Materials and methods for treating cancer

Abstract

This document provides methods and materials involved in treating cancer. For example, chimeric antigen receptor (CAR) T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide and methods of using such CAR T cells are provided. For example, CAR T cells provided herein can be used in an adoptive cell therapy (e.g., a CAR T cell therapy) to treat a mammal (e.g., a human) having cancer.

Description

MATERIALS AND METHODS FOR TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Patent Application Serial No. 63 / 548,465, filed on November 14, 2023, and claims the benefit of U.S. Patent Application Serial No. 63 / 636,427, filed on April 19, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.SEQUENCE LISTINGThis application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2274W01_SL.xml.” The XML file, created on November 9, 2024, is 33,013 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.TECHNICAL FIELDThis document relates to methods and materials involved in treating cancer. For example, this document provides methods and materials for using chimeric antigen receptor (CAR) T cells having one or more of (1) a reduced level of a neurofilament medium chain (NEFM) polypeptide, (2) a reduced level of an uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA) polypeptide, (3) a reduced level of a TNF receptor superfamily member 9 (TNFRSF9) polypeptide, (4) a reduced level of an A-kinase anchoring protein 12 (AKAP12) polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a granulysin (GNLY) polypeptide, (7) an elevated level of a major histocompatibility complex, Class II, DR Beta 5 (HLA-DRB5) polypeptide, and (8) an elevated level of a CTD small phosphatase 1 (CTDSP1) polypeptide. This document also provides methods and materials for using such CAR T cells in an adoptive cell therapy (e.g., a CAR T cell therapy) to treat a mammal (e.g., a human) having cancer.BACKGROUNDCD19-directed chimeric antigen receptor T (CART 19) cells have emerged as a potentially curative immunotherapy in a subset of patients with hematological malignancies.While initial responses are impressive, the majority of responsive patients relapse within a year. Recent studies suggest that CAR T cells are susceptible to states of dysfunction, such as T cell senescence.SUMMARYThis document provides methods and materials for generating T cells (e.g., CAR T cells) having one or more of (1) an altered (e.g., a reduced) level of a NEFM polypeptide, (2) an altered (e.g., a reduced) level of a UACA polypeptide, (3) an altered (e.g., a reduced) level of a TNFRSF9 polypeptide, (4) an altered (e.g., a reduced) level of a AKAP12 polypeptide, (5) an altered (e.g., a reduced) level of a MYC polypeptide, (6) an altered (e.g., an elevated) level of a GNLY polypeptide, (7) an altered (e.g., an elevated) level of a HLA-DRB5 polypeptide, and (8) an altered (e.g., an elevated) level of a CTDSP1 polypeptide. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have reduced NEFM polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to knock out (KO) a nucleic acid encoding a NEFM polypeptide to reduce NEFM polypeptide expression in that T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have reduced UACA polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a UACA polypeptide to reduce UACA polypeptide expression in that T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have reduced TNFRSF9 polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a TNFRSF9 polypeptide to reduce TNFRSF9 polypeptide expression in that T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have reduced AKAP12 polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding an AKAP12 polypeptide to reduce AKAP12 polypeptide expression in that T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have elevated GNLY polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to include nucleic acid (e.g., exogenous nucleic acid) encoding a GNLY polypeptide to increase GNLY polypeptide expression in that T cell. Insome cases, a T cell (e.g., a CAR T cell) can be engineered to have elevated HLA-DRB5 polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to include nucleic acid (e.g., exogenous nucleic acid) encoding an HLA-DRB5 polypeptide to increase HLA-DRB5 polypeptide expression in that T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to have elevated CTDSP1 polypeptide expression (e.g., for use in adoptive cell therapy). For example, a T cell (e.g., a CAR T cell) can be engineered to include nucleic acid (e.g., exogenous nucleic acid) encoding a CTDSP1 polypeptide to increase CTDSP1 polypeptide expression in that T cell.This document also provides methods and materials for using T cells (e.g., CAR T cells) having one or more of (1) an altered (e.g., a reduced) level of a NEFM polypeptide, (2) an altered (e.g., a reduced) level of a UACA polypeptide, (3) an altered (e.g., a reduced) level of a TNFRSF9 polypeptide, (4) an altered (e.g., a reduced) level of a AKAP12 polypeptide, (5) an altered (e.g., a reduced) level of a MYC polypeptide, (6) an altered (e.g., an elevated) level of a GNLY polypeptide, (7) an altered (e.g., an elevated) level of a HLA-DRB5 polypeptide, and (8) an altered (e.g., an elevated) level of a CTDSP1 polypeptide. For example, T cells (e.g., CAR T cells) having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA- DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be administered (e.g., in an adoptive cell therapy) to a mammal (e.g., a human) having cancer to treat the mammal’s cancer.As demonstrated herein, NEFM polypeptides, UACA polypeptides, TNFRSF9 polypeptides, AKAP12 polypeptides can promote CAR T cell senescence, and GNLY polypeptides, HLA-DRB5 polypeptides, and CTDSP1 polypeptides can reduce CAR T cell senescence. Also as demonstrated herein, CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide are lesssusceptible to T cell senescence, and can exhibit enhanced efficacy of CAR T cell function and antitumor activity. In some cases, CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be incorporated into adoptive T cell therapies (e.g., CAR T cell therapies) to treat, for example, mammals having cancer.In general, one aspect of this document features T cells having reduced susceptibility to T cell senescence. The methods T cells having reduced susceptibility to T cell senescence can comprises (a) a reduced level of a NEFM polypeptide, a reduced level of a UACA polypeptide, a reduced level of a TNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, an elevated level of a GNLY polypeptide, an elevated level of a HLA-DRB5 polypeptide, or an elevated level of a CTDSP1 polypeptide, and (b) nucleic acid encoding a chimeric antigen receptor, and where the T cell expresses the chimeric antigen receptor. The T cell can include the reduced level of the NEFM polypeptide. The T cell can include the reduced level of the UACA polypeptide. The T cell can include the reduced level of the TNFRSF9 polypeptide. The T cell can include the reduced level of the AKAP12 polypeptide. The T cell can include the elevated level of the GNLY polypeptide. The T cell can include the elevated level of the HLA-DRB5 polypeptide. The T cell can include the elevated level of the CTDSP1 polypeptide. The T cell can include the reduced level of the NEFM polypeptide, the reduced level of the UACA polypeptide, the reduced level of the TNFRSF9 polypeptide, the reduced level of the AKAP12 polypeptide, the elevated level of the GNLY polypeptide, the elevated level of the HLA-DRB5 polypeptide, and the elevated level of the CTDSP1 polypeptide. The chimeric antigen receptor can target a tumor-associated antigen. The tumor- associated antigen can be CD 19. The chimeric antigen receptor can include a BBi^ signaling domain or a 28^ signaling domain. The T cell can be obtained from a human. The T cell does not exhibit T cell senescence as rapidly as a comparable T cell lacking the reduced level of the NEFM polypeptide, the reduced level of the UACA polypeptide, the reduced level of the TNFRSF9 polypeptide, the reduced level of the AKAP12 polypeptide, the elevated level ofthe GNLY polypeptide, the elevated level of the HLA-DRB5 polypeptide, and the elevated level of the CTDSP1 polypeptide.In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, administering, to a mammal having cancer, a composition comprising a T cell comprising (a) a reduced level of a NEFM polypeptide, a reduced level of a UACA polypeptide, a reduced level of a TNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, an elevated level of a GNLY polypeptide, an elevated level of a HLA-DRB5 polypeptide, or an elevated level of a CTDSP1 polypeptide, and (b) nucleic acid encoding a chimeric antigen receptor, and where the T cell expresses the chimeric antigen receptor. The mammal can be identified as being in need of T cells having reduced susceptibility to CAR T cell senescence. The mammal can be a human. The composition can include from about 100,000 to about 1,000,000,000 of the T cells. The cancer can be a mantle cell lymphoma (MCL), a diffuse large B cell lymphoma (DLBCL), a Hodgkin’s lymphoma, a non-Hodgkin lymphoma, an acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a germ cell tumor, a hepatocellular carcinoma, a bowel cancer, a lung cancer, a breast cancer, an ovarian cancer, a melanoma, a brain cancers, or a multiple myeloma. The chimeric antigen receptor can target a tumor-associated antigen expressed by a cancer cell of the cancer. The tumor-associated antigen can be CD 19.