Methods, compositions and systems for immune monitoring

Abstract

Disclosed are methods, kits, and systems for the monitoring of T cells using a panel for assessing at least one marker characteristic of a T cell population and optionally an adoptive cell therapy (ACT)-specific detection reagent for flow cytometric analysis. The method may be used to monitor T cells in a subject who will undergo or is receiving ACT, or to monitor expansion of T cells during the generation of an ACT T cell product. Also disclosed are compositions, kits and systems for performing any portion of the disclosed methods.

Description

METHODS, COMPOSITIONS AND SYSTEMS FOR IMMUNE MONITORINGTECHNICAL FIELD

[0001] The present disclosure relates generally to immune monitoring response to adoptive cell therapies (ACT) using flow cytometry.BACKGROUND

[0002] Adoptive cell therapies (ACT) have emerged as a promising frontier in cancer and autoimmune disorders, harnessing the power of the immune system to combat diseases. However, the full potential of ACT remains elusive, often hindered by challenges such as variable patient responses and limited persistence of infused cells. It is imperative to understand the deep cellular immune phenotypes of these therapies pre- and post-infusion to elucidate what aspects of the infused cells correlate with persistence and positive clinical outcome. Deep phenotyping facilitates the identification of key cellular biomarkers influencing ACT efficacy and helps guide optimal cellular manufacturing processes.SUMMARY

[0003] Disclosed is a method for monitoring T cells. The method may be used to monitor T cells in a subject who will undergo or is undergoing adoptive cell therapy (ACT) or to monitor expansion of T cells during the generation of an ACT T cell product.

[0004] In an embodiment, the method may comprise the steps of: (i) obtaining a sample from a subject; (ii) treating the sample with at least one reagent to identify a marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker; and (iii) assessing the presence or absence and / or amount of T cells having the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (ii) by flow cytometry. In some aspects, surface staining the sample with the reagent is used to identify the at least one marker characteristic of a T cell population and / or ACT-specific T cell population in the sample.

[0005] Also, in some aspects, the step of assessing the presence or absence and / or amount of T cells and / or ACT-specific T cells having the at least one marker is performed by flow cytometry. The ACT-specific T cell may be a CAR T cell. In alternate embodiments, the at least one marker used to identify viable T cell populations present in the sample may be usedwith a variety of different ACT-specific detection markers. In some aspects, the marker characteristic of a T cell population assesses viability, persistence, activation, and / or exhaustion of T cells in the sample from the subject.

[0006] Also disclosed are compositions, including kits, and systems for performing the disclosed methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The disclosure may be better understood by reference to the following non-limiting drawings.

[0008] FIG. 1 depicts a block diagram of an embodiment of a method of the invention.

[0009] FIG. 2 depicts a 19-color panel layout of antibody combinations for surface staining donor cells infused with a commercially available CD 19 CAR product in accordance with an embodiment of the present disclosure.

[0010] FIG. 3 depicts a chart of the overall assay precision in CD3+ / CD19+ CAR- T cells from donor cells infused with a commercially available CD 19 CAR product in accordance with an embodiment of the present disclosure.

[0011] FIGs. 4A-4C depict representative flow cytometric dot plots of staining of T cell subsets within the native compartment and CAR-T compartment from donor cells infused with a commercially available CD 19 CAR T cell product in accordance with an embodiment of the present disclosure.

[0012] FIG. 5 depicts a block diagram of an analysis system used for detection and / or quantification markers specific to T cells and / or ACT T cells.DETAILED DESCRIPTION

[0013] The following description recites various aspects and embodiments of the present methods, compositions, kits and systems. No particular embodiment is intended to define the scope of the methods, compositions, kits and systems. Rather, the embodiments merely provide non-limiting examples of various methods, compositions, kits and systems that are at least included within the scope of the methods, compositions, kits and systems. Thedescription is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.Definitions

[0014] The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section 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 in this section prevails over the definition that is incorporated herein by reference.

[0015] Illustrative examples are given to introduce the general subject matter discussed herein and are not intended to limit the scope of the disclosed concepts. Figure descriptions in the detailed description describe various additional features and examples with reference to the drawings in which like numerals indicate like elements, and directional descriptions are used to describe the illustrative aspects, but, like the illustrative aspects, should not be used to limit the present disclosure.

[0016] When introducing elements of the present disclosure or the embodiment(s) thereof, the articles "a," "an," "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. It is understood that aspects and embodiments of the disclosure described herein include "consisting" and / or "consisting essentially of' aspects and embodiments.

[0017] The term "and / or" when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression "A, B and / or C" isintended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0018] Various aspects of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0019] Embodiments of the present disclosure may be used to analyze a sample or biological sample. The sample may be in liquid, solid, and / or semi-solid form. The biological sample may include tissue, blood, biofluids, biosolids and the like as well as combinations thereof. Thus, the term “biological sample” includes, by way of example and without limitation, whole blood, plasma or serum, tissue such as tissue from the spleen, bone marrow, or thymus, urine, cerebral spinal fluid (CSF), lymph samples, saliva, sputum, stool samples, lavages, semen, and / or body fluids and chemical constituents thereof in raw form and / or in preparations. In certain embodiments, the sample comprises blood, or tissue from the spleen, bone marrow, or thymus.

