 >10-fold. ix. 0.5x106-1.0x106cells were aliquoted in 100 µL / well of a 96-well V-bottom plate for flow cytometric analysis to assess functional potential of iγδ T cells following Step 1.4.2.a. x. iγδ T cell functional potential flow panel is shown in Table 1.19 and Table 1.20. Large scale SFFF 2D culture method to redifferentiate iCD34 cells into iγδ T cells a. DLL4 / VCAM-1 / RN-coated well plates were prepared. i. The Recombinant proteins were reconstituted in 1X DPBS following Step 1.3.2.b-c. ii. Coating reagents of DLL4 (10 μg / mL), VCAM-1 (5 μg / mL), and RN (10 μg / mL) were prepared based on the number of plates needed for large-scale redifferentiation according to Table 1.11.Attorney Docket No: JBI6895WOPCT1 Table 1.11: Reagents needed for coating the large-scale DLL4 / VCAM-1 and RN plate. Stock centration Number o Final Reagent Con f Volume for a 48- mL) P Concentration (µg / late(s) well Plate (µL) (µg / mL) DLL4250 1 192 10VCAM-1100 1 240 5RN1000 1 48 101X DPBSN / A 1 4500 N / Aiii. Each well of the 48-well plate(s) was coated with 100 µL of the coating solution and the plate(s) were manually rocked to ensure the solution covers the surface of the well. iv. The plate(s) were incubated at 37 °C for one hour. a) Note: plate(s) can be stored overnight at 4 °C. Upon use, follow Step 1.3.2.e.vi.a. b. Day 0: iCD34 cells derived from the 2D-adherent, 3D-single cell or 3D-cluster fractions and meeting exemplary criteria (Step 1.4.1.a.ii.v) were redifferentiated to iγδ T cells using the large scale SFFF 2D culture method. i. For fresh iCD34 cells, the method in Step 1.4.1.a.i. was used to assess purity of the Lin‒ CD34+ cell population. If of sufficient purity, the cells were counted as in Step 1.3.2.e.iv and then the concentration was adjusted as in Step 1.3.3.b.iii. ii. For cryopreserved iCD34 cells, cells were thawed using the method outlined in Step 1.4.1.a.i.b. If of sufficient purity, the cells were counted as in Step 1.3.2.e.iv and then the concentration was adjusted as in Step 1.3.3.b.iii. iii. Based on cell counts, the volume of iγδ T cell redifferentiation medium was adjusted (Table 1.9) such that the iCD34 cells were at a final cell concentration of 4.8x104cells / mL before plating. iv. DLL4 / VCAM-1 / RN solution was removed from the pre-coated plate, and the plate was washed with 0.5 mL 1X DPBS / well. v. 1.2x104live cells / 250µL / well of the pre-coated 48-well plate were seeded. vi. The plate was incubated at 37 °C 5% CO2, 18% O2 to initiate redifferentiation.Attorney Docket No: JBI6895WOPCT1 c. Day 3: The cells were fed by adding iγδ T cell redifferentiation medium (Table 1.9) to the wells. a. The iγδ T cell differentiation medium was brought to RT. b. 250 µL of the iγδ T cell differentiation medium was gently added to each well containing cells. c. The plate was incubated at 37 °C, 5% CO2, 18% O2. d. Day 7: Each well of redifferentiating cells was replated into a freshly coated DLL4 / VCAM-1 / RN well in RT iγδ T cell redifferentiation medium + IL-15 (Table 1.10). i. A freshly coated well plate was prepared with DLL4 / VCAM-1 / RN according to Step 1.3.3.a. ii. iγδ T cell redifferentiation medium was prepared with exogenous IL-15 as in Table 1.10. iii. Using a P1000, cells were gently pipetted up and down (4-6 times) and then the iγδ T cells were collected in a 15 mL conical tube. iv. The wells were rinsed with 250 µL of IMDM wash medium 3 times and the cell suspensions were collected in the 15 mL conical tube. v. The cell suspensions were centrifuged at 300 x g for 5 minutes at RT. vi. The cell pellet was resuspended in 1 mL of iγδ T cell redifferentiation medium + IL-15 listed in Table 1.10. vii. From the new pre-coated plate, VCAM-1 / DLL4 / RN coating solution was removed and the plate was washed once with 0.5 mL 1X DPBS / well. viii. 500 µL / well of resuspended cells were pipetted onto the side of each well. The plate was incubated at 37 °C, 5% CO2, 18% O2. e. Day 10: 50% medium exchange iγδ T cell redifferentiation medium + IL-15 (Table 1.10) to wells. i. The medium was exchanged by gently removing half of the volume of culture medium and adding the same volume of fresh RT iγδ T cell redifferentiation medium + IL-15 (Table 1.10). ii. The plate was incubated at 37 °C 5% CO2, 18% O2.Attorney Docket No: JBI6895WOPCT1 f. Day 14: iγδ T cells were harvested and cell purity, phenotype, and maturity were evaluated by flow cytometry. i. The cells were collected in a 50 mL conical tube by using P1000, and pipetted up and down 4-6 times, gently. ii. The wells were rinsed with IMDM wash medium and the cells were collected into the 50 mL conical tube. iii. The cells were centrifuged at 300 x g for 5 minutes at RT. iv. The supernatant was removed and the cell pellets were resuspended to 1-1.5 mL of iγδ T cell redifferentiation medium + IL-15 depending on the size of the cell pellet. v. The cells were counted using Moxi Go II (Step 1.3.2.e.iv.). vi. 0.5 x106to 1.0 x106cells were aliquoted for flow cytometric analysis to assess the purity and phenotype of iγδ T cells in 100 µL / well of a 96-well V-bottom plate as in Step 1.4.1.b. iγδ T cell redifferentiation panel is shown in Table 1.18 and Table 1.19. vii. 0.5 x106to 1.0 x106cells in 100 µL / well were aliquoted into two wells of a 96-well U-bottom plate and the method of Step 1.3.4 was used to perform a functional potential assay (panels are shown in Table 1.19 and Table 1.20). viii. Exemplary criteria of iγδ T cell redifferentiation step: cell purity (defined by percentage of CD3+ Vγ9+ Vδ2+ population) >60% and cell fold expansion per input iCD34 cells >10-fold. ix. Large scale redifferentiated iγδ T cells were ready for expansion. Functional Potential Assay a. From cells seeded in Step 1.3.3.e.vii., one