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1. T cells at day 0 (DO) were collected from normal donors to generate CART D8 cells. CART D8 cells were activated by co-culturing them with lethally irradiated CD I9 JeKo-1 cell line to generate CART D15 (after one activation cycle) and CART D22 (after two activation cycles). Cells were collected after each activation cycle to measure the percentages of the cells that expressed p 16, p21, and p53. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates.Figure 2. NSG mice were transplanted with 10A6 Luciferase and CD19+JeKo-1 cells. Once the engraftment of JeKo-1 cells was confirmed by bioluminescence, the mice were randomized for administration of 10A6 untransduced (UTD) cells, CART19-BB^ (BBz) cells, or CART19-28 (28z) cells at D8, DI 5, and D22 transplantations. The survival of the mice was plotted. *p < 0.05, **p < 0.01, ***p < 0.001. Log-rank (Mantel-Cox) test.Figures 3A-3C. CART cell functions decreased upon repeated stimulation. Figure3 A) Summary of CART cell production (D0-D8) and activation protocol (D8-D15 and DI 5- D22). T cells from normal donors (ND) were activated with anti-CD3 / CD28 Dynabeads for 24 hours and divided into three groups for UTD (untransduced) control cells, BBz cells (T cells transduced with CAR19-4-1BB virus), and 28z cells (T cells transduced with CAR19- CD28 virus). The CART cells were fed until D6 when the magnetic beads were removed to rest the CART cell for two days. The cells were co-cultured with lethally irradiated CD 19+ JeKo-1 cell line twice in a week with resting intervals to generate D15 and D22 CART cells. Figure 3B) Cytotoxicity assay: CART cells at different activation cycles were co-cultured with Luc+JeKo-1 cells with varying E:T ratios. This figure shows results from the 0.32: 1 E:T ratio. The viability of the Luc+JeKo-1 was measured by bioluminescence 10 minutes after addition of the substrate (*p < 0.05, Mixed-effects analysis; Tukey's multiple comparisons test, error bars, SEM; up to seven biological and three technical replicates). Figure 3C) Proliferation assay: The same cells as in Figure 3B were co-cultured with JeKo-1 at 1 : 1 E:T ratio. The co-cultures were fed at D3 with fresh media and CART cells were counted by volumetric assays with flow cytometer (*p < 0.05, two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates).Figures 4A-4D. CART cell exhaustion was not responsible for impaired T cell function upon recurrent activation cycle. Figure 4A) Expression of CTLA-4 in 28z cells increased over time (*p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates). Figure 4B) LAG-3 expression didn’t change over time in any of the CART groups tested. Figure 4C) PD-1 expression stayed similar in BBz cells over time but decreased in 28z cells (*p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates). Figure 4D) TIM-3 was the only exhaustion marker whose expression was increased in both CARTs and remained elevated over time (*p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates).Figures 5A-5D. T cell senescence associated phenotypes changes were more noticeable in BBz cells. Figures 5A and 5B) Senescent T cells are known to lose CD28 and CD27 expression. Both marker expressions were decreased in BBz cells over time. Figure 5C) CART cells upon activation cycles were assayed for SA-Bgal activity (measured in mean fluorescent intensity) of each type of CART cell at different time points. The signal intensity increased in both CART cells at D8 and at DI 5 compared to DO (*p < 0.05, Mixed- effects analysis; Tukey's multiple comparisons test, error bars, SEM; up to seven biological and three technical replicates). Figure 5D) CART cells were co-cultured with lethally irradiated JeKo-1 cells in the presence of 2 pM EdU. The percentage of CART cells that were positive for EdU (indicative of cells entering cell cycle) was plotted. EdU positivity stayed similar at D8 and at D15 but decreased at D22 in BBz cells. Cycling cells increased at D15 but decreased again at D22 for 28z cells. (*p < 0.05, **p < 0.01, ***p < 0.001, Two- way ANOVA; Tukey’s multiple comparisons test, error bars, SEM; seven biological and three technical replicates).Figures 6A-6F. Recurrent activation induced impaired CART cell activity in vivo. Figure 6A) Luc+JeKo-1 cells were transplanted into NSG mice. The mice were randomized based on the tumor load (measured by bioluminescence) while the tumor burden was low. Mice groups received indicated CART cells. The tumor burden 16 days after the CART cell infusion was plotted. Tumor levels were lower in mice that were administered D8 CARTcells or DI 5 CART cells than in mice that were administered UTD cells while the tumor levels in mice that were administered D22 CART cells were not lower than mice that were administered UTD cells for both BBz cells and 28z cells. Tumor levels in mice that were administered D22 BBz cells were lower than tumor levels in mice that were administered D22 28z cells. Figure 6B) Blood samples from mice in Figure 6A were collected and the number of CART cells in circulating in blood was used as a measurement of CART cell persistency. The mice that were administered D8 BBz cells had detectable levels of T cells in circulating blood while mice that were administered D15 or D22 BBz cells did not, showing the impairing effect of recurrent activation on CART cells. Figure 6C) Similar experiment to Figure 6A, with the exception of infusing CART cells while the tumor burden in mice was high (stress model). In this experiment only mice that were administered D8 BBz cells or D8 28z cells had a lower tumor burden as compared to mice that were administered UTD cells, indicating impaired CART cell activity in DI 5 CART cells as well. Figure 6D) Blood samples from mice in Figure 6C were to count CART cells in circulating blood. The mice that were administered D8 BBz cells had more T cells in the circulating blood from mice that were administered D15 BBz cells or D22 BBz cells. Mice that were administered D8 28z cells had detectable levels of T cells. Figure 6E and 6F) Peripheral blood samples were collected from the mice 21 days after CART cell infusion. The cytokine analysis from the blood samples indicated increased TNF-a and IL-10 levels for recurrently activated BBz cells while only IL-10 levels were increased in recurrently activated 28z cells. Increased TNF-a and IL-10 levels were reported in senescent T cells. (*p < 0.05, ****p < 0.0001, Two-way ANOVA; Tukey’s multiple comparisons test, error bars, SEM).Figures 7A-7E. Transcriptome analysis suggested senescence like changes in CART cells upon recurrent activation. Figure 7A) RNA from both CART cell types (BBz vs 28z) at indicated time points from 3 normal donors are sequenced. The table shows number of genes whose expression changed significantly Figure 7B) Gene set enrichment analysis (GSEA) indicated that senescence related genes were enriched in DI 5 BBz cells compared to DO T cells and DI 5 28z cells. Figure 7C) Further GSEA analysis identified changes in senescent T cell associated phenotypes such as DNA damage response (DDR) and oxidative stress (OS). Figure 7D) Immunoblotting for DNA damage markers was done to investigate if the GSEAresults had relevance at the molecular level. As the GSEA suggested, DNA damage marker levels changed based on the signaling domain or on the activation cycle. p-H2AX (Serl39), p-ATM (Seri 981) and p-Chk2 (Thr68) levels were higher in D15 BBz cells as compared to D8BBz cells, but their levels were similar in D8 28z cells and DI 5 28z cells. Overall level of p-BRCAl (Seri 524) was higher in BBz cells while levels of p-p53 (Seri 5) and p-Chk2 (Thr68) levels were higher in 28z cells, indicating that the preferential DNA damage response was based on the CSD present in the CAR. Figure 7E) Recurrently activated CART cells from two separate donors were immunoblotted against OS response markers catalase and SOD1. The level of both polypeptides increased consistently upon recurrent activation. The increase was independent of the CSD present in the CART cells.Figures 8A-8G. CART cell response to senescence induced by irradiation (IR). Figure 8A) Senescence was induced during CART cell generation by IR. The CART cells generation protocol was similar to that shown Figure 3 A except for CART cells were exposed to 3 Gy IR. Figures 8B-8E) Expression levels of indicated senescence markers upon IR at D8 were plotted by qPCR. Expression levels of senescence markers were increased compared to untreated CART cell controls. Figures 8F-8G) IR or untreated CART cells were cocultured with JeKo-1 cells. CART cell proliferation (Figure 8F) and cytotoxicity (Figure 8G) were plotted. Proliferation of CART cells upon IR did not increase. Interestingly, irradiated 28z cells (28z-IR cells) killed JeKo-1 more successfully while no increase in cytotoxicity was detected in irradiated BBz cells (BBz-IR cells).Figures 9A-9D. Identification of targets in CART cell senescence. Figures 9A and 9B) According to the transcriptome analysis, expression of both UACA and NEFM was downregulated in D15 BBz cells as compared to D8 BBz cell (3 normal donors). Figure 9C) UACA gene was knocked out with CRISPR / Cas9 technologies. Extra bands in cells transduced with single-guide RNA (sgRNA) targeting UACA (sgUACA) and with the indicated cleavage enzymes suggested generation of mutations in the UACA gene. Figure 9D) CART cells with a knocked-out UACA gene had improved CAR specific killing target cells (representative of 3 experiments with different donors).Figure 10. Jurkat cells were transduced with lentiviral particles containing nontargeting control sgRNA (sgCont), sgUACA, sgRNA targeting NEFM (sgNEFM), or bothsgUACA and sgNEFM, along with Cas9. Genomic DNA was analyzed by cleavage assay to detect mutations.Figures 11A-1 IF. Jurkat cells from Figure 10 were cultured for four days and expression of exhaustion markers (Figures 11 A-l ID) and expression of senescence markers (Figures 1 IE and 1 IF) were monitored. UACA KO Jurkat cells expressed less exhaustion and senescence markers. Measurements were done in three replicates.Figure 12. UACA KO Jurkat cells duplicate faster. The Jurkat cells from Figure 10 were cultured in normal growth media and were followed for four days. The cells were counted at each day and the fold change as compared to DI was plotted. UACA KO Jurkat cells duplicated fastest while NEFM KO cells duplicated slowest. Dual KO Jurkat cells duplicated at similar rates with sgControl treated cells.Figures 13A-13D. Knocking out UACA and NEDM in Jurkat-Lucia™ NFAT(- CD28) cells (JNFAT cells). Figures 13A) JNFAT cells were transduced with lentiviral particles expressing sgCont, sgUACA, or dual (sgUACA+sgNEFM). The extra bands in the presence of cleavage enzyme indicated the intended mutations created in each gene. Figure 13B) JNFAT cells with indicated mutations were cultured and counted daily. UACA KO cells proliferated more compared to control and dual KO JNFAT cells. Figure 13C) The absolute cell number was plotted. UACA KO cells proliferated faster compared to control and dual KO JNFAT cells. Figure 13D) The fold change in the cell number was plotted. UACA KO cells duplicated faster compared to control and dual KO JNFAT cells.Figures 14A-14B. JNFAT activation with beads upon knocking out UACA & NEFM. JNFAT cells from Figure 13 were cultured with CD3 / CD28 activating dynabeads. The activation of UACA KO cells was similar to control cells after 24 hours of activation (Figure 14A), however after 48 hours of coculture the activation increased dramatically compared to control cells (Figure 14B). Dual KO JNFAT cells were overactivated in both time points suggesting overactivation was inducing impairments in T cells.Figures 15A-15F. Recurrent activation induced impaired CART cell activity in vivo. Figure 15A) Mice were intravenously engrafted with 1 x 106luciferase+ JeKo-1 cells. After JeKo-1 tumor burden reached ~107photons / second, mice received 1 x 106UTD or CART cells intravenously as indicated in each group. Tumor burden was serially assessed viabioluminescence (n=5-6 mice / group). Figure 15B) Tumor burdens 16 days post-CART cell infusion are shown (one-way ANOVA). Figure 15C) Kaplan-Meier survival curve is shown (Log-rank test). Figure 15D) In the stress model, mice were intravenously engrafted with 1 x 106luciferase+ JeKo-1 cells. After JeKo-1 tumor burden reached ~108photons / second, mice received 1 x 106UTD or CART cells intravenously as indicated in each group (n=5 mice / group). Tumor burdens 16 days post-CART cell are shown (one-way ANOVA). Figures 15E-15F) Peripheral blood samples from mice in the stress model were collected 21 days after CART cell infusion. Serum levels of TNF- a (Figure 15E) and IL-10 (Figure 15F) are plotted (one-way ANOVA). Error bars, SEM. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Figures 16A-16F. Recurrent activation / rest differentially induced senescence in CART cells with alternative costimulatory domains. Figures 16A-16B) The percentage of CD3+T cells that were positive for pl6 (Figure 16A) and p53 (Figure 16B) as measured by flow cytometer were plotted (n=7 donors; two-way ANOVA). Figure 16C) Recurrently activated CART cells were immunoblotted for OS response proteins catalase and SOD1. Figures 16D-16E) Recurrently activated CART cells were assessed for CD27 (Figure 16D) and CD28 (Figure 16E) expression by flow cytometry (n=7 donors; two-way ANOVA). Figure 16F) Recurrently activated CART cells were immunoblotted for DNA damage markers p-H2AX (Seri 39), p-ATM (Seri 981), and p-Chk2 (Thr68). Error bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001.Figures 17A-17G. Recurrent activation induced senescence and MYC activation in BB^. Figure 17A) senMayo gene set enrichment is shown for the indicated CART groups. Figure 17B) Significantly enriched or depleted MYC-associated gene sets (p<0.1) from GO and curated databases are shown. Figure 17C) DNA damage and senescence makers were measured by immunoblotting of the oncogenic MYC T58A and matched control CART groups (representative of two donors). Figure 17D) p53 levels are shown in the indicated CART groups as measured by flow cytometer (t-Test, error bars, SEM, three technical replicates). Figures 17E-17G) MYC T58A OE CART cells were cocultured with JeKo-1 cells. Cycling cells were measured by EDU (Figure 17E), cytotoxicity (Figure 17F), andproliferation (Figure 17G) (t-Test, each point represents the average of three technical replicates from individual biological donors). *p < 0.05, **p < 0.01, ***p < 0.001.Figures 18A-18H. Wild-type MYC overexpression induced dysfunction in only BB^. Figure 18A) Human WT MYC was overexpressed in CART cells made from 4 normal donors and MYC expression was measured by qPCR. Figures 18B-18C). Each CART cell was cocultured with JeKo-1 and the killing was plotted in various E:T ratios for BB(^ (Figure 18B) and 28C, (Figure 18C). Each dot represents a different donor. Figure 18D) JeKo-1 bearing mice received control or wildtype MYC-overexpressing CART cells. Peripheral blood samples were collected from mice to measure the number of circulating CART cells (t- test). Figures 18E-18F) The tumor burdens of the mice that received control or MYC overexpressing BB^ or 28c, measured by bioluminescence were plotted weekly (two-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001. Figure 18G) Tumor burden of the mice that received MYC overexpressed or matched control CART cells was serially assessed via bioluminescence (n=4-5 mice / group). Figure 18H) The survival of the mice in Figure 18F and Figure 18F.Figures 19A-19J. Irradiation (IR)-induced senescence had opposing impact on CART cells with differing costimulatory domains in vitro and in the clinic. Figures 19A-19D) Senescent CART cells were produced from irradiated T cells (3 Gy). RNA isolated from D8 CART cells was used in qPCR for indicated senescence markers (One-way ANOVA; error bars, SEM, three technical replicates). Fold change was calculated by 2" (-delta delta CT) method and each CART cell was normalized to control group separately. Figures 19E-19F) Irradiated CART cells were cocultured with IeKo-1 cells and assessed for target cell killing (Figure 19E), cell cycle (Figure 19F), and proliferation (Figure 19G). n=6 donors, t-Test, each point represents the average of three technical replicates from an individual biological donor. Figure 19H Axi-cel- and tisa-cel -treated patients were clustered as responsive and nonresponsive. senMayo gene set enrichment is shown. Figure 191) Genes belonging to senMayo gene sets were converted to gene score based on the expression of genes in senMayo, and then patients were divided into senMayo high and low groups. Kaplan-Meier progression-free survival curve is shown (Log-rank test). Figure 19J) MYC-related gene setenrichment is shown in non-responsive tisa-cel -treated patients. (R: responsive, NR: nonresponsive). *p < 0.05, **p < 0.01, ****p < 0.0001.Figures 20A-20F. Recurrent activation / resting cycle did not alter T cell exhaustion markers. Figure 20A) Summary of CART cell production (D0-D8) and activation protocol (D8-D15 and D15-D22). T cells from normal donors were activated with anti-CD3 / CD28 Dynabeads for 24 hours and divided into three groups for UTD (untransduced control), BB(transduced with CAR19-4-1BB virus), and 28(^ (transduced with CAR19-CD28 virus). The CART cells were fed with fresh media until D6 when the magnetic beads were removed to rest the CART cell for two days. The cells were co-cultured with JeKo-1 twice in a week with resting intervals to generate DI 5 and D22 CART cells. Figure 20B) Exhaustion markers during the CART manufacturing process (D0-D8) were assessed by flow cytometry at the indicated timepoints (two-way ANOVA, comparisons to DO). Figures 20C-20F) Exhaustion markers upon recurring activation / resting cycles (D0-D22) were assessed by flow cytometry at the indicated timepoints (Two-way ANOVA; Tukey's multiple comparisons test). Error bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Figures 21A-21C. Recurrent activation / resting induces CART cell dysfunction. Figure 21A) Summary of the in vivo experiments. Figure 21B) Mice were intravenously engrafted with 1 x 106luciferase+JeKo-1 cells. After JeKo-1 tumor burden reached ~108photons / second, mice received 1 x 106UTD or CART cells intravenously as indicated in each group. Tumor burden was serially assessed via bioluminescence (n=5 mice / group). Figure 21C) Peripheral blood samples from stress model mice were collected to assess CART cell proliferation by absolute counts of human CD3+cells by flow cytometry (oneway ANOVA). Error bars, SEM. *p < 0.05, **p < 0.01.Figures 22A-22G. RNA-seq provided additional support for emergence of senescence upon recurrently activated BB(^. Figure 22A) RNA was sequenced from CART cells after recurrent activation / resting cycles. Principal component analysis (PCA) is shown. n=3 biological replicates. Figure 22B) Unsupervised hierarchical clustering analysis of the indicated CART groups is shown. Figure 22C) Selected senescence-associated gene sets that showed significant changes are plotted. Figures 22D-22F) T cell exhaustion marker expression was assessed by flow cytometry after MYC overexpression (three technicalreplicates, t-test). Figure 22G) OS markers were assessed by immunoblotting after MYC overexpression. Error bars, SEM. *p < 0.05, **p < 0.01.Figures 23A-23H. Irradiation induced senescence but not overt exhaustion phenotype. Figure 23 A) Irradiated CART cells were generated similar to Fig. 20A (D0-D8), but here the cells were irradiated with 3 Gy on D2. Figures 23B-23E) Immunophenotypical markers associated with senescent T cells were assessed by