[0020] As used herein, “CAR T cells” or “CAR-T cells” are a type of immune cell is genetically modified ex vivo to recognize and attack target cells, such as cancer cells, tumor cells, cells associated with autoimmune diseases, or other cells associated with diseases or pathological conditions. T cells (e.g., from a patient’s blood) are genetically modified to express a receptor that binds to a specific ligand (e.g., proteins) on a target cell, such as a cancer cell. The receptor is termed a “chimeric antigen receptor” or “CAR”.

[0021] As used herein, the terms “adoptive cell therapy” or “ACT” refers to a type of immunotherapy wherein immune cells, often T cells, are harvested from a subject (e.g., patient) and modified ex vivo to enhance the ability of the T cells to target cancer cells and / or cells associated with a disease or pathological condition. As used herein, “ACT cell product” refers to a cell (e.g., a T cell) modified and used in ACT.

[0022] As used herein, the term “reagent to identify at least one marker characteristic of a T cell population” is a reagent that recognizes (e.g., binds to) markers expressed on T cells. In some embodiments, the reagent to identify at least one marker characteristic of a T cell population is an antibody which binds to a protein (e.g., CD3) on T cells.

[0023] As used herein, the term “reagent to identify an ACT-specific detection marker” is a reagent that recognizes (e.g., binds to) markers expressed by an ACT cell product, for example an ACT T cell product such as a CAR T cell. By way of example, the ACT-specific detection marker may be an antibody which binds to a protein (e.g., CD 19) targeted by the ACT cell product, or an antibody which binds to a linker (e.g., a G4S or Whitlow / 218 linker) component of the ACT cell product.

[0024] As used herein, the terms “subject” and “individual” may be used interchangeably. A subject may comprise an animal. Thus, in some embodiments, the sample is obtained from a mammalian animal, including, but not limited to a dog, a cat, a horse, a rat, a monkey, and the like. In some embodiments, the sample is obtained from a human subject. In some embodiments, the subject is a patient, that is, a living person presenting themselves in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, the subject is an individual who will undergo or is undergoing ACT.Methods, Compositions and Systems for Immune Monitoring

[0025] Disclosed herein are methods, compositions (including kits), and systems for immune monitoring. The methods, compositions and systems may be embodied in a variety of ways.Methods

[0026] In an embodiment, disclosed is a method for monitoring T cells. The method may be used to monitor T cells of a subject who will undergo or is undergoing ACT. The method may be used to monitor T cells in a subject receiving ACT or to monitor expansion of T cells during the generation of an ACT T cell product.

[0027] In certain embodiments, the method for monitoring T cells comprises the steps of: ((i) obtaining a sample from a subject; (ii) treating the sample with at least one reagent to identify a marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker; and (iii)assessing the presence or absence and / or amount of T cells having the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (ii) by flow cytometry. In an embodiment, assessing the presence or absence and / or amount of T cells having the at least one marker characteristic of a T cell population provides the phenotype of the T cell population.

[0028] The method may be used to monitor T cells of a subject who will undergo or is undergoing ACT. The method may be used to monitor T cells in a subject receiving ACT or to monitor expansion of T cells during the generation of an ACT T cell product. In certain embodiments, the ACT T cell product is an ACT CAR T cell product. The sample may be taken from the subject prior to ACT, and at least one time after the initiation of ACT. Additionally, and / or alternatively, the sample may be blood cells that are to be used to make an ACT T cell product. In certain embodiments, The ACT T cell product may be an ACT CAR T cell product. In these embodiments, the sample used to make the ACT T cell product may be assessed prior to ACT and / or during expansion of the cells to make the ACT T cell product.

[0029] In some embodiments, the sample is a biological sample taken from a subject. In some instances, the biological sample is taken from the blood, spleen, bone marrow, or thymus of a subject. In some embodiments, the biological sample comprises native T cells. In certain embodiments, the biological sample comprises native T cells and / or adoptively transferred T cells.

[0030] In some embodiments, the sample is taken from the subject prior to treatment with the ACT and at least one time after the initiation of treatment with ACT. For example, the sample may be taken from the subject at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times after the initiation of treatment with ACT. In certain embodiments, the sample is taken at regular intervals following treatment with ACT. Samples may be taken weekly, monthly, and / or yearly. For example, the subject may be monitored monthly after ACT for about one year and then monitored at six month intervals for several years thereafter.

[0031] In some embodiments, the sample may be blood cells that are to be used to make an ACT T cell product. In an embodiment, the ACT T cell product is an ACT CAR T cell product. In these embodiments, the sample used to make the ACT T cell product may be assessed prior to ACT and / or during expansion of the cells to make the ACT T cell product.Such characterization can provide valuable assessment of T cell viability, memory, effector T cell subsets, regulatory T cells, and activation / exhaustion for the ACT T cell product. Such monitoring may be performed daily, weekly or monthly as needed during the expansion process.

[0032] The panel of markers used in the present methods may include reagents to detect biomarkers that identify T cell populations present in the sample, and / or are related to T cell function and the immune response thereby providing a phenotype for the T cell. The marker to identify T cell populations present in the sample may comprise at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. Or a plurality of these markers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 may be assessed.

[0033] Optionally, a reagent to assess a marker for T cell function (e.g., viability, persistence, activation, and / or exhaustion) is used. The marker for T cell function may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR.

[0034] Optionally, a reagent to determine T cell viability is used. For example, commercially available reagents, such as VIVAFIX™ dye, can be used to assess the viability of mammalian cells by flow cytometry. Or other dyes used to determine cell viability may be used.