well was assigned as “unstimulated” and a second well was assigned as “PMA + Ionomycin stimulated.” b. The plate(s) were centrifuged at 400 x g for 3 minutes at RT. c. The medium was sterilely removed without disturbing the cell pellet. d. Diluted brefeldin A was prepared at 1:1000 with iγδ T cell differentiation medium (Table 1.9) for non-activated sample. i. Note: A volume of solution necessary for the number of unstimulated cell samples was prepared. For example, if there are 10 unstimulated cell samples,Attorney Docket No: JBI6895WOPCT1 1 mL of iγδ T cell differentiation medium can be mixed with 1 µL brefeldin A. e. A diluted cell activation cocktail was prepared by diluting Cell Activation Cocktail (with brefeldin A) at 1:100 with iγδ T cell differentiation medium (Table 1.9). i. Note: A volume of solution necessary for the number of PMA + Ionomycin stimulated cell samples was prepared. For example, if there are 10 PMA + Ionomycin stimulated samples, 1 mL of iγδ T cell differentiation medium can be mixed with 10 µL Cell Activation Cocktail. ii. Note: Cell Activation Cocktail (with brefeldin A) was prepared with DMSO. The reagent was fully thawed prior to preparing dilution. f. To set up the unstimulated sample, with a P200, 100 µL / well of diluted brefeldin A from Step 1.3.4.d. was pipetted into the assigned well and gently pipetted up and down 4-6 times. g. To set up PMA + Ionomycin sample, with a P200, 100 µL / well of diluted cell activation cocktail from Step 1.3.4.e. was pipetted into the assigned well and gently pipetted up and down 4-6 times. h. The plate was incubated at 37 °C, 5% CO2, 18% O2 in an incubator for 5 hours. i. The plate was removed from the incubator. j. Surface staining was performed as in Step 1.4.1.b. Flow Cytometry Procedure and Data Analysis 1.4.1 Flow cytometry procedure a. A percentage (i.e., purity) of Lin- CD34+ cell population was determined via flow cytometry. i. iCD34 cells used as starting material for iγδ T cell redifferentiation can be either freshly redifferentiated or thawed cryopreserved cells. a) When freshly harvested iCD34 cells were used, 1.0x105cells / well in 100 µL were plated into a 96-well, V-Bottom plate. The cells were then stained as in Step 1.4.1.a.ii. b) When thawed cryopreserved iCD34 cells were used, the following procedure was used.Attorney Docket No: JBI6895WOPCT1 i) A cryovial of frozen iCD34 cells was thawed in a 37 °C water bath for 1 minute or until small pieces of frozen floating cells were visible. ii) The thawed iCD34 cells were transferred from the vial slowly in a drop-wise manner into a 15 mL conical tube containing 9 mL of RT IMDM wash medium. The thawed iCD34 cells were transferred from the vial slowly in a drop-wise manner into a 15 mL conical tube containing 9 mL of RT IMDM wash medium. The total volume was 10 mL. iii) The 15 mL conical tube containing the thawed iCD34 cells was centrifuged at 300 x g for 3 minutes at RT. iv) Without disturbing the cell pellet, medium was aspirated from 15 mL conical tube leaving behind 0.5 mL of wash medium. v) The conical tube was gently tapped to loosen the pellet. vi) With a P1000, a volume of IMDM wash medium was addded to bring to 1 mL and gently pipetted 4-6 times to break up the cell pellet and resuspend the cells. vii) A 10 µL aliquot was removed to perform cell count. See Step 1.3.2.e.iv. for cell counting procedure. c) 1x105cells / well were seeded in 100 µL into a 96-well, V-Bottom plate and the procedure of Step 1.4.1.a.ii.i was followed to stain the cells. ii. Cell surface staining of iCD34 cells: a) 100 µL / well of Stain Buffer was added and the plate was centrifuged at 400 x g for 3 minutes. To include the Kasumi-1 cell line as a staining control, the process in Step 1.4.1.a.i.b)-c) was followed to thaw cryopreserved cells and the Kasumi cells were seeded for staining. b) The plate was flicked to remove supernatant. c) The Near IR Live / Dead stain working solution and Fc Block reagents were prepared following the table list below.Attorney Docket No: JBI6895WOPCT1 Table 1.12: Reagents needed for live / dead and Fc blocking. Reagents Stock concentration Working Dilution Stain Buffer Near IR Live / Dead 1000 X 1:1000 Stain Buffer Fc Block 20 X 1:20 Stain Buffer d) The cell pellets were resuspended in 100 µL / well of pre-diluted Near IR Live / Dead and diluted Fc block reagent. e) The plate was incubated at RT for 10 minutes in the dark. f) 100 µL of Stain Buffer was added to each well and the cells were centrifuged at 400 x g for 3 minutes. g) The plate was flicked to remove the supernatant, and the cell pellets were resuspended in 200 µL Stain Buffer added to each well. h) The cells were centrifuged at 400 x g for 3 minutes. i) The Antibody Master Mix was prepared according to the panel of interest in Stain Buffer. i) If iCD34 cells do not express a CAR, the iCD34 Panel (no CAR detection) from Table 1.14 can be used. (a) In a 1.8 mL Eppendorf tube, the dump channel containing Lineage Marker Master Mix was prepared by combining 10 μL of each antibody conjugated with FITC, Alexa 488, or BB515. This Lineage Marker Master Mix was treated as a single antibody and was stained at 1:100 dilution. ii) If iCD34 cells do express a CAR, iCD34 Panel (with CAR detection) shown in Table 1.15 can be used. (a) In a 1.8mL Eppendorf tube, the dump channel containing Lineage Marker Master Mix was prepared by combining 10 μL of each antibody conjugated with Alexa 700. This Lineage Marker Master Mix was treated as a single antibody and stained at 1:100 dilution. iii) To determine the identity of the Lineage marker-positive cells post iCD34 cell redifferentiation, the 22-color Multilineage Panel was used as shown in Table 1.16.Attorney Docket No: JBI6895WOPCT1 j) Total volume of Antibody Master Mix was calculated as follows: (number of wells + 3) x 100 µL. k) Antibody Master Mix incorporated Brilliant Stain Buffer at a dilution of 1:10. l) The Antibody Master Mix in Stain Buffer was prepared for antibodies detecting surface antigens in a reagent reservoir and mixed well by pipetting up and down 4-6 times with a P200 multichannel pipette. m) The plate was flicked to remove supernatant. n) The cell pellets were resuspended in 100 µL / well of Antibody Master Mix. o) The plate was incubated at 4 °C for at least 30 minutes in the dark. p) Step 1.4.1.a.ii.e)-g) was repeated for washing with Stain Buffer. q) To fix the stained samples, pre-diluted fixative was prepared according to Table 1.13. Table 1.13: Preparation of 1X Stabilizing fixative buffer. Vol. of 3X Solvent Vol. of Total Reagents Stabilizing used to dH2O working Working fixative (mL) dilute (mL) Vol. (mL) concentration Stabilizing Fixative 3X Concentrate 1 dH2O 2 3 1X r) The cell pellets were resuspended in 200 µL / well of pre-diluted stabilizing fixative. s) The stained cells were acquired on the flow cytometer. t) Stained samples were run within a week after fixation. u) Exemplary criteria for iCD34 redifferentiation step: purity of >70% Lin‒ CD34+ cells, >100-fold expansion (for 2D process of iPSC to iCD34 redifferentiation) and >0.5-fold expansion (for 3D process of iPSC to iCD34 redifferentiation) per input iPSC. Table 1.14: iCD34 panel (without CAR detection) Laser Filter Fluorochrome Antibody(UV) 100 mW379 / 28 BUV395 CD45(violet) 200 mW410LP, 431 / 28 BV421 CD38Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody CD3, CD2 e) 100 mW 505LP, 530 / 30 F 35a, CD11b, CD11c, (blu ITC, Alexa 488, BB515 CD14, CD56, CD19, CD20, CD16, CD2 (green) 150 mW 575 / 26 PE CD34 (red) 140 mW 650LP, 670 / 30 APC, Alexa 647 CD43 750LP, 780 / 60- high performance PMT APC-Cy7 Live / dead Near IR Table 1.15: iCD34 panel (with CAR detection) Laser Filter Fluorochrome Antibody (UV) 100 mW 379 / 28 BUV395 CD45 410LP, 431 / 28 BV421 CD34 (violet) 200 mW 690LP, 710 / 50 BV711 CD43 505LP, 530 / 30 FITC, Alexa 488, B CAR detection reagent (blue) 100 mW B515 690P, 710 / 50 PerCP-Cy5.5 CD38 CD3, CD235a, CD11b, CD11c, 690LP, 730 / 45 Alexa 700 CD14, CD56, CD19, CD20, (red) 140 mW CD16, CD2 750LP, 780 / 60- high performance PMT APC-Cy7 Live / dead Near IR Table 1.16: 22-color Multilineage Panel Laser Filter Fluorochrome Antibody 379 / 28 BUV395 CD2 450LP, 515 / 30 BUV496 CD11b 550LP, 586 / 15 BUV563 Notch-1(UV) 100 mW600LP, 610 / 20 BUV615 CD14 635LP, 670 / 30 BUV661 CD20 690LP, 740 / 35 BUV737 CD45 770LP, 820 / 60 BUV805 CD3 410LP, 431 / 28 BV421 CD41 450LP, 470 / 14 BV480 CD56 (violet) 200 mW 550LP, 586 / 15 BV570 Live / Dead 600LP, 610 / 20 BV605 SSEA4 630LP, 670 / 30 BV650 CD11c 685LP, 710 / 50 BV711 CD16 710LP, 740 / 35 BV750 CD33 750LP, 780 / 60- high performance PMT BV786 CD7 (blue) 100 mW 505LP, 515 / 20 BB515 SSEA3Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody 690LP, 710 / 50 PerCP-Cy5.5, BB700 CD19 (green) 150 mW 586 / 15 PE CD34 750LP, 780 / 60 PE-Cy7 CD38 670 / 30 APC, Alexa 647 CD43 (red) 140 mW 690LP, 710 / 50 R718 CD235a 750LP, 780 / 60- high perf PMT APC / Fire 750 CD326 b. Cell surface staining of iγδ T cells i. 100 µL of Stain Buffer / well was added and the cells were centrifuged at 400 x g for 3 minutes at 4 °C. ii. The plate was flicked to remove supernatant, 200 µL of Stain Buffer was added and the plate was centrifuged at 400 x g for 3 minutes. iii. Steps 1.4.1.a.ii.a)-h) were followed for washing and live / dead staining. iv. The Antibody Master Mix was prepared in Stain Buffer. a) The iγδ T cell redifferentiation panel listed in Table 1.17 or Table 1.18 can be used to stain for cell phenotype. b) The iγδ T cell functional potential panels listed in Table 1.19 or Table 1.20 can be used to stain for functional potential assay. i) Note: antibodies listed in Table 1.19 or Table 1.20 that detect surface antigens were used. ICS antibodies were not included in the surface staining step, eg, anti-TNF-α, IFNγ, and Granzyme B antibodies. v. Steps 1.4.1.a.ii.i)-t) were followed to stain for cell phenotyping. vi. Steps 1.4.1.a.ii.i)-q) were followed to stain for functional potential assay and Step 1.4.1.c was followed for intracellular staining of iγδ T cells with cytokine detection. Table 1.17: iγδ T cell redifferentiation panel (without CAR detection) Laser Filter Fluorochrome Antibody 379 / 28 BUV395 CD3 450LP, 515 / 30 BUV496 CD25 (UV) 100 mW 550LP, 586 / 15 BUV563 CD45RA 600LP, 610 / 20 BUV615 CD2 635LP, 670 / 30 BUV661 CD277 (BTN3) 690LP, 740 / 35 BUV737 CD56Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody 770LP, 820 / 60 BUV805 CD11a 410LP, 431 / 28 BV421 CD197 (CCR7) 450LP, 470 / 14 BV480 CD95 600LP, 610 / 20 BV605 CD5 (violet) 200 mW 630LP, 670 / 30 BV650 CD16 685LP, 710 / 50 BV711 HLA-ABC 750LP, 780 / 60- high performance PMT BV786 CD122(blue) 100 mW505LP, 515 / 20 BB515 TCR Vγ9586 / 15 PE Vδ1 TCR 600LP, 610 / 20 PE-CF594 NKG2D (green) 150 mW 635LP, 670 / 30 PE-Cy5 CD1a 750LP, 780 / 60 PE-Cy7 CD27 670 / 30 APC, Alexa 647 Vδ2TCR 690LP, 710 / 5p0 APC-R700 CD127 (red) 140 mW 750LP, 780 / 60- high performance PMT APC-Cy7 Live / dead Near IR Table 1.18. iγδ T cell redifferentiation panel (with CAR detection). Laser Filter Fluorochrome Antibody 379 / 28 BUV395 CD3 450LP, 515 / 30 BUV496 CD25 550LP, 586 / 15 BUV563 CD45RA (UV) 100 mW 600LP, 610 / 20 BUV615 CD2 635LP, 670 / 30 BUV661 CD4 690LP, 740 / 35 BUV737 CD56 770LP, 820 / 60 BUV805 CD11a 410LP, 431 / 28 BV421 CD161 450LP, 470 / 14 BV480 CD95 600LP, 610 / 20 BV605 CD5 (violet) 200 mW 630LP, 670 / 30 BV650 CD16 685LP, 710 / 50 BV711 HLA-ABC 710LP, 740 / 35 BV750 CD8 750LP, 780 / 60 BV786 CD122 (blue) 100 mW 505LP, 515 / 20 FITC, Alexa 488, BB515 CAR detection reagent 586 / 15 PE LDLR 600LP, 610 / 20 PE-CF594 NKG2D (green) 150 mW 635LP, 670 / 30 PE-Cy5 CD1a 750LP, 780 / 60 PE-Cy7 CD27Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody 670 / 30 APC, Alexa 647 Vδ2TCR (red) 140 mW 690LP, 710 / 5p0 Alexa 700 CD7 750LP, 780 / 60 APC-Cy7 Live / dead Near IR c. Intracellular staining of iγδ T cells to detect soluble effectors i. Step 1.3.4 was followed to set up the activation assay. The iγδ T cell functional potential panel listed in Table 1.19 or Table 1.20 was used to perform the functional potential assay. ii. Surface marker staining was performed following Step 1.4.1.b. prior to intracellular staining for cytokine detection in this section. iii. BD Perm / Wash 1:10 was diluted with dH2O. a) Note: The amount of wash was calculated to prepare based on number of samples x volume. iii. Cells were resuspended with 100 µL Fixation / Permeabilization solution per well for 20 minutes at RT. iv. 100 µL of diluted 1X BD Perm / Wash buffer was added per well. v. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. vi. The plate was flicked and 200 µL of diluted 1X BD Perm / Wash buffer was added per well. vii. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. viii. Diluted ICS antibody (anti-TNFα, IFNγ, and Granzyme B antibodies) master mix was prepared according to Table 1.19 or Table 1.20 in diluted 1X BD Perm / Wash buffer. a) Total volume of diluted ICS antibodies was calculated as follows: (number of wells + 3) x 100 µL. ix. The cells were resuspended in 100 µL / well. x. The plate was incubated at 4 °C for 30 minutes in the dark. xi. 100 µL of 1X BD Perm / Wash buffer was added per well. xii. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. xiii. The plate was flicked and 200 µL of 1X BD Perm / Wash buffer was added per well.Attorney Docket No: JBI6895WOPCT1 xiv. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. xv. A diluted Stabilizing Fixative was prepared (Table 1.13). xvi. The plate was flicked and 200 µL of diluted Stabilizing Fixative was added per well. Table 1.19: iγδ T cell functional potential panel (without CAR detection). Laser Filter Fluorochrome Antibody 379 / 28 BUV395CD3450LP, 515 / 30 BUV496CD8β(UV) 100 mW550LP, 586 / 15 BUV563CD7600LP, 610 / 20 BUV615CD161690LP, 740 / 35 BUV737CD56410LP, 431 / 28 BV421IFN-γ (ICS)550LP, 586 / 15 BV570CD45(violet) 200 mW630LP, 670 / 30 BV650CD122685LP, 710 / 50 BV711CD4710LP, 740 / 35 BV750CD16(blue) 100 mW690LP, 710 / 50 PerCP-Cy5.5, BB700CD8α586 / 15 PETNF-α (ICS)635LP, 670 / 30 PE-Cy5CD5(green) 150 mW 750LP, 780 / 60 PE-Cy7Granzyme B (ICS)670 / 30 APC, Alexa 647TCR Vδ2(red) 140 mW690LP, 710 / 50 AF700TCR Vγ9750LP, 780 / 60- high APC-Cy7 performance PMT Live / dead Near IR Table 1.20: iγδ T cell functional potential panel (with CAR detection). Laser Filter Fluorochrome Antibody 379 / 28 BUV395 CD3 (UV) 100 mW 450LP, 515 / 30 BUV496 CD8β 550LP, 586 / 15 BUV563 CD7 600LP, 610 / 20 BUV615 CD161 690LP, 740 / 35 BUV737 CD56 410LP, 431 / 28 BV421 IFN-γ (ICS) (violet) 200 mW 550LP, 586 / 15 BV570 CD45 630LP, 670 / 30 BV650 CD122 685LP, 710 / 50 BV711 CD4Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody 710LP, 740 / 35 BV750 CD16 505LP, 530 / 30 FITC, Alexa 488, CAR detection rea blue) 100 mW B gent ( B515 690LP, 710 / 50 PerCP-Cy5.5, BB700 CD8α (green) 150 mW 586 / 15 PE TNF-α (ICS) 635LP, 670 / 30 PE-Cy5 CD5 750LP, 780 / 60 PE-Cy7 Granzyme B (ICS) 670 / 30 APC, Alexa 647 TCR Vδ2 (red) 140 mW 690LP, 710 / 50 AF700 TCR Vγ9 750LP, 780 / 60- high performance PMT APC-Cy7 Live / dead Near IR 1.4.2 Gating strategy of Flow Cytometry Data a. Gating strategy to identify Lin‒ CD34+ cell population. To identify the Lin‒ CD34+ cell population using panels listed in Table 1.14 or Table 1.15, the gating strategy outlined in Figure 2 was followed. i. Lineage markers include: CD3, CD235a, CD11b, CD11c, CD14, CD56, CD19, CD20, CD16 and CD2 ii. To calculate the purity of the Lin‒ CD34+ cell population (ie, iCD34 cells), the following formula was used: [(% of Lin- population) x (% of (CD34+CD43+) + (CD34+CD43- ))] b. Gating strategy to identify which hematopoietic lineages are present after iCD34 redifferentiation step using 22-color multilineage panel. To identify the Lin+ cell populations (Table 13), the gating strategy outlined in Figure 3 was followed. c. Gating strategy to identify iγδ T cells. To identify iγδ T cells, the gating strategy outlined in Figure 4 was followed. i. To calculate the purity of the CD3+ Vγ9+ Vδ2+ cell population (ie, iγδ T cells), the following formula was used: [(% of CD3+population) x (% of TCRVγ9+ Vδ2+)] ii. To calculate iγδ T cell fold expansion per iCD34 input, the following formula was used:Attorney Docket No: JBI6895WOPCT1 [(Purity of iγδ T cells) x (Total cell yield / well)] / [(Purity of iCD34 cells seeded) x (Cell seeding numbers / well)] iii. To calculate iγδ T cell fold expansion per iPSC input, the following formula was used: [(Purity of iCD34 cells) x (Total cell yield / well)] / [(number of iPSC cells seeded / well)] x [(Purity of iγδ T cells) x (Total cell yield / well)] / [(Purity of iCD34 cells seeded) x (Cell seeding numbers / well)] iv. To evaluate the surface phenotype, gate on viable CD3+ Vγ9+ Vδ2+ cell population and then assess: CD5 vs CD11a, CD1a vs HLA-ABC, CD56 vs NKG2D, and CD27 vs CD45RA d. Gating strategy to measure functional potential of unstimulated and PMA + Ionomycin stimulated iγδ T cells The gating strategy for measuring functional potential of unstimulated and PMA + Ionomycin stimulated iγδ T cells is shown in Figure 5. Example 2: Flow-based assay to measure the functional potential of redifferentiated iγδ T cells Table 2.1: Abbreviations used