flow cytometry (two-way ANOVA). Figures 23F-23H) T cell exhaustion marker expression was assessed by flow cytometry (two- way ANOVA). Error bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.DETAILED DESCRIPTIONThis document provides methods and materials for generating T cells (e.g., CAR T cells) having one or more of (1) an altered (e.g., a reduced) level of a NEFM polypeptide, (2) an altered (e.g., a reduced) level of a UACA polypeptide, (3) an altered (e.g., a reduced) level of a TNFRSF9 polypeptide, (4) an altered (e.g., a reduced) level of a AKAP12 polypeptide, (5) an altered (e.g., a reduced) level of a MYC polypeptide, (6) an altered (e.g., an elevated) level of a GNLY polypeptide, (6) an altered (e.g., an elevated) level of a HLA-DRB5 polypeptide, and (7) an altered (e.g., an elevated) level of a CTDSP1 polypeptide. In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a NEFM polypeptide to reduce NEFM polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to KO nucleic acid encoding a NEFM polypeptide). A T cell that is engineered to KO nucleic acid encoding a NEFM polypeptide can also be referred to herein as a NEFM KO T cell, a NEFM' / _T cell, a NEFM1'0T cell, or a NEFMK0T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding an UACA polypeptide to reduce UACA polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to KO nucleic acid encoding an UACA polypeptide). A T cell that is engineered to KO nucleic acid encoding an UACA polypeptide can also be referred to herein as an UACA KO T cell, an UACA_ / ' T cell, an UACA1'0T cell, or an UACAKOT cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a TNFRSF9 polypeptide to reduce TNFRSF9 polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is notengineered to KO nucleic acid encoding a TNFRSF9 polypeptide). A T cell that is engineered to KO nucleic acid encoding a TNFRSF9 polypeptide can also be referred to herein as a TNFRSF9 KO T cell, a TNFRSF9AT cell, a TNFRSF9k 0T cell, or a TNFRSF9K0T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding an AKAP12 polypeptide to reduce AKAP12 polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to KO nucleic acid encoding an AKAP12 polypeptide). AT cell that is engineered to KO nucleic acid encoding an AKAP12 polypeptide can also be referred to herein as an AKAP12 KO T cell, an AI< AP I 2' ' T cell, an AKAPn1^0T cell, or an AKAP12KOT cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a MYC polypeptide to reduce MYC polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to KO nucleic acid encoding a MYC polypeptide). AT cell that is engineered to KO nucleic acid encoding a MYC polypeptide can also be referred to herein as a MYC KO T cell, a MYC ’ T cell, MYCk 0T cell, or a MYCKOT cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a GNLY polypeptide to elevate GNLY polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to contain nucleic acid encoding a GNLY polypeptide). AT cell that is engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a GNLY polypeptide can also be referred to herein as a GNLY+T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding an HLA-DRB5 polypeptide to elevate HLA-DRB5 polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to contain nucleic acid encoding an HLA-DRB5 polypeptide). A T cell that is engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding an HLA-DRB5 polypeptide can also be referred to herein as an HLA-DRB5+T cell. In some cases, a T cell (e.g., a CAR T cell) can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a CTDSP1 polypeptide to elevate CTDSP1 polypeptide expression in that T cell (e.g., as compared to a comparable T cell that is not engineered to contain nucleic acid encoding a CTDSP1 polypeptide). A T cell that is engineered to contain nucleic acid (e.g., exogenousnucleic acid) encoding a CTDSP1 polypeptide can also be referred to herein as a CTDSPU T cell.In some cases, a T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) an altered (e.g., a reduced) level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be less likely to undergo senescence (e.g., as compared to a CAR T cell that is not engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide). For example, reducing a level of a NEFM polypeptide, reducing a level of a UACA polypeptide, reducing a level of a TNFRSF9 polypeptide, reducing a level of a AKAP12 polypeptide, elevating a level of a GNLY polypeptide, elevating a level of a HLA-DRB5 polypeptide, and / or elevating a level of a CTDSP1 polypeptide in a T cell can be effective to delay the onset of senescence in the T cell by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, a T cell having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can maintain one or more T cell functions for at least 2 weeks (e.g., about 15 days, about 18 days, about 22 days, or more). In some cases, a T cell having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can maintain one or more T cell functions for from about 2 weeks to about 4 weeks.Any appropriate method can be used to determine whether or not one or more T cells (e.g., T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide) are senescent. Examples of methods that can be used to evaluate T cell (e.g., CAR T cell) senescence include, without limitation, methods that include measuring expression of one or more senescence markers (e.g., reduced expression of CD28 and / or reduced expression of CD27), measuring expression of one or more DNA damage markers, measuring expression of a pl 6 polypeptide, measuring expression of a p21 polypeptide, measuring expression of a p53 polypeptide, measuring expression of telomerase polypeptides, measuring expression of one or more surface polypeptides (e.g., a CD57 polypeptide, a CD366 polypeptide, and a KLRG1 polypeptide), and measuring expression of one or more senescence associated secretory phenotype (SASP) polypeptides (e.g., a IL -6 polypeptide, a IL-8 polypeptide, a IFN-g polypeptide, and a TNF polypeptide).In some cases, a T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can have enhanced CAR T cell function such as improved antitumor activity, improved proliferation, improved cell killing (e.g., improved killing of tumor cells), improved cytokine secretion, improved persistence, memory T cell differentiation (e.g., as compared to a CAR T cell that is not engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide). Any appropriate method can be used to assess one or more functions of T cells (e.g., T cells having one or more of (1) a reducedlevel of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide). Examples of methods that can be used to evaluate T cell (e.g., CAR T cell) functions include, without limitation, cytotoxicity assays (e.g., to evaluate whether or not T cells (e.g., CAR T cells) are effective at killing target cells), cell number determinations, proliferation assays, and degranulation assays (e.g., to measure proinflammatory cytokine release from T cells).A T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a ARAP 12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be any appropriate T cell. AT cell can be a naive T cell. Examples of T cells that can be engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide as described herein include, without limitation, cytotoxic T cells (e.g., CD4+CTLs and / or CD8+CTLs). For example, a T cell that can be engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be a CAR T cell. In some cases, one or more T cells designed to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be T cells that were obtained from a mammal(e.g., a mammal having cancer) that is to be treated with those T cells designed to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide. For example, T cells can be obtained from a mammal to be treated with the materials and method described herein.In some cases, as an alternative to using T cells, the methods and materials provided herein can be applied to NK cells. For example, the methods and materials provided herein can be used for generating NK cells (e.g., CAR NK cells) having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide.The term “reduced level” as used herein with respect to a level of a NEFM polypeptide, an UACA polypeptide, a TNFRSF9 polypeptide, a AKAP12 polypeptide, or a MYC polypeptide refers to any level that is lower than a reference level of that polypeptide in control T cells or any level of that polypeptide that is lower in the post-engineered / treated T cells as compared to the level of that polypeptide in the pre-engineered / treated version of those T cells. The term “reference level” as used herein with respect to a NEFM polypeptide, an UACA polypeptide, a TNFRSF9 polypeptide, aAKAP12 polypeptide, or a MYC polypeptide refers to the level of that polypeptide typically observed in control T cells from one or more healthy mammals (e.g., humans) not engineered to have a reduced level of that polypeptide as described herein. Control T cells can include, without limitation, T cells that are wild-type T cells obtained from a healthy mammal. In some cases, a reduced level of a NEFM polypeptide, a reduced level of an UACA polypeptide, a reduced level of a TNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, or a reduced level of a MYC polypeptide can be an undetectable level of that polypeptide. In some cases, a reduced level of a NEFM polypeptide, a reduced level of an UACA polypeptide, a reduced level of aTNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, or a reduced level of a MYC polypeptide can be an undetectable level of that polypeptide.A T cell having (e.g., engineered to have) one or more of a reduced level of a NEFM polypeptide, a reduced level of a UACA polypeptide, a reduced level of a TNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, and a reduced level of a MYC polypeptide can be generated using any appropriate method. In some cases, a T cell (e.g., a CAR T cell) can be treated with one or more inhibitors to reduce polypeptide expression in that T cell (e.g., as compared to a T cell that was not treated with the one or more inhibitors). For example, a T cell (e.g., a CAR T cell) can be treated with one or more NEFM polypeptide inhibitors to reduce a level of NEFM polypeptides in that T cell (e.g., as compared to a T cell that was not treated with the one or more NEFM polypeptide inhibitors). For example, a T cell (e.g., a CAR T cell) can be treated with one or more UACA polypeptide inhibitors to reduce a level of UACA polypeptides in that T cell (e.g., as compared to a T cell that was not treated with the one or more UACA polypeptide inhibitors). For example, a T cell (e.g., a CAR T cell) can be treated with one or more TNFRSF9 polypeptide inhibitors to reduce a level of TNFRSF9 polypeptide in that T cell (e.g., as compared to a T cell that was not treated with the one or more TNFRSF9 polypeptide inhibitors). For example, a T cell (e.g., a CAR T cell) can be treated with one or more AKAP12 polypeptide inhibitors to reduce a level of AKAP12 polypeptides in that T cell (e.g., as compared to a T cell that was not treated with the one or more AKAP12 polypeptide inhibitors). For example, a T cell (e.g., a CAR T cell) can be treated with one or more MYC polypeptide inhibitors to reduce a level of MYC polypeptides in that T cell (e.g., as compared to a T cell that was not treated with the one or more MYC polypeptide inhibitors).A NEFM polypeptide inhibitor can be any appropriate NEFM polypeptide inhibitor. A NEFM polypeptide inhibitor can be an inhibitor of NEFM polypeptide expression. In some cases, an inhibitor of NEFM polypeptide activity can be used to reduce the level of NEFM polypeptide activity instead of or in addition to reducing the level of a NEFM polypeptide expression by a T cell (e.g., a CAR T cell). Examples of compounds that can inhibit NEFM polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to a NEFM polypeptide, and small molecules that target (e.g.,target and bind) to a NEFM polypeptide. Examples of compounds that can inhibit of NEFM polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of a NEFM polypeptide (e.g., a siRNA molecule or a shRNA molecule), nucleic acid molecules designed to induce CRISPR-mediated interference of polypeptide expression of a NEFM polypeptide (e.g., sgRNAs), antisense molecules, and miRNAs.An UACA polypeptide inhibitor can be any appropriate UACA polypeptide inhibitor. An UACA polypeptide inhibitor can be an inhibitor of UACA polypeptide expression. In some cases, an inhibitor of UACA polypeptide activity can be used to reduce the level of UACA polypeptide activity instead of or in addition to reducing the level of a UACA polypeptide expression by a T cell (e.g., a CAR T cell). Examples of compounds that can inhibit UACA polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to an UACA polypeptide, and small molecules that target (e.g., target and bind) to an UACA polypeptide. Examples of compounds that can inhibit of UACA polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of an UACA polypeptide (e.g., a siRNA molecule or a shRNA molecule), nucleic acid molecules designed to induce CRISPR-mediated interference of polypeptide expression of an UACA polypeptide (e.g., sgRNAs), antisense molecules, and miRNAs.A TNFRSF9 polypeptide inhibitor can be any appropriate TNFRSF9 polypeptide inhibitor. A TNFRSF9 polypeptide inhibitor can be an inhibitor of TNFRSF9 polypeptide expression. In some cases, an inhibitor of TNFRSF9 polypeptide activity can be used to reduce the level of TNFRSF9 polypeptide activity instead of or in addition to reducing the level of a TNFRSF9 polypeptide expression by a T cell (e.g., a CAR T cell). Examples of compounds that can inhibit TNFRSF9 polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to a TNFRSF9 polypeptide, and small molecules that target (e.g., target and bind) to a TNFRSF9 polypeptide. Examples of compounds that can inhibit of TNFRSF9 polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of a TNFRSF9 polypeptide (e.g., a siRNA molecule or a shRNAmolecule), nucleic acid molecules designed to induce CRISPR-mediated interference of polypeptide expression of a TNFRSF9 polypeptide (e.g., sgRNAs), antisense molecules, and miRNAs.An AKAP12 polypeptide inhibitor can be any appropriate AKAP12 polypeptide inhibitor. An AKAP12 polypeptide inhibitor can be an inhibitor of AKAP12 polypeptide expression. In some cases, an inhibitor of AKAP12 polypeptide activity can be used to reduce the level of AKAP12 polypeptide activity instead of or in addition to reducing the level of a AKAP12 polypeptide expression by a T cell (e.g., a CAR T cell). Examples of compounds that can inhibit AKAP12 polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to an AKAP12 polypeptide, and small molecules that target (e.g., target and bind) to an AKAP12 polypeptide. Examples of compounds that can inhibit of AKAP12 polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of an AKAP12 polypeptide (e.g., a siRNA molecule or a shRNA molecule), nucleic acid molecules designed to induce CRISPR-mediated interference of polypeptide expression of an AKAP12 polypeptide (e.g., sgRNAs), antisense molecules, and miRNAs.AMYC polypeptide inhibitor can be any appropriate MYC polypeptide inhibitor. A MYC polypeptide inhibitor can be an inhibitor of MYC polypeptide expression. In some cases, an inhibitor of MYC polypeptide activity can be used to reduce the level of MYC polypeptide activity instead of or in addition to reducing the level of a MYC polypeptide expression by a T cell (e.g., a CAR T cell). Examples of compounds that can inhibit MYC polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to a MYC polypeptide, and small molecules that target (e.g., target and bind) to a MYC polypeptide. Examples of compounds that can inhibit of MYC polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of a MYC polypeptide (e.g., a siRNA molecule or a shRNA molecule), nucleic acid molecules designed to induce CRISPR-mediated interference of polypeptide expression of a MYC polypeptide (e.g., sgRNAs), antisense molecules, and miRNAs.In some cases, instead of or in addition to a MYC polypeptide inhibitor being an inhibitor of MYC polypeptide activity and / or being an inhibitor of MYC polypeptide expression, a MYC polypeptide inhibitor can target another polypeptide in a MYC signaling pathway (e.g., a polypeptide upstream of a MYC polypeptide in a MYC signaling pathway) to reduce the level of MYC polypeptide. Examples of polypeptides in a MYC pathway include, without limitation, CTNNB1 polypeptides, BRD2 polypeptides, BRD3 polypeptides, BRD4 polypeptides, CDK2 polypeptides, ERK polypeptides, SRC polypeptides, PP2A polypeptides, GSK-3B polypeptides, MAX polypeptides, CCND1 polypeptides, and CDC42 polypeptides. For example, a MYC polypeptide inhibitor can increase a CDC42 polypeptide, thereby reducing the level of a MYC polypeptide expression by a T cell (e.g., a CAR T cell). For example, a MYC polypeptide inhibitor can decrease one or more of a BRD2 polypeptide, a BRD3 polypeptide, a BRD4 polypeptide, and a CCND1 polypeptide, thereby reducing the level of a MYC polypeptide expression by a T cell (e.g., a CAR T cell).In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a NEFM polypeptide to reduce NEFM polypeptide expression in that T cell, can be engineered to KO nucleic acid encoding an UACA polypeptide to reduce UACA polypeptide expression in that T cell, can be engineered to KO nucleic acid encoding a TNFRSF9 polypeptide to reduce TNFRSF9 polypeptide expression in that T cell, can be engineered to KO nucleic acid encoding an AKAP12 polypeptide to reduce AKAP12 polypeptide expression in that T cell, and / or can be engineered to KO nucleic acid encoding a MYC polypeptide to reduce MYC polypeptide expression in that T cell.In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a NEFM polypeptide to reduce NEFM polypeptide expression in that T cell. For example, at least one endogenous allele of a nucleic acid encoding a NEFM polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a NEFM polypeptide. In another example, both endogenous alleles of a nucleic acid encoding a NEFM polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a NEFM polypeptide.In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding an UACA polypeptide to reduce UACA polypeptide expression in that T cell. Forexample, at least one endogenous allele of a nucleic acid encoding an UAC A polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of an UACA polypeptide. In another example, both endogenous alleles of a nucleic acid encoding an UACA polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of an UACA polypeptide.In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a TNFRSF9 polypeptide to reduce TNFRSF9 polypeptide expression in that T cell. For example, at least one endogenous allele of a nucleic acid encoding a TNFRSF9 polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a TNFRSF9 polypeptide. In another example, both endogenous alleles of a nucleic acid encoding a TNFRSF9 polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a TNFRSF9 polypeptide.In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding an AKAP12 polypeptide to reduce AKAP12 polypeptide expression in that T cell. For example, at least one endogenous allele of a nucleic acid encoding an AKAP12 polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of an AKAP12 polypeptide. In another example, both endogenous alleles of a nucleic acid encoding an AKAP12 polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of an AKAP12 polypeptide.In some cases, a T cell (e.g., a CAR T cell) can be engineered to KO nucleic acid encoding a MYC polypeptide to reduce MYC polypeptide expression in that T cell. For example, at least one endogenous allele of a nucleic acid encoding a MYC polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a MYC polypeptide. In another example, both endogenous alleles of a nucleic acid encoding a MYC polypeptide can be disrupted (e.g., knocked out) to generate a T cell (e.g., a CAR T cell) having a reduced level of a MYC polypeptide.When a T cell (e.g., a CAR T cell) is engineered to KO nucleic acid encoding a NEFM polypeptide to reduce NEFM polypeptide expression in that T cell, is engineered toKO nucleic acid encoding an UACA polypeptide to reduce UACA polypeptide expression in that T cell, is engineered to KO nucleic acid encoding a TNFRSF9 polypeptide to reduce TNFRSF9 polypeptide expression in that T cell, is engineered to KO nucleic acid encoding an AKAP12 polypeptide to reduce AKAP12 polypeptide expression in that T cell, and / or is engineered to KO nucleic acid encoding a MYC polypeptide to reduce MYC polypeptide expression in that T cell, any appropriate method can be used to KO nucleic acid. Examples of techniques that can be used to knock out a nucleic acid encoding a NEFM polypeptide, a nucleic acid encoding an UACA polypeptide, a nucleic acid encoding a TNFRSF9 polypeptide, a nucleic acid encoding an AKAP12 polypeptide, and / or a nucleic acid encoding a MYC polypeptide include, without limitation, gene editing, homologous recombination, non-homologous end joining, microhomology end joining, prime editing, and base editing. For example, gene editing (e.g., with engineered nucleases) can be used to knock out a nucleic acid encoding a NEFM polypeptide, a nucleic acid encoding an UACA polypeptide, a nucleic acid encoding a TNFRSF9 polypeptide, a nucleic acid encoding an AKAP12 polypeptide, and / or a nucleic acid encoding a MYC polypeptide, e g., such that a full length polypeptide is no longer expressed. Examples of nucleases that can be used for genome editing include, without limitation, CRISPR-associated (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, homing endonucleases (HE; also referred to as meganucleases), base editors, and prime editors.In some cases, a clustered regularly interspaced short palindromic repeat (CRISPR) I Cas system can be used (e.g., can be introduced into one or more T cells) to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide. A CRISPR / Cas system used to KO nucleic acid can include a guide RNA(gRNA) that is complementary to the target nucleic acid (e.g., nucleic acid encoding a NEFM polypeptide, nucleic acid encoding an UACA polypeptide, nucleic acid encoding a TNFRSF9 polypeptide, nucleic acid encoding an AKAP12 polypeptide, and / or nucleic acid encoding a MYC polypeptide). Examples of nucleic acids that can encode a gRNA that is specific to nucleic acid encoding a NEFM polypeptide include, without limitation, CCGAGCTGTAAGCGAGGCGC (SEQ ID NO: 1),CTCGGTTACCCGCCGGTAGG (SEQ ID N0:2), and TCGTCATTTGCGCGAATACC (SEQ ID N0:3). In some cases, a gRNA can be designed based on a sequence of nucleic acid encoding a NEFM polypeptide. Examples of nucleic acids encoding a NEFM polypeptide sequence include, without limitation, those set forth in National Center for Biotechnology Information (NCBI) accession no. NM_005382 (e.g., version NM_005382.2), accession no. XM_015144993 (e.g., version XM_015144993.2), and accession no. XM_016959243 (e.g., version XM_016959243.3).Examples of nucleic acids that can encode a gRNA that is specific to nucleic acid encoding an UACA polypeptide include, without limitation, GACCTCAATCCTTGCTAAAA (SEQ ID NO:4), ATTACAACCAGTGACACTGC (SEQ ID NO: 5), and TAATATCAACTCCATGTATA (SEQ ID NO: 6). In some cases, a gRNA can be designed based on a sequence of nucleic acid encoding an UACA polypeptide. Examples of nucleic acids encoding an UACA polypeptide sequence include, without limitation, those set forth in NCBI accession no. NM_001008224 (e.g., version NM_001008224.3), accession no. NM_001357407 (e.g., version NM_001357407.1), and accession no. XM_001088091 (e.g., version XM_001088091.4).Examples of nucleic acids that can encode a gRNA that is specific to nucleic acid encoding a TNFRSF9 polypeptide include, without limitation, CCTGCGCTGGAGAAACTATT (SEQ ID NO: 7), CCTTGTAGTAACTGCCCAGC (SEQ ID NO:8), and CATAGTAGCCACTCTGTTGC (SEQ ID NON). In some cases, a gRNA can be designed based on a sequence of nucleic acid encoding a TNFRSF9 polypeptide. Examples of nucleic acids encoding a TNFRSF9 polypeptide sequence include, without limitation, those set forth in NCBI accession no. NM_001561 (e.g., version NM_001561.6), accession no. NM_001077508 (e.g., version NM_001077508.1), and accession no. NM_001025773 (e.g., version NM_001025773.1).Examples of nucleic acids that can encode a gRNA that is specific to nucleic acid encoding an AKAP12 polypeptide include, without limitation GGGCGACACGGTCGTTAGTG (SEQ ID NO: 10), TGTCAAGCCGAAACCTTAGC (SEQ ID NO: 11), and TTAGGGCTCCTTGACCGTTC (SEQ ID NO: 12). In some cases, a gRNA can be designed based on a sequence of nucleic acid encoding an AKAP12 polypeptide.Examples of nucleic acids encoding an AKAP12 polypeptide sequence include, without limitation, those set forth in NCBI accession no. NM_001370346 (e.g., version NM_001370346.1), accession no. NM_O31185 (e.g., version NM_031185.3), and accession no. NM_001278476 (e.g., version NM_001278476.1).Examples of nucleic acid molecules designed to induce RNA interference of polypeptide expression of a MYC polypeptide (e.g., a siRNA molecule or a shRNA molecule) include, without limitation AACGTTGAGGGGCATCGTCG (SEQ ID NO:29), GCCGTATTTCTACTGCGACG (SEQ ID NO:30), and CGCCGTCGTTGTCTCCCCGA (SEQ ID NO: 31). In some cases, a gRNA can be designed based on a sequence of nucleic acid encoding a MYC polypeptide. Examples of nucleic acids encoding a MYC polypeptide sequence include, without limitation, those set forth in NCBI accession no. NM_002467.6 (e.g., version NM_002467.6), CCDS87627, and P01106-1.A CRISPR / Cas system used to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can include any appropriate Cas nuclease. Examples of Cas nucleases include, without limitation, Casl, Cas2, Cas3, Cas9, Casio, Cpfl, Casl2, and Casl2a. In some cases, a Cas component of a CRISPR / Cas system designed to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can be a Cas9 nuclease. For example, the Cas9 nuclease of a CRISPR / Cas9 system described herein can be as described elsewhere (see, e.g., Nidhi et al., Int. J. Mol. Sci., 22(7):3327 (2021); Hillary et al., Mol. Biotechnol., 65(3):311-325 (2023); Li et al., Signal Transduct. Target Ther., 8(1):36 (2023); Thakore et al., Nat. Methods, 2(12): 1143-9 (2015); and Barger et al., eLife, 10:e55070 (2021)).Components of a CRISPR / Cas system (e.g., a gRNA and a Cas nuclease) used to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptidecan be introduced into one or more T cells (e.g., CAR T cells) in any appropriate format. In some cases, a component of a CRISPR / Cas system can be introduced into one or more T cells as a nucleic acid encoding a gRNA and / or a nucleic acid encoding a Cas nuclease. For example, a nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease (e g., a Cas9 nuclease) can be introduced into one or more T cells. In some cases, a component of a CRISPR / Cas system can be introduced into one or more T cells as a gRNA and / or as a Cas nuclease. For example, at least one gRNA and at least one Cas nuclease (e.g., a Cas9 nuclease) can be introduced into one or more T cells.In some cases, a ZFN system can be used (e.g., can be introduced into one or more T cells) to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide. A ZFN system used to KO nucleic acid can include a polypeptide including (a) a DNA-binding domain (e.g., zinc fingers) that is complementary to a target nucleic acid (e.g., nucleic acid encoding a NEFM polypeptide, nucleic acid encoding an UACA polypeptide, nucleic acid encoding a TNFRSF9 polypeptide, nucleic acid encoding an AKAP12 polypeptide, and / or nucleic acid encoding a MYC polypeptide), and (b) a nuclease domain (e.g., a nuclease domain that can created double-strand breaks). A ZFN system used to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can include any appropriate nuclease domain. In some cases, a nuclease domain of a ZFN system designed to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can be a Fokl nuclease domain.In some cases, a TALEN system can be used (e.g., can be introduced into one or more T cells) to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYCpolypeptide. A TALEN system used to KO nucleic acid can include a polypeptide including (a) a transcription activator-like (TAL) effector DNA-binding domain directing a nuclease to a target nucleic acid (e.g., nucleic acid encoding a NEFM polypeptide, nucleic acid encoding an UACA polypeptide, nucleic acid encoding a TNFRSF9 polypeptide, nucleic acid encoding an AKAP12 polypeptide, and / or nucleic acid encoding a MYC polypeptide), and (b) a nuclease domain (e.g., a nuclease domain that can created double-strand breaks). A TALEN system used to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can include any appropriate nuclease. In some cases, a nuclease can be a nonspecific nuclease. In some cases, a nuclease can function as a dimer. In some cases, a nuclease of a TALEN system designed to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can be a Fokl nuclease.Components of a gene-editing system (e.g., a CRISPR / Cas system) used to KO nucleic acid encoding a NEFM polypeptide, to KO nucleic acid