[0035] In some embodiments, a reagent to identify an ACT-specific marker is used in addition to the reagent to identify a marker characteristic of a T cell population. In an embodiment, up to a 21 -marker flow cytometry panel (including, e.g., reagents to detect 20 T cell markers and a reagent to determine T cell viability) can be used with an ACT-specific detection reagent to provide up to a 22-marker flow cytometry panel. In some embodiments, more than one ACT-specific detection reagents may be used. In some embodiments, the flow cytometry panel may comprise more than 22 markers. In some embodiments, the ACT- specific detection reagent may be varied depending upon the ACT cell product being assessed. In some embodiments, the ACT cell product is a CD 19 CAR T cell product and the ACT-specific detection reagent is a CD19 CAR T cell detection reagent. Or another ACT- specific detection reagent may be used to assess a different ACT cell product. For example,the ACT-specific detection reagent may detect a linker (e.g., a G4S or Whitlow / 218 linker) component of the ACT cell product.

[0036] In some embodiments, the panel comprises reagents to detect at least CD3, a viability marker, and an ACT-specific marker. In some embodiments, the panel comprises reagents to detect at least CD3, CD4, CD8, a viability marker, and an ACT-specific marker.

[0037] In some embodiments, native T cells are assessed by flow cytometry using the methods described herein. In certain embodiments, adoptively transferred T cells are assessed by flow cytometry using the methods described herein. In some embodiments, native T cells are phenotyped by flow cytometry using antibodies for one or more of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. In certain embodiments, adoptively transferred T cells are phenotyped by flow cytometry using antibodies for one or more of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR.

[0038] In some embodiments, adoptively transferred T cells are phenotyped at least for ACT cell detection. For example, in a subject infused with the ACT, CD19-targeted CAR T-cell therapeutics (i.e., CD19 CAR products), CAR T-cells in a sample from the subject may be phenotyped by flow cytometry using antibodies for CD 19, such as the FMC63 antibody. Or other reagents that detect CD 19 CAR T cells may be used. As a further example, in subjects infused with the ACT, if the CD19-targeted CAR T-cell therapeutics comprise a G4S or Whitlow / 218 linker, CAR T-cells may be phenotyped by flow cytometry using antibodies for the G4S or Whitlow / 218 linker, such as the E3U7Q or E7O2V antibodies.

[0039] FIG. 1 illustrates an embodiment of a method of the invention. Thus, as shown in FIG. 1, in some embodiments, the method of monitoring T cells described herein comprises isolating cells from a subject being treated with CAR T cell therapy, treating an aliquot of cells with at least one reagent that detects T-cell specific markers and optionally at least one reagent that detects least one CAR T marker, assessing the presence or absence of various T cell populations and / or CAR T cells by flow cytometry, and reporting results.

[0040] In some embodiments, cells are stained with a plurality of one or more fluorophore- conjugated antibodies targeting surface markers. For example, FIG. 2 depicts a 19-color layout of a 22-biomarker panel suitable for use with the methods disclosed herein. Themarkers in FIG. 2 were assessed in isotype controls (Isotype-01 and Isotype-02) and the panel used to assess two experimental samples (Experimental -03 and Experimental-04). In FIG. 2 anti -CD 19 antibody was used as the ACT detection reagent. Detection dyes (Color / Format) were selected to control for known levels of expression for the various markers shown and to allow for multiplex analysis of multiple markers. For example, detection agents that have high staining may be used to detect markers that are present in low amounts and vice versa.

[0041] In an embodiment, the overall assay precision may be monitored and a precision cutoff selected. For example, FIG. 3 shows the overall assay precision in CD3+ / CD19+ CAR-T cells showing 20% CV as the cut-off and staining for various T cell populations in donor samples spiked with the CAR-T product.

[0042] The panel may include biomarkers that allow the monitoring of naive, memory, effector T cell subsets, regulatory T cells, activation / exhaustion biomarkers and quiescent cells, which are known to have proliferative capacity. For example, CD3, CD4 and CD8 are main lineage markers for the identification of T cells. CD62L, CD45RA, CD197, CD95 and CD28 are markers relating to memory and naive T cell markers. CD45 is a pan lymphocyte marker. CD25, CD127 and CD39 are markers that identify regulatory T cells. HLA-DR, CD69, CD137, CD278 and CD134 are immune cell activation markers. CD223, CD366 and CD279, in combination, immune cell exhaustion markers. FIGs. 4A-4C show T cell populations in native T cells as compared to CAR-T cell subsets for markers associated with activation (e.g., 41BB (i.e., CD137)), memory (CD45RA), exhaustion (HLA, LAG), and other T-cell characteristics.

[0043] Thus, in some embodiments, the method further comprises assessing viability, persistence, activation and / or exhaustion of T cells. In some embodiments, the assessed T cells are native T cells and / or adoptively transferred T cells. Thus, the panel can allow for the phenotyping of the native cells in conjunction with that of the adoptively transferred cell therapy pre- and post-infusion with the ACT as well as to monitor the ACT product during expansion.

[0044] In some embodiments, red blood cells are lysed following surface staining with antibody combinations. For example, an ammonium chloride lysis buffer may be used to lyse red blood cells. Other conventional techniques known in the art may be use for red blood celllysis. In certain embodiments, samples are fixed prior to acquisition on a flow cytometer. Conventional techniques known in the art may be used for cell fixation.