in Example 2 Abbreviation Definition CAR Chimeric antigen receptor CoA Certificate of analysis Fc Fc region of an immunoglobulin DPBS Dulbecco’s Phosphate-buffered Saline iγδ T cell iPSC-derived γδ T cell IFNγ Interferon γ FBS Fetal bovine serum RPMI 1640 Roswell Park Memorial Institute 1640 cell culture medium IL-2 Interleukin-2 IL-15 Interleukin-15 PMA Phorbol 12-myristate 13-acetate iPSC induced pluripotent stem cell ICS Intracellular staining RT Room temperature rh Recombinant human TCR T cell receptorAttorney Docket No: JBI6895WOPCT1 Abbreviation Definition TC Tissue culture TNFα Tumor necrosis factor α Vγ9 Variable region domain γ9 Vδ2 Variable region domain δ2 Materials Cells: a. Redifferentiated iγδ T cells derived from unedited or gene-edited, research-grade or GMP-grade, γδ T-iPSC clones. b. Zoledronate-expanded primary γδ T cells are used as a positive control and are expanded following the protocol outlined in ‘Evaluating the cytotoxic potential of expanded iγδ T cells using a long-term cytotoxicity assay’. Reagents: Table 2.2: Reagents for iγδ T cell and primary γδ T cell culture. Reagent Vendor, Product No. Storage condition StemSpan™ SFEM II StemCellcat.# -20 °C, upon thaw use within two 09655 weeks and store at 4°C Lymphoid Progenitor StemCell Technologies, cat.# -20 °C, upon thaw use within two Expansion Supplement 09915 weeks and store at 4°C (10X) RPMI 1640 Medium ThermoFisher Scientific, cat.# store at 4°C 11875135 FBS ThermoFisher Scientific, cat.# -20 °C, upon thaw use within one A3840001 month and store at 4°C rhIL-2, premium grade Miltenyi Biotec, cat.# 130- Store lyophilized rhIL-2 at 097-748 -20 °C. Reconstitute according to CoA to achieve biological activity of 500 IU / μL and store at -20 °C in 50 μL aliquots. rhIL-15, premium grade Miltenyi Biotec, cat.# 130- Store lyophilized rhIL-15 at -20 095-765 °C. Reconstitute according to CoA to achieve biological activity of 2000 IU / μL and store at -20 °C in 50 μL aliquots. Table 2.3: Supplies and consumables. Item Product No. 96-well Conical (U)-Bottom Plate, TC SurfaceThermo Scientific, cat.# 1633206-well Nunc™ Cell-Culture Treated platesThermo Scientific, cat.# 140675Fisherbrand™ Sterile Polystyrene Disposable Thermo Scientific, Serological Pipets with Magnifier Stripe (various cat.# 13-676-10H, sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11F Pipette Tips RT LTS 20 µL F 960A / 10 (P20)Rainin, cat.# 30389225Attorney Docket No: JBI6895WOPCT1 Item Product No. Pipette Tips RT LTS 200 µL F 960A / 10 (P200)Rainin, cat.# 30389239Pipette Tips RT LTS 1000 µL F 768A / 8 (P1000)Rainin, cat.# 3038921250 mL conical tubesCorning, cat.# 352098Falcon® 15 mL Polystyrene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Corning, cat.# 352095 Sterile Reagent ReservoirVWR, cat.# 633001271.8 mL cryovialsThermo Scientific, cat.# 3754181.5 mL Eppendorf tubesGreiner-bio-one cat.# 616261Cell liftersCorning, cat.# 3008Moxi GO Cassette Type S+ Orflo, cat.# MXC030 Orflo Moxi Cyte Viability Reagent Orflo, cat.# MX055 Table 2.4: Reagents for Flow Panels. Reagent Vendor Product Dilution Storage No. Flow staining Reagents Stain Buffer BD Biosciences 554656 1X4 °CBrilliant Stain Buffer BD Biosciences 361651 1:104 °CStabilizing Fixative 3X BD Biosciences 3380 4 °C Concentrate 36 1:3 Human TruStain FcX™ (Fc BioL 4 °C Receptor Blocking Solution) egend 422302 1:20 ICS Reagents BD Cytofix / Cytoperm kit 1:10 for 4 °C Perm / (Perm / Wash buffer and Wash Fixation / Permeabilization BD Bioscie Buffer solution) nces 554714 1X for Fix / Perm solution Cell Activation Reagents Cell Activation Cocktail (a 4 °C pre-mixed cocktail with optimized concentrations of BioLegend 423304 1:100 PMA and Ionomycin) with Brefeldin A -20°C, aliquot Brefeldin A Solution (1,000X) BD Biosciences 420601 1:1000 to avoid freeze / thaw Table 2.5: Antibodies in iγδ T cell functional capacity panel. Bolded antibodies are added during the ICS stage. Fluorochrome Marker Clone Vendor Product No. BUV395 CD3 HIT3a BD 740283 BUB563 CD7 M-T701 BD 741355Attorney Docket No: JBI6895WOPCT1 Fluorochrome Marker Clone Vendor Product No. BUV496 CD8β 2ST8.5H7 BD 749837 BUV615 CD161 DX12 BD 751151 BV421 IFNγ 4S.B3 BioLegend 502532 BV570 CD45 HI30 BioLegend 304034 BV650 CD122 Mik-β3 BD 743117 BV711 CD4 OKT4 BioLegend 317440 BV750 CD16 3G8 BioLegend 302082 BB700 CD8α RPA-T8 BD 566452 PE TNFα MAb11 BioLegend 502909 PE-Cy5 CD5 L17F12 BioLegend 364032 PE-Cy7 Granzyme B QA16A02 BioLegend 372214 APC Vδ2 123R3 Miltenyi Biotec 130-121-339 AF700 Vγ9 B3 BioLegend 331318 APC-Cy7 Live / dead NearThermo FisherIR Scientific L10119 Equipment Moxi GO II Cell Counter CO2Incubator 4 °C Refrigerator -20 °C Freezer -80 °C Freezer Centrifuge Configuration of BD FACSymphony A5 cytometer Table 2.6: Configuration of BD FACSymphony A5 cytometer Lasers Filter SetFluorochrome379 / 28 BUV395 450LP, 515 / 30 BUV496 550LP, 586 / 15 BUV563 354 (UV) 100 mW 600LP, 610 / 20 BUV615 635LP, 670 / 30 BUV661 690LP, 740 / 35 BUV737Attorney Docket No: JBI6895WOPCT1 Lasers Filter SetFluorochrome770LP, 820 / 60 BUV805 410LP, 431 / 28 BV421 450LP, 470 / 14 BV480 550LP, 586 / 15 BV570 600LP, 610 / 20 BV605 405 (violet) 200 mW 630LP, 670 / 30 BV650 685LP, 710 / 50 BV711 710LP, 740 / 35 BV750 750LP, 780 / 60 BV786 505LP, 515 / 20 BB515, FITC, AF488 600LP, 610 / 20 BB630 635LP, 670 / 30 BB660 488 (blue) 100 mW 690LP, 710 / 50 PerCP-Cy5.5, BB700 750LP, 780 / 60 BB790 Additional FSC-A, H, W and SSC-A, H, W 586 / 15 PE 600LP, 610 / 20 PE-Texas Red 561 (green) 150 mW 635LP, 670 / 30 PE-Cy5 690LP, 710 / 50 PE-Cy5.5 750LP, 780 / 60 PE-Cy7 670 / 30 APC, Alexa 647 640 (red) 140 mW 690LP, 710 / 50 Alexa 700 750LP, 780 / 60 APC-Cy7 Exemplary Procedure Day -1: Thaw ex vivo expanded primary γδ T cells. a. 10 mL of primary