encoding an UACA polypeptide, to KO nucleic acid encoding a TNFRSF9 polypeptide, to KO nucleic acid encoding an AKAP12 polypeptide, and / or to KO nucleic acid encoding a MYC polypeptide can be introduced into one or more T cells (e.g., CAR T cells) using any appropriate method. A method of introducing components of a gene-editing system into a T cell can be a physical method. A method of introducing components of a gene-editing system into a T cell can be a chemical method. A method of introducing components of a gene-editing system into a T cell can be a particle-based method. Examples of methods that can be used to introduce components of a gene-editing system into one or more T cells include, without limitation, electroporation, transfection (e.g., lipofection), transduction (e.g., viral vector mediated transduction such as adenovirus mediated transduction), microinjection, nucleofection, transposons (e.g., Sleeping Beauty transposon systems), and mRNA-mediated delivery systems.The term “elevated level” as used herein with respect to a level of a polypeptide (e.g., one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide) refers to any level that is higher than a reference level of that polypeptide in control T cells or any level of that polypeptide that is higher in the post-engineered / treated T cells as compared to the level of that polypeptide in the pre-engineered / treated version of those T cells. The term “reference level” as used herein with respect to a polypeptide (e.g., one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide) refers to the level of that polypeptide typically observed in control T cells from one or more healthy mammals (e.g., humans) not engineered to have an elevated level of that polypeptide as described herein. Control T cells can include, without limitation, T cells that are wild-type T cells obtained from a healthy mammal.A T cell having (e.g., engineered to have) an elevated level of one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide can be generated using any appropriate method. In some cases, a T cell (e.g., a CAR T cell) can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a NEFM polypeptide to increase NEFM polypeptide expression in that T cell (e.g., as compared to the level in a control T cell that was not engineered to contain nucleic acid encoding a NEFM polypeptide), can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a HLA-DRB5 polypeptide to increase HLA-DRB5 polypeptide expression in that T cell (e.g., as compared to the level in a control T cell that was not engineered to contain nucleic acid encoding a HLA-DRB5 polypeptide), and / or can be engineered to contain nucleic acid (e.g., exogenous nucleic acid) encoding a CTDSP1 polypeptide to increase CTDSP1 polypeptide expression in that T cell (e.g., as compared to the level in a control T cell that was not engineered to contain nucleic acid encoding a CTDSP1 polypeptide).Any appropriate method can be used to introduce nucleic acid (e.g., nucleic acid encoding a GNLY polypeptide, nucleic acid encoding a HLA-DRB 5 polypeptide, and / or nucleic acid encoding a CTDSP1 polypeptide) into a T cell. For example, an exogenous nucleic acid encoding one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide can be introduced into a T cell by transduction (e.g., viral transduction) or transfection.In some cases, a nucleic acid (e.g., an endogenous nucleic acid) encoding one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide within a T cell can be modified to increase a level of expression of one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide in the T cell. For example, an endogenous nucleic acid encoding a GNLY polypeptide, a HLA-DRB5 polypeptide, or a CTDSP1 polypeptide within a T cell can be modified by gene-editing techniques (e.g., CRISPR / Cas systems, TALENs systems, and base pair editing) to increase the level of expression of one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide in the T cell.Any appropriate method can be used to modify endogenous nucleic acid encoding a GNLY polypeptide, a HLA-DRB5 polypeptide, or a CTDSP1 polypeptide within a T cell to increase a level of one or more one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide within the T cell. For example, components of a gene-editing system (e.g., a CRISPR / Cas system) can be used to increase promotor and / or enhancer activity to drive increased expression of one or more of a GNLY polypeptide, a HLA-DRB5 polypeptide, and a CTDSP1 polypeptide. Any appropriate method can be used to introduce a gene-editing system into one or more T cells (e.g., CAR T cells). A method of introducing components of a gene-editing system into a T cell can be a physical method. A method of introducing components of a gene-editing system into a T cell can be a chemical method. A method of introducing components of a gene-editing system into a T cell can be a particle-based method. Examples of methods that can be used to introduce components of a gene-editing system into one or more T cells include, without limitation, electroporation, transfection (e.g., lipofection), transduction (e.g., viral vector mediated transduction such as adenovirus mediated transduction), microinjection, nucleofection, transposons (e.g., Sleeping Beauty transposon systems), and mRNA-mediated delivery systems.Examples of GNLY polypeptides that can be elevated in a T cell provided herein (e.g., a CAR T cell) and examples of nucleic acids encoding a GNLY polypeptide that can be elevated in a T cell provided herein (e.g., a CAR T cell) include those set forth in the NCBI databases at, for example, accession no. NM_006433 (e.g., version NM_006433.5), accession no. NM_001044680 (e.g., version no. NM_001044680.2), accession no.NM_001278755 (e.g., version NM_001278755.1), and accession no. NP_006424 (e.g., version NP_006424.2). In some cases, a GNLY polypeptide that can be elevated in a T cell provided herein can have an amino acid sequence set forth in SEQ ID NO: 13 (see, e.g., Example 3).Examples of HLA-DRB5 polypeptides that can be elevated in a T cell provided herein (e.g., a CAR T cell) and examples of nucleic acids encoding a HLA-DRB5 polypeptide that can be elevated in a T cell provided herein (e.g., a CAR T cell) include those set forth in the NCBI databases at, for example, accession no. NM_002125 (e.g., version NM_002125.4) and accession no. NP_002116 (e.g., version NP_002116.2). In some cases, a HLA-DRB5 polypeptide that can be elevated in a T cell provided herein can have an amino acid sequence set forth in SEQ ID NO: 14 (see, e.g., Example 3).Examples of CTDSP1 polypeptides that can be elevated in a T cell provided herein (e.g., a CAR T cell) and examples of nucleic acids encoding a CTDSP1 polypeptide that can be elevated in a T cell provided herein (e.g., a CAR T cell) include those set forth in the NCBI databases at, for example, accession no. NM_001206878 (e.g., version NM_001206878.2), accession no. XM_036164466 (e.g., version no. XM_036164466.1), accession no. NM_001128079 (e.g., version no. NM_001128079.1), and accession no. NP_001193807 (e.g., version NP_001193807.1). In some cases, a CTDSP1 polypeptide that can be elevated in a T cell provided herein can have an amino acid sequence set forth in SEQ ID NO: 15 (see, e.g., Example 3).A T cell (e.g., a CAR T cell) having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can express (e.g., can be engineered to express) any appropriate antigen receptor. In some cases, an antigen receptor can be a heterologous antigen receptor. In some cases, an antigen receptor can be a CAR. In some cases, an antigen receptor can be a tumor-specific antigen receptor. For example, a T cell can be engineered to express a tumor-specific antigen receptor that targets a tumor-specific antigen (e.g., a cell surface tumor-specific antigen)expressed by a cancer cell in a mammal having cancer. In some cases, an antigen receptor can be a tumor-associated antigen receptor. For example, a T cell can be engineered to express a tumor-associated antigen receptor that targets a tumor-associated antigen (e.g., a cell surface tumor-associated antigen) expressed by a cancer cell in a mammal having cancer. Examples of antigens that can be recognized by an antigen receptor expressed by a T cell having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a U AC A polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide as described herein include, without limitation, cluster of differentiation 19 (CD 19), mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), epidermal growth factor receptor (EGFR), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), CD33, CD123, CLL-1, E-Cadherin, folate receptor alpha, folate receptor beta, IL13R, EGFRviii, CD22, CD20, kappa light chain, lambda light chain, desmopressin, CD44v, CD45, CD30, CD5, CD7, CD2, CD38, BCMA, CD138, FAP, CS-1, and C-met. For example, a T cell having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA- DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be designed to express an antigen receptor targeting CD 19.When an antigen receptor is a CAR, the CAR can be any appropriate CAR. A CAR can include an antigen-binding domain, an optional hinge, a transmembrane domain, and one or more signaling domains. Examples of antigen-binding domains include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), and domains from growth factors that bind to a cancer cell receptor (e.g., domains from EGF, PDGR, FGF, TGF, or derivatives thereof). In some cases, an antigen-binding domain of a CAR can target (e.g., can target and bind to) a cancer antigen or a cancer-specific antigen. Insome cases, an antigen-binding domain of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No. 2017 / 0183418 at paragraph