[0045] As disclosed herein, frequency and event counts may be analyzed by flow cytometry. This allows for the assay to be quantitative as well as qualitative. With the addition of a dualplatform CD45 Trucount and quantitation standards, both absolute cells per microliter and / or equivalent reference fluorophores (ERF) for checkpoint markers can be determined. The panel may be built so the ACT-specific detection reagents can be switched with detection reagents directed against new products without disrupting performance of the panel with regards to maintaining specificity, sensitivity, and spillover characteristics.Compositions and Kits

[0046] Also disclosed herein are compositions and / or kits for monitoring T cells. As described above, the sample may be from a subject who will undergo or is undergoing ACT.

[0047] Thus, disclosed herein are compositions for monitoring T cells. The compositions may comprise at least one reagent to identify a marker characteristic of a T cell population present in a sample from a subject and, optionally, at least one reagent to identify an ACT- specific detection marker. The marker characteristic of a T cell population may comprise at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. The composition may further comprise a reagent for determination of T cell viability. The marker for viability may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. Where the ACT comprises administration of a CD 19 CAR T cell product, the at least one reagent to identify the adoptive cell therapy (ACT)-specific detection reagent may be a CD 19 CAR T cell detection reagent.

[0048] Optionally, a reagent to assess a marker for T cell function (e.g., viability, persistence, activation, and / or exhaustion) is used. The marker for T cell function may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR.

[0049] Optionally, a reagent to determine T cell viability is used. For example, commercially available reagents, such as VIVAFIX™ dye, can be used to assess the viability ofmammalian cells by flow cytometry. Or other dyes used to determine cell viability may be used.

[0050] In some embodiments, the compositions comprises reagents to detect at least CD3, a T cell viability marker, and an ACT-specific marker. In some embodiments, the composition comprises reagents to detect at least CD3, CD4, CD8, a T cell viability marker, and an ACT- specific marker.

[0051] Also disclosed are kits for monitoring T cells. The kits may comprise at least one reagent to identify at least one marker characteristic of a T cell population present in a sample obtained from a subject, and optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker in the sample obtained from the subject; and instructions for use. The marker characteristic of a T cell population may comprise at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. The kit may further comprise a reagent for determination of T cell viability. Where the ACT comprises administration of a CD 19 CAR T cell product, the at least one reagent to identify the adoptive cell therapy (ACT)-specific detection reagent may be a CD 19 CAR T cell detection reagent. As disclosed above for the methods, the compositions and / or kits may include a plurality of reagents to detect a plurality of markers (e.g., 2, 3, 4, 5, 6, 7, 8, 9., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more). Optionally, a reagent to detect a marker for T cell viability is used. The marker for T cell viability may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. Or commercially available reagents, such as VIVAFIX™ dye, can be used to assess the viability of mammalian cells by flow cytometry. Or other dyes used to determine cell viability may be used.

[0052] In an embodiment, the reagents of the compositions and / or kits are used to assess the presence or absence and / or amount of T cells having the markers so as to provide a phenotype for the T cell population. The reagents of the compositions and / or kits may be used to monitor T cells in a subject receiving ACT or to monitor expansion of T cells during the generation of an ACT CAR T cell product as disclosed herein.

[0053] In some embodiments, a reagent to identify an ACT-specific marker is used in the disclosed compositions and / or kits in addition to the reagent to identify a markercharacteristic of a T cell population. For example, the compositions and / or kits may comprise up to a 21 -marker flow cytometry panel (including, e.g., reagents to detect 20 T cell markers and a reagent to determine T cell viability) can be used with an ACT-specific detection reagent to provide up to a 22-marker flow cytometry panel. In some embodiments, the compositions and / or kits may comprise a flow cytometry panel with more than 22 markers. In some embodiments, the ACT-specific detection reagent may be varied depending upon the ACT cell product being assessed. In some embodiments, the ACT cell product is a CD 19 CAR T cell product and the ACT-specific detection reagent is a CD 19 CAR T cell detection reagent. Or another ACT-specific detection reagent may be used to assess a different ACT cell product. For example, the ACT-specific detection reagent may detect a linker (e.g., a G4S or Whitlow / 218 linker) component of the ACT cell product. Thus, markers characteristic of a T cell population and ACT-specific detection markers described above and useful in the present compositions and / or kits.

[0054] Thus, in certain embodiments, the plurality markers used with either the composition of the kit comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR, and a marker to assess T cell viability. The marker for T cell viability may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR. Or commercially available reagents, such as VIVAFIX™ dye, can be used to assess the viability of mammalian cells by flow cytometry. Or other reagents used to determine cell viability may be used. Thus, the panel may include biomarkers that identify viable T cell populations present in the sample, and / or are related to T cell function and the immune response. In an embodiment, a 21 -marker flow cytometry panel can be used with an ACT-specific detection reagent to provide a 22-marker flow cytometry panel. In some embodiments, the flow cytometry panel may comprise more than 22 markers. In certain embodiments, adoptively transferred T cells are assessed by flow cytometry.

[0055] In some embodiments, the panel comprises reagents to detect at least CD3, a viability marker, and an ACT-specific marker. In some embodiments, the panel comprises reagents to detect at least CD3, CD4, CD8, a viability marker, and an ACT-specific marker.