γδ T cellwas prepared (Table 2.7). The medium was brought to room temperature (RT, 15-25 °C) prior to use. The medium should not be warmed up using water bath. Table 2.7: Primary γδ T cell medium. ReagentStock Concentration Final Concentration Volume for 10 mL medium RPMI 1640 medium1X 1X 9 mLFBS 100 % 10% 1 mL IL-2 500 IU / μL 100 IU / mL 2 μL IL-15 2000 IU / μL 200 IU / mL 1 μLAttorney Docket No: JBI6895WOPCT1 b. A cryovial of frozen expanded primary γδ T cells was thawed in a 37 °C water bath for 1 minute or until small pieces of frozen floating cells were visible. c. The thawed expanded primary γδ T cells material from the vial was slowly transferred in a drop-wise manner into a 15 mL conical tube containing 9 mL of RT RPMI 1640 medium. The thawed expanded primary γδ T cells from the vial was slowly transferred in a drop-wise manner into a 15 mL conical tube containing 9 mL of RT RPMI 1640 medium. The total volume was 10 mL. d. The 15 mL conical tube containing thawed expanded primary γδ T cells was centrifuged at 300 x g for 3 minutes at RT. e. Without disturbing the cell pellet, the medium from 15 mL conical tube was slowly aspirated and 0.5 mL of medium was left behind. f. The conical tube was gently tapped to loosen the pellet. g. With a P1000, a volume of the primary γδ T cell medium was added to bring to 1 mL and the suspension was gently pipetted 4-6 times to break up the cell pellet. h. A 10 µL aliquot was removed for cell counting. i. The cells were counted using Moxi GO II cell counter. i. 10 μL of cell suspension was mixed with 90 μL of Orflo Moxi Cyte Viability Reagent. ii. Cells were counted on Moxi GO II following program “Cell Count and Viability.” iii. Prompts were followed within the program and 60 μL of diluted cell reagent was loaded. iv. The display calculated the total live cell count. v. Cell diameter gates were adjusted to permit a cell size between 6 to 14 μm and the cell viability gate was adjusted based on live / dead population separation. vi. The formula below was used to calculate the final cell count: vii. [(Live cell density (cell no. / mL)) x (Dilution factor of 10) x (Volume of cell resuspension (mL))] j. Cells were plated at a density of 1x106-2x106cells / mL in γδ T cell medium into one well of a 6-well plate. The volume in a well was not allowed to exceed 3 mL. k. The plated were incubated overnight in a 37 °C, 5% CO2, 18% O2 incubator.Attorney Docket No: JBI6895WOPCT1 Day 0: Seed redifferentiated iγδ T cells and primary γδ T cells for cell activation assay. a. Primary γδ T cells were harvested and plateda 96-well U-bottom plate. i. With a 5 mL serological pipette, primary γδ T cells were collected and transferred to a 15 mL conical tube. ii. The wells were rinsed with 1 mL of primary γδ T cell medium. iii. A 10 µL aliquot for cell count was removed as in Step 2.3.1.i. iv. 0.5x106to 1.0x106cells were plated into two wells of a 96-well U-bottom plate. The volume was not allowed to exceed 200 µL / well. v. One well was assigned as “unstimulated” and the second well was assigned as “PMA + Ionomycin stimulated.” b. Redifferentiated iγδ T cells were plated into two wells of the 96-well U-bottom plate from Step 2.3.2.a. The concentration of cells / well was 0.5x106to 1.0x106cells / well. One well was assigned as “unstimulated” and a second well was assigned as “PMA + Ionomycin stimulated.” c. The plate was centrifuged at 400 x g for 3 minutes at RT. d. The medium was sterilely removed without disturbing the cell pellet. e. 10 mL of fresh iγδ T cell redifferentiation medium was prepared (Table 2.8). Table 2.8: iγδ T cell redifferentiation medium. Reagent Stock Final Volume for 10 mL Concentration Concentration medium StemSpan™ SFEM II1X 1X 4.5 mLLymphoid Progenitor Expansion Supplement (10X) 10X 1X 0.5 mL f. Diluted Brefeldin A at 1:1000 in iγδ T cell differentiation medium was prepared (Table 6) for unstimulated samples. Note: A volume necessary for the number of unstimulated cell samples was prepared. For example, if there are 10 unstimulated cell samples, 1 mL of iγδ T cell differentiation medium with 1 µL Brefeldin A can be prepared. g. 500 µL of diluted Brefeldin A at 1:1000 in primary γδ T cell medium was prepared (Table 5).Attorney Docket No: JBI6895WOPCT1 h. A diluted Cell Activation Cocktail (with Brefeldin A) was prepared by diluting Cell Activation Cocktail (with Brefeldin A) at 1:100 in iγδ T cell differentiation medium (Table 6). i. Note: A volume necessary for the number of PMA + Ionomycin stimulated samples was prepared. Example, if there are 10 PMA + Ionomycin stimulated cell samples, 1 mL of iγδ T cell differentiation medium with 10 µL Cell Activation cocktail (with Brefeldin A) can be prepared. ii. Note: Cell Activation Cocktail (with Brefeldin A) was prepared with DMSO. The reagent was fully thawed prior to preparing dilution. i. 500 µL of diluted Cell Activation Cocktail (with Brefeldin A) was prepared in primary γδ T cell medium (Table 5). j. To set up the unstimulated iγδ T cell sample, 100 µL / well of diluted Brefeldin A from Step 2.3.2.f was pipetted into each assigned well and gently pipetted up and down 4-6 times. k. To set up the unstimulated primary γδ T cell sample, 100 µL / well of diluted Brefeldin A from Step 2.3.2.g was pipetted into the assigned well and gently pipetted up and down 4-6 times. l. To set up the PMA + Ionomycin stimulated iγδ T cell sample, 100 µL / well of diluted cell activation cocktail from Step 2.3.2.h was pipetted into the assigned well and gently pipetted up and down 4-6 times. m. To set up the PMA + Ionomycin stimulated primary γδ T cell sample, 100 µL / well of diluted cell activation cocktail was pipetted from Step 2.3.2.i into the assigned well and gently pipetted up and down 4-6 times. n. The plate was incubated in a 37 °C, 5% CO2, 18% O2 incubator for 5 hours. o. The plate was removed from the incubator to initiate antibody staining. Measuring functional potential via flow cytometry. a. 100 µL of Stain Buffer / well was added and the plate was centrifuged at 400 x g for 3 minutes at 4 °C. b. The plate was flicked to remove supernatant, 200 µL of Stain Buffer was added and the plate was centrifuged at 400 x g for 3 minutes.Attorney Docket No: JBI6895WOPCT1 c. The Near IR Live / Dead stain working solution and Fc Block reagents were prepared following the table list below. Table 2.9: Reagents needed for live / dead and Fc blocking. Reagents Stock concentration Working Dilution Stain Buffer Near IR Live / Dead 1000X 1:1000 Stain Buffer Fc Block 20X 1:20 Stain Buffer d. The plate was flicked to remove supernatant and the cell pellets were resuspended in 100 µL / well of pre-diluted Near IR Live / Dead and diluted Fc block reagent. e. The plate was incubated at RT for 10 minutes. f. 100 µL of Stain Buffer was added to each well and cells were centrifuged at 400 x g for 3 minutes. g. The plate was flicked to remove the supernatant, and the cell pellets were resuspended by adding 200 µL Stain Buffer to each well. h. The cells were centrifuged at 400 x g for 3 minutes. i. The Antibody Master Mix was prepared in Stain Buffer for antibodies detecting surface antigens in iγδ T Cell Functional Potential panel (Table 2.10 or Table 2.11) in a reagent reservoir. i. Note: ICS antibodies, namely anti-TNFα, IFNγ, and Granzyme B antibodies, should not be included during the surface staining step. Table 2.10: iγδ T cell functional potential flow panel (without CAR detection). Laser Filter Fluorochrome Antibody354 (UV) 100 mW379 / 28 BUV395 CD3450LP, 515 / 30 BUV496CD8β550LP, 586 / 15 BUV563CD7600LP, 610 / 20 BUV615CD161690LP, 740 / 35 BUV737CD56405 (violet) 200 mW410LP, 431 / 28 BV421IFNγ*550LP, 586 / 15 BV570CD45630LP, 670 / 30 BV650CD122685LP, 710 / 50 BV711CD4710LP, 740 / 35 BV750CD16488 (blue) 100 mW690LP, 710 / 50 PerCP-Cy5.5, BB700 CD8α561 (green) 150 mW586 / 15 PE TNFα*Attorney Docket No: JBI6895WOPCT1 Laser Filter Fluorochrome Antibody 635LP, 670 / 30 PE-Cy5CD5750LP, 780 / 60 PE-Cy7Granzyme B*640 (red) 140 mW670 / 30 APC, Alexa 647Vδ2690LP, 710 / 50 AF700Vγ9* Indicated antibodies are added ONLY during ICS in1X BD Perm / Wash buffer (Step 2.3.3.u). Table 2.11: iγδ T cell functional potential flow panel (with CAR detection). Laser Filter Fluorochrome Antibody 354 (UV) 100 mW379 / 28 BUV395 CD3450LP, 515 / 30 BUV496CD8β550LP, 586 / 15 BUV563CD7600LP, 610 / 20 BUV615CD161690LP, 740 / 35 BUV737CD56405 (violet) 200 mW410LP, 431 / 28 BV421IFNγ* 550LP, 586 / 15 BV570CD45630LP, 670 / 30 BV650CD122685LP, 710 / 50 BV711CD4710LP, 740 / 35 BV750CD16488 (blue) 100 mW505LP, 515 / 20 BB515, FITC, AF488 CAR detection reagent690LP, 710 / 50 PerCP-Cy5.5, BB700CD8α561 (green) 150 mW586 / 15 PE TNFα*635LP, 670 / 30 PE-Cy5CD5750LP, 780 / 60 PE-Cy7Granzyme B*640 (red) 140 mW670 / 30 APC, Alexa 647Vδ2690LP, 710 / 50 AF700Vγ9* Indicated antibodies were added ONLY during ICS in1X BD Perm / Wash buffer (Step 2.3.3.u). ii. Total volume of Antibody Master Mix was calculated as followed: (number of wells + 3) x 100 µL. iii. Antibody Master Mix incorporated Brilliant Stain Buffer at a dilution of 1:10. iv. In a reagent reservoir, surface antibodies, Brilliant Stain Buffer and Stain Buffer were combined. The combined preparation was mixed 4-6 times with a pipette avoiding bubbles. j. The plate was flicked to remove supernatant. k. The cell pellets were resuspended in 100 µL / well of Antibody Master Mix. l. The plate was incubated at 4 °C for at least 30 minutes in the dark.Attorney Docket No: JBI6895WOPCT1 m. The plate was washed as in Steps 2.3.3.f-h. n. The cells were resuspended in 100 µL Fixation / Permeabilization solution per well for 20 minutes at RT in the dark. o. BD Perm / Wash 1:10 was diluted with dH2O. i. Note: The amount of wash to prepare was calculated based on the number of samples x volume. p. The cells were resuspended with 100 µL Fixation / Permeabilization solution per well for 20 minutes at RT in the dark. q. 100 µL of diluted 1X BD Perm / Wash buffer was added per well. r. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. s. The plate was flicked and 200 µL of diluted 1X BD Perm / Wash buffer was added per well. t. The plate was centrifuged at 400 x g for 3 minutes at 4 °C. u. Diluted ICS antibodies (anti-TNFα, IFNγ, and Granzyme B antibodies) were prepared from Table 2.10 or Table 2.11 in diluted 1X BD Perm / Wash buffer. v. The cells were resuspended in 100 µL / well. w. The plate was incubated at 4 °C for 30 minutes in the dark. x. The plate was washed as in Steps 2.3.3.f-h. y. Diluted Stabilizing Fixative was prepared (Table 2.12). Table 2.12: Preparation of 1X Stabilizing fixative buffer. Reagents Vol. of 3X Vol. of Total Working Solvent used Stabilizing dH2O working concentration to dilute fixative (mL) (mL) Vol. (mL) Stabilizing Fixative 1 dH2O 2 3 1X 3X Concentrate z. The plate was flicked and 200 µL of Stabilizing Fixative was added per well. aa. Proceeded to acquiring stained cells on flow cytometer. Gating strategy a. Gating strategy for measuring functional potential of redifferentiated iγδ T cells and expanded primary γδ T cells. To gate on iγδ T cells and primary γδ T cells, the gating strategy as depicted in Figure 5 was followed.Attorney Docket No: JBI6895WOPCT1 b. Gating strategy for measuring functional potential of redifferentiated iγδ T cells and expanded primary γδ T cells. To assess functional potential of iγδ T cell, the gating strategy as depicted in Figure 6 was followed. Cells were gated on Vγ9+ Vδ2+ T cells population.