[0056] In some instances, the composition and / or kit further comprises reagents for performing flow cytometry. Thus, the composition and / or kit may comprise reagents forperforming surface staining of cells using a plurality of one or more fluorophore-conjugated antibodies targeting surface markers. In some embodiments, the ACT-specific detection reagent is an antibody for detecting an antigen associated with a specific ACT. For example, in subjects infused with an ACT, e.g., CD19-targeted CAR T-cell therapeutics (i.e., CD19 CAR products), CAR-T cells may be detected, phenotyped, and / or quantified by flow cytometry using antibodies for CD 19. In certain embodiments, the composition and / or kit further comprises a reagent for lysing red blood cells (e.g., an ammonium chloride lysis buffer or other reagents known in the art). In some instances, the composition and / or kit further comprises a reagent for cell fixation. In some embodiments, the composition and / or kit further comprises a reagent for assessing cell viability. For example, commercially available dyes, such as VIVAFIX™ dye, can be used to assess the viability of mammalian cells by flow cytometry. The reagents of the compositions and / or kits provided herein may be provided in a liquid form or in a dried form.Systems

[0057] Also disclosed are systems for performing any of the steps of the disclosed methods and / or using any of the disclosed compositions and / or kits.

[0058] For example, disclosed is a system comprising: (i) at least one reagent to identify at least one marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an ACT-specific detection marker; and (ii) a component or station to detect the marker characteristic of a T cell population and optionally the ACT- specific detection marker of (i). In an embodiment, assessing the presence or absence or amount of T cells having the marker characteristic of a T cell population present in the sample and, optionally, the ACT-specific detection marker provides a phenotype for the T cell population and / or ACT T cell product. The systems may be used to monitor T cells in a subject receiving ACT or to monitor expansion of T cells during the generation of an ACT T cell product as disclosed herein. In certain embodiments, the ACT T cell product is an ACT CAR T cell product. Also disclosed is a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to run any of the stations and / or components of the system and / or perform a step or steps of any of the disclosed method steps.

[0059] Markers characteristic of a T cell population and ACT-specific detection markers described above and useful in the present systems. For example, in certain embodiments, the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366 and / or HLA-DR. Additionally, or alternatively, the at least one reagent to identify the ACT-specific detection marker is a CD19 CAR T cell detection reagent. The panel may include biomarkers that identify viable T cell populations present in the sample, and / or are related to T cell function and the immune response.

[0060] Optionally, a reagent to assess a marker for T cell function (e.g., viability, persistence, activation, and / or exhaustion) is used. The marker for T cell function may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR.

[0061] Optionally, a reagent to determine T cell viability is used. For example, commercially available reagents, such as VIVAFIX™ dye, can be used to assess the viability of mammalian cells by flow cytometry. The marker for viability may comprise at least one of CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD34, CD137, CD 197, CD223, CD278, CD279, CD366, and / or HLA-DR. Or other reagents used to determine cell viability may be used.

[0062] In an embodiment, a 21 -marker flow cytometry panel can used with an ACT-specific detection reagent to provide a 22-marker flow cytometry panel. In some embodiments, the flow cytometry panel may comprise more than 22 markers. The ACT-specific detection reagent may be varied depending upon the ACT T cell product being assessed. In some embodiments, the ACT is a CD19 CAR product, and the ACT-specific detection reagent is a CD 19 CAR T cell detection reagent. Or another ACT-specific detection reagent may be used to assess a different ACT T cell product. In some embodiments, native T cells are assessed by flow cytometry. In certain embodiments, adoptively transferred T cells are assessed by flow cytometry.

[0063] In some embodiments, the panel comprises reagents to detect at least CD3, a viability marker, and an ACT-specific marker. In some embodiments, the panel comprises reagents to detect at least CD3, CD4, CD8, a viability marker, and an ACT-specific marker.

[0064] Any of the stations and / or components of the system may be automated, robotically controlled, and / or controlled at least in part by a computer and / or programmable software.

[0065] Thus, the system may comprise a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to run the system or any part (e.g., station or component) of the system and / or perform a step or steps of the methods of any of the disclosed embodiments. In some embodiments, a system is provided that includes one or more data processors and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform part or all of one or more methods or processes disclosed herein and / or run any of the parts of the systems disclosed herein.

[0066] Also disclosed is a computer-program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to cause one or more data processors to run any of the components of a system or to perform any of the steps of the disclosed methods. Any suitable computer readable medium may be utilized including hard disks, CD-ROMs, optical storage devices, or magnetic storage devices.

[0067] Thus, in an embodiment, disclosed a computer-program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to perform at least one of the steps of treating the sample with at least one reagent to identify a marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an adoptive cell therapy (ACT)-product, thus assessing the presence or absence and / or amount of T cells having the marker characteristic of a T cell population and optionally the ACT-specific detection marker.

[0068] Also disclosed a computer-program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to run at least one station of a system comprising a component or station to detect a marker characteristic of a T cell population and optionally an ACT-specific detection marker as measured by at least one reagent to identify at least one marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an ACT-specific detection marker.

[0069] Markers characteristic of a T cell population and ACT-specific detection markers described above and useful in the present computer program products. For example, incertain embodiments, the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366 and / or HLA-DR. Additionally, or alternatively, the at least one reagent to identify the ACT-specific detection marker is a CD19 CAR T cell detection reagent. The panel may include biomarkers that identify viable T cell populations present in the sample, and / or are related to T cell function and the immune response. In some embodiments, the plurality of markers comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, and / or HLA-DR and a marker to assess viability. In an embodiment, a 21-marker flow cytometry panel can be used with an ACT- specific detection reagent to provide a 22-marker flow cytometry panel. In some embodiments, the flow cytometry panel may comprise more than 22 markers. The ACT- specific detection reagent may be varied depending upon the ACT T cell product being assessed. In some embodiments, the ACT is a CD19 CAR product, and the ACT-specific detection reagent is a CD19 CAR T cell detection reagent. Or another ACT-specific detection reagent may be used to assess a different ACT T cell product. In some embodiments, native T cells are assessed by flow cytometry. In certain embodiments, adoptively transferred T cells are assessed by flow cytometry. In certain embodiments, both native and adoptively transferred T cells are assessed by flow cytometry.

[0070] The systems and computer products may perform any of the methods disclosed herein. One or more embodiments described herein can be implemented using programmatic modules, engines, or components. A programmatic module, engine, or component can include a program, a sub-routine, a portion of a program, a software component, or a hardware component capable of performing one or more stated tasks or functions. As used herein, a module or component can exist on a hardware component independently of other modules or components. Alternatively, a module or component can be a shared element or process of other modules, programs, or machines.

[0071] FIG. 5 shows a block diagram of an analysis system 500 used for detection and / or quantification markers specific to T cells and / or ACT T cells. As illustrated in FIG. 5, modules, engines, or components (e.g., program, code, or instructions) executable by one or more processors may be used to implement the various subsystems of an analyzer system according to various embodiments. The modules, engines, or components may be stored on anon-transitory computer medium. As needed, one or more of the modules, engines, or components may be loaded into system memory (e.g., RAM) and executed by one or more processors of the analyzer system. In the example depicted in FIG. 5, modules, engines, or components are shown for implementing the methods or running any of the systems of the disclosure.

[0072] Thus, FIG. 5 illustrates an example computing device 500 suitable for use with systems and the methods according to this disclosure. The example computing device 500 includes a processor 505 which is in communication with the memory 510 and other components of the computing device 500 using one or more communications buses 515. The processor 505 is configured to execute processor-executable instructions stored in the memory 510 to perform one or more methods or operate one or more stations for detecting native T-cell and / or ACT markers according to different examples, such as those in FIGS. 1-4 or disclosed elsewhere herein. In this example, the memory 510 may store processorexecutable instructions 525 that can analyze 520 results for sample as discussed herein.

[0073] The computing device 500 in this example may also include one or more user input devices 530, such as a keyboard, mouse, touchscreen, microphone, etc., to accept user input. The computing device 500 may also include a display 535 to provide visual output to a user such as a user interface. The computing device 500 may also include a communications interface 540. In some examples, the communications interface 540 may enable communications using one or more networks, including a local area network (“LAN”); wide area network (“WAN”), such as the Internet; metropolitan area network (“MAN”); point-to- point or peer-to-peer connection; etc. Communication with other devices may be accomplished using any suitable networking protocol. For example, one suitable networking protocol may include the Internet Protocol (“IP”), Transmission Control Protocol (“TCP”), User Datagram Protocol (“UDP”), or combinations thereof, such as TCP / IP or UDP / IP.

[0074] The computer-usable or computer-readable medium may be, but is not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples (a non-exhaustive list) of the computer-readable medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a random-access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, and a portable compact disc read-only memory (CD-ROM). The computer-usable or computer-readable medium could even be paper or another suitable medium, upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner if necessary, and then stored in a computer memory.

[0075] Computer program code for carrying out operations of the present disclosure may be written in an object-oriented programming language such as Java7, Smalltalk, Python, Labview, C++, or VisualBasic. However, the computer program code for carrying out operations of the present disclosure may also be written in conventional procedural programming languages, such as the “C” programming language or even assembly language. The program code may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer. In the latter scenario, the remote computer may be connected to the user's computer through a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0076] Various embodiments of the disclosure have been described herein. It should be recognized that these embodiments are merely illustrative of the present disclosure.Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. It is expected that skilled artisans can employ such variations as appropriate, and the disclosure is intended to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated or otherwise clearly contradicted by context.EXAMPLESExample 1 - Method development and validation

[0077] Healthy donor samples were surfaced stained with the antibody combinations described in FIG. 2. After surface staining, the red blood cells were lysed with an ammonium chloride lysis buffer and the cells were fixed prior to acquisition on a 5-Laser BioRad ZE5.

[0078] Additionally, the samples were assessed for intra-assay precision, inter-operator precision, inter-instrument precision, whole blood stability and post-fixation stability. The panel was monitored for stability as infused CAR cells are best monitored for true stability. During method development and validation, a commercially available CD 19 CAR product was utilized for precision experiments. Samples were phenotyped by flow cytometry according to the 19-color panel layout depicted in FIG. 2.

[0079] FIG. 3 shows the phenotypes assessed by flow cytometry in CD3+ / CD19+ CAR-T cells and the overall assay precision in samples from four donors (DI, D2, D3, and D7) each spiked with 5% CAR-T cells. For each sample and each reportable result, the mean, SD and %CV were calculated. An acceptance criterion of <20% CV was applied. The data used to generate FIG. 3 is reported in TABLE 1.TABLE 1. Phenotypes of CD3+ / CD19+ CAR-T cells and the overall assay precision in samples from four donors (DI, D2, D3, and D7).