[0045] List of Embodiments

[0046] The following is a non-exhaustive exemplary list of embodiments encompassed in the claimed invention. A1. A method of de novo generation of human iPSC-derived γδ (iγδ) T cells under normoxic, serum-free and feeder-free culture conditions from iPSC-derived hematopoietic stem cells (iHSCs), said method comprising: a) seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; b) culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC-derived cell intermediates; c) replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with the coating comprising at least one Notch ligand and at least one α4β1 ligand; d) culturing the iHSC-derived cell intermediates in the first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) harvesting redifferentiated iγδ T cells at about day 14 of culture. A2. The method of embodiment A1, wherein the at least one Notch ligand is Delta-like ligand 4 (DLL4). A3. The method of embodiment A1, wherein the the at least one α4β1 ligand comprises vascular cell adhesion molecule 1 (VCAM1) and RetroNectin. A4. The method of embodiment A1, wherein the coating comprises DLL4, VCAM1, and RetroNectin. A5. The method of embodiment A1, wherein step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture. A6. The method of embodiment A1, wherein step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture.Attorney Docket No: JBI6895WOPCT1 A7. The method of embodiment A1, wherein the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential. A8. The method of embodiment A7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13-acetate and ionomycin. A9. The method of embodiment A7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining. B1. A method of de novo generation of human iPSC-derived γδ (iγδ) T cells under normoxic, serum-free and feeder-free culture conditions from iPSC-derived hematopoietic stem cells (iHSCs), said method comprising: a) a step for performing a function of seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; b) a step for performing a function of culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC- derived cell intermediates; c) a step for performing a function of replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with the coating comprising at least one Notch ligand and at least one α4β1 ligand; d) a step for performing a function of culturing the iHSC-derived cell intermediates in the first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) a step for performing a function of harvesting redifferentiated iγδ T cells at about day 14 of culture. B2. The method of embodiment B1, wherein the at least one Notch ligand is Delta-like ligand 4 (DLL4). B3. The method of embodiment B1, wherein the the at least one α4β1 ligand comprises vascular cell adhesion molecule 1 (VCAM1) and RetroNectin. B4. The method of embodiment B1, wherein the coating comprises DLL4, VCAM1, and RetroNectin.Attorney Docket No: JBI6895WOPCT1 B5. The method of embodiment B1, wherein step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture. B6. The method of embodiment B1, wherein step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture. B7. The method of embodiment B1, wherein the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential. B8. The method of embodiment B7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13-acetate and ionomycin. B9. The method of embodiment B7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining.

[0047] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0048] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

Attorney Docket No: JBI6895WOPCT1 Claims 1. A method of de novo generation of human iPSC-derived γδ (iγδ) T cells under normoxic, serum-free and feeder-free culture conditions from iPSC-derived hematopoietic stem cells (iHSCs), said method comprising: a) seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; b) culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC-derived cell intermediates; c) replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with the coating comprising at least one Notch ligand and at least one α4β1 ligand; d) culturing the iHSC-derived cell intermediates in the first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) harvesting redifferentiated iγδ T cells at about day 14 of culture.

2. The method of claim 1, wherein the at least one Notch ligand is Delta-like ligand 4 (DLL4).

3. The method of claim 1, wherein the the at least one α4β1 ligand comprises vascular cell adhesion molecule 1 (VCAM1) and RetroNectin.

4. The method of claim 1, wherein the coating comprises DLL4, VCAM1, and RetroNectin.

5. The method of claim 1, wherein step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture.

6. The method of claim 1, wherein step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture.

7. The method of claim 1, wherein the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential.

8. The method of claim 7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13- acetate and ionomycin.Attorney Docket No: JBI6895WOPCT1 9. The method of claim 7, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining.

10. A method of de novo generation of human iPSC-derived γδ (iγδ) T cells under normoxic, serum-free and feeder-free culture conditions from iPSC-derived hematopoietic stem cells (iHSCs), said method comprising: a) a step for performing a function of seeding iHSCs into a first tissue culture vessel coated with a coating comprising at least one Notch ligand and at least one α4β1 ligand; b) a step for performing a function of culturing the iHSCs in a first culture medium containing interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L) for about 7 days thereby generating iHSC-derived cell intermediates; c) a step for performing a function of replating the iHSC-derived cell intermediates into a second tissue culture vessel coated with the coating comprising at least one Notch ligand and at least one α4β1 ligand; d) a step for performing a function of culturing the iHSC-derived cell intermediates in the first culture medium further comprising interleukin-15 (IL-15) for about 7 days; and e) a step for performing a function of harvesting redifferentiated iγδ T cells at about day 14 of culture.

11. The method of claim 10, wherein the at least one Notch ligand is Delta-like ligand 4 (DLL4).

12. The method of claim 10, wherein the the at least one α4β1 ligand comprises vascular cell adhesion molecule 1 (VCAM1) and RetroNectin.

13. The method of claim 10, wherein the coating comprises DLL4, VCAM1, and RetroNectin.

14. The method of claim 10, wherein step b) further comprises adding additional first culture medium to the first tissue culture vessel about on about day 3 of culture.

15. The method of claim 10, wherein step d) further comprises exchanging the first culture medium further comprising IL-15 on about day 10 of culture.Attorney Docket No: JBI6895WOPCT1 16. The method of claim 10, wherein the redifferentiated iγδ T cells are assayed for one or more of purity, fold expansion, and functional potential.

17. The method of claim 16, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises stimulation of the redifferentiated iγδ T cells with phorbol 12-myristate 13- acetate and ionomycin.

18. The method of claim 16, wherein the assay for the functional potential of the redifferentiated iγδ T cells comprises cell surface staining and intracellular staining.

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