[0080] FIG. 4A-4C show T cell populations in native T cells as compared to CAR-T cell subsets for markers associated with activation (e.g., 41BB) memory (CD45RA), exhaustion (HLA, LAG) and other T-cell characteristics. TABLE 2 lists the markers defining each T cell subset phenotype.TABLE 2. T cell subset phenotypes.

[0081] TABLES 3 through 5 demonstrate that the majority of the populations successfully passed validation acceptance criteria, with 448 of 480 reportables meeting intra-assay precision, 395 of 483 reportables meeting inter-operator precision and 343 of 384 reportables meeting inter-instrument performance. Of those reportables that failed to meet acceptance, greater than 50% were from exceedingly low frequency subset populations. Assay stability was determined to be 48 hours with a post-fixation stability time of 6 hours and a minimal event count of 50,000 total CD45+ events.TABLE 3. Intra-assay performanceTABLE 4. Inter-operator performanceTABLE 5. Inter-instrument performance

[0082] Deep phenotyping by flow cytometry is not merely a research tool but an imperative for discovering cellular signatures of immune protection from disease. The standardized method described herein allows measurement of potential cellular attributes across multiple therapies and indications, helping to further define cellular biomarkers during manufacturing and immune monitoring associated with protection.Example 2 - EmbodimentsAl . A method for monitoring T cells comprising the steps of(i) obtaining a sample from a subject;(ii) treating the sample with at least one reagent to identify a marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker; and(iii) assessing the presence or absence and / or amount of T cells having the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (ii) by flow cytometry.A2. The method of embodiment Al, wherein the sample is from a subject who will undergo or is undergoing ACT.A3. The method of any of the preceding embodiments, wherein the sample is taken from the subject prior to ACT, and at least one time after the initiation of ACT.A4. The method of any of the preceding embodiments, wherein the sample is used to make an ACT T cell product.A5. The method of any of the preceding embodiments, wherein the sample used to make the ACT T cell product is assessed prior to ACT.A6. The method of any of the preceding embodiments, wherein the ACT T cell product is assessed during expansion of the cells.A7. The method of any of the preceding embodiments, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD 197, CD223, CD278, CD279, CD366, or HLA-DR.A8. The method of any of the preceding embodiments, wherein the ACT comprises administration of CD 19 CAR T cell product and the at least one reagent to identify the ACT-specific detection marker is a CD19 CAR T cell detection reagent.A9. The method of any of the preceding embodiments, wherein the CD 19 CAR T cell detection reagent is an antibody to CD 19.A10. The method of any of the preceding embodiments, wherein the method further comprises a reagent for determining viability of the T cell population.Al 1. The method of any of the preceding embodiments, wherein the marker characteristic of a T cell population comprises CD3.A12. The method of any of the preceding embodiments, wherein a reagent to identify an ACT-specific detection marker is used.Al 3. The method of any of the preceding embodiments, wherein the marker characteristic of a T cell population assesses viability, persistence, activation, and / or exhaustion of T cells in the sample from the subject.Bl. A composition for monitoring T cells comprising at least one reagent to identify a marker characteristic of a T cell population present in a sample from a subject and, optionally, at least one reagent to identify an ACT-specific detection marker.B2. The composition of embodiment Bl, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, or HLA-DR.B3. The composition of embodiments Bl or B2, wherein the at least one reagent to identify the adoptive cell therapy (ACT)-specific detection reagent is a CD 19 CAR T cell detection reagent.B4. The composition of any of the preceding embodiments, wherein the method further comprises a reagent for determining viability of the T cell population.B5. The composition of any of the preceding embodiments, wherein the marker characteristic of a T cell population comprises CD3.B6. The composition of any of the preceding embodiments, wherein a reagent to identify an ACT-specific detection marker is used.Cl . A kit for monitoring T cells comprising:(i) at least one reagent to identify at least one marker characteristic of a T cell population present in a sample obtained from a subject, and optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker in the sample obtained from the subject; and(ii) instructions for use.C2. The kit of embodiment Cl, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278,CD279, CD366, or HLA-DR.C3. The kit of embodiments Cl or C2, wherein the at least reagent to identify the ACT-specific detection marker is a CD 19 CAR T cell detection reagent.C4. The kit of any of the preceding embodiments, wherein the method further comprises a reagent for determining viability of the T cell population.C5. The kit of any of the preceding embodiments, wherein the marker characteristic of a T cell population comprises CD3.C6. The kit of any of the preceding embodiments, wherein a reagent to identify an ACT-specific detection marker is used.DI . A system for monitoring T cells comprising a component and / or a station performing any of the steps of embodiments A1-A10, or using the composition of embodiments B1-B3, or using the kit of embodiments C1-C3.D2. The system of embodiment D2 comprising:(i) at least one reagent to identify at least one marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an ACT-specific detection marker; and(ii) a component or station to detect the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (i).D3. The system of embodiment DI or D2, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366 or HLA-DR.D4. The system of any of the preceding embodiments, wherein the at least one reagent to identify the ACT-specific detection marker is a CD19 CAR T cell detection reagent.D5. The system of any of the preceding embodiments, wherein the method further comprises a reagent for determining viability of the T cell population.D6. The system of any of the preceding embodiments, wherein the marker characteristic of a T cell population comprises CD3.D7. The system of any of the preceding embodiments, wherein a reagent to identify an ACT-specific detection marker is used.D8. The system of any of the preceding embodiments, further comprising a computer and / or a computer-program tangibly embodied in a non-transitory machine- readable storage medium, including instructions configured to run any of the stations and / or components of the system.El . A computer-program product tangibly embodied in a non-transitory machine- readable storage medium, including instructions configured to perform a step or steps of any of embodiments A1-A13, and / or use any of the compositions of embodiments B1-B6, and / or use any of the kits of embodiments C1-C6, and / or run any of the stations and / or components of the system of embodiments D1-D8.

Claims

CLAIMSWhat is claimed is:

1. A method for monitoring T cells comprising the steps of:(i) obtaining a sample from a subject;(ii) treating the sample with at least one reagent to identify a marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker; and(iii) assessing the presence or absence and / or amount of T cells having the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (ii) by flow cytometry.

2. The method of claim 1, wherein the sample is from a subject who will undergo or is undergoing ACT.

3. The method of claim 2, wherein the sample is taken from the subject prior to ACT, and at least one time after the initiation of ACT.

4. The method of claim 1, wherein the sample is used to make an ACT T cell product.

5. The method of claim 4, wherein the sample used to make the ACT T cell product is assessed prior to ACT.

6. The method of claim 4, wherein the ACT T cell product is assessed during expansion of the cells.

7. The method of claim 1, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, or HLA-DR.

8. The method of claim 2, wherein the ACT comprises administration of CD19 CAR T cell product and the at least one reagent to identify the ACT-specific detection marker is a CD 19 CAR T cell detection reagent.

9. The method of claim 8, wherein the CD19 CAR T cell detection reagent is an antibody to CD 19.

10. The method of claim 1, wherein the method further comprises a reagent for determining viability of the T cell population.

11. The method of claim 1, wherein the marker characteristic of a T cell population comprises CD3.

12. The method of claim 10 or 11, wherein a reagent to identify an ACT-specific detection marker is used.

13. The method of claim 1, wherein the marker characteristic of a T cell population at least one of CD3, CD4, or CD8.

14. The method of claim 1, wherein the marker characteristic of a T cell population assesses viability, persistence, activation, and / or exhaustion of T cells in the sample from the subject.

15. A composition for monitoring T cells comprising at least one reagent to identify a marker characteristic of a T cell population present in a sample from a subject and, optionally, at least one reagent to identify an ACT-specific detection marker.

16. The composition of claim 15, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, or HLA-DR.

17. The composition of claim 15, wherein the at least one reagent to identify the adoptive cell therapy (ACT)-specific detection reagent is a CD 19 CAR T cell detection reagent.

18. The composition of claim 15, wherein the method further comprises a reagent for determining viability of the T cell population.

19. The composition of claim 15, wherein the marker characteristic of a T cell population comprises CD3.

20. The composition of claim 18 or 19, wherein a reagent to identify an ACT- specific detection marker is used.

21. The composition of claim 20, wherein the marker characteristic of a T cell population at least one of CD3, CD4, or CD8.

22. A kit for monitoring T cells comprising:(i) at least one reagent to identify at least one marker characteristic of a T cell population present in a sample obtained from a subject, and optionally, at least one reagent to identify an adoptive cell therapy (ACT)-specific detection marker in the sample obtained from the subject; and(ii) instructions for use.

23. The kit of claim 22, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69,CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366, or HLA-DR.

24. The kit of claim 22, wherein the at least reagent to identify the ACT-specific detection marker is a CD 19 CAR T cell detection reagent.

25. The kit of claim 22, wherein the method further comprises a reagent for determining viability of the T cell population.

26. The kit of claim 22, wherein the marker characteristic of a T cell population comprises CD3.

27. The kit of claim 25 or 26, wherein a reagent to identify ACT-specific detection marker is used.

28. A system for monitoring T cells comprising a component and / or a station performing any of the steps of claims 1-14, or using the composition of claims 15-21, or using the kit of claims 22-27.

29. The system of claim 28 comprising:(i) at least one reagent to identify at least one marker characteristic of a T cell population present in the sample and, optionally, at least one reagent to identify an ACT-specific detection marker; and(ii) a component or station to detect the marker characteristic of a T cell population and optionally the ACT-specific detection marker of (i).

30. The system of claim 28, wherein the marker characteristic of a T cell population comprises at least one of CD3, CD4, CD8, CD25, CD28, CD39, CD62L, CD69, CD45, CD45RA, CD95, CD127, CD134, CD137, CD197, CD223, CD278, CD279, CD366 or HLA-DR.

31. The system of claim 28, wherein the at least one reagent to identify the ACT- specific detection marker is a CD 19 CAR T cell detection reagent.

32. The system of claim 28, wherein the method further comprises a reagent for determining viability of the T cell population.

33. The system of claim 28, wherein the marker characteristic of a T cell population comprises CD3.

34. The system of claim 32 or 33, wherein a reagent to identify ACT-specific detection marker is used.

35. The system of claim 28, further comprising a computer and / or a computerprogram tangibly embodied in a non-transitory machine-readable storage medium,including instructions configured to run any of the stations and / or components of the system.

36. A computer-program product tangibly embodied in a non-transitory machine- readable storage medium, including instructions configured to perform a step or steps of any of claims 1-14, and / or use any of the compositions of claims 15-21, and / or use any of the kits of claims 22-27, and / or run any of the stations and / or components of the system of claims 28-35.

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