[0015] and the sequence listing; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No. 2017 / 0183413 at paragraph

[0049] , Figure 2, Table 9, and the sequence listing; U.S. Patent Application Publication No. 2018 / 0291079 such as U.S. Patent Application Publication No. 2018 / 0291079 at paragraphs

[0041] -

[0045] , and Table 4; U.S. Patent Application Publication No. 2020 / 0289563 such as U.S. Patent Application Publication No.2020 / 0289563 at paragraphs

[0006] -

[0053] ,

[0186] -

[0189] , and Table 1; and U.S. Patent Application Publication No. 2003 / 0211097 such as U.S. Patent Application Publication No. 2003 / 0211097 at paragraphs

[0081] and [0211-0215] and the sequence listing).In some cases, a CAR can include an optional hinge region. In some cases, a hinge region can be located between an antigen-binding domain and a transmembrane domain of a CAR. In some cases, a hinge region can provide a CAR with increased flexibility for the antigen-binding domain. For example, a hinge region can reduce spatial limitations of an antigen-binding domain of a CAR and its target antigen (e.g., to increase binding between an antigen-binding domain of a CAR and its target antigen). Examples of hinge regions that can be used as described herein include, without limitation, a membrane-proximal region from an IgG, a membrane-proximal region from CD8, and a membrane-proximal region from CD28. In some cases, a hinge region of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No. 2018 / 0000914 at paragraph

[0168] , and Table 1; U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No. 2017 / 0183418 at paragraphs

[0034] ,

[0037] ,

[0040] , and Table 2; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No. 2017 / 0183413 at paragraph

[0116] ; and U.S. Patent Application Publication No. 2017 / 0145094 such as U.S. Patent Application Publication No. 2017 / 0145094 at paragraph

[0104] ).A CAR described herein can include any appropriate transmembrane domain. A transmembrane domain can be located between an antigen-binding domain and a signaling domain of a CAR and / or located between a hinge and a signaling domain of a CAR. In somecases, a transmembrane domain can provide structural stability for the CAR. For example, a transmembrane domain can include a structure (e.g., a hydrophobic alpha helix structure) that can span a cell membrane and can anchor the CAR to the plasma membrane. Examples of transmembrane domains that can be used as described herein include, without limitation, CD3^ transmembrane domains, CD4 transmembrane domains, CD8 (e.g., a CD8a) transmembrane domains, CD28 transmembrane domains, CD 16 transmembrane domains, and erythropoietin receptor transmembrane domains. In some cases, a transmembrane domain of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2016 / 0120906 such as U.S. Patent Application Publication No. 2016 / 0120906 at paragraphs

[0155] ,

[0161] ,

[0269] , Figure 4, and Figure 11; U.S. Patent Application Publication No. 2019 / 0209616 such as U.S. Patent Application Publication No. 2019 / 0209616 at paragraph

[0026] ; U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No. 2018 / 0000914 at paragraphs

[0168] -

[0171] ; U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No. 2017 / 0183418 at paragraphs

[0116] -

[0118] ; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No. 2017 / 0183413 at paragraphs

[0116] -

[0118] ; and U.S. Patent Application Publication No. 2017 / 0145094 such as U.S. Patent Application Publication No. 2017 / 0145094 at paragraphs

[0104] -

[0107] ).A CAR described herein can include any appropriate signaling domain or combination of signaling domains (e.g., a combination of two, three, or four signaling domains). In some cases, a signaling domain of a CAR can be an intracellular signaling domain normally found within T cells or NK cells. In some cases, a CAR can include a signaling domain that renders a T cell expressing the CAR susceptible to senescence (e.g., susceptible to senescence upon reactivation or recurrent activation). Examples of signaling domains that can be used as described herein include, without limitation, BB(^ signaling domains, 28^ signaling domains, CD2 signaling domains, CD3 signaling domains, CD28 signaling domains, Toll-like receptor (TLR) signaling domains (e.g., TLR3 or TLR4 signaling domains), CD27 intracellular signaling domains, 0X40 (CD 134) intracellular signaling domains, 4-1BB (CD137) intracellular signaling domains, CD278 intracellularsignaling domains, DAP 10 intracellular signaling domains, DAP 12 intracellular signaling domains, FceRly intracellular signaling domains, CD278 intracellular signaling domains, CD122 intracellular signaling domains, CD132 intracellular signaling domains, CD70 intracellular signaling domains, cytokine receptor intracellular signaling domains, and CD40 intracellular signaling domains. In some cases, a CAR for use as described herein can be designed to be a first generation CAR having a CD3(^ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a second generation CAR having a CD28 intracellular signaling domain or 4-1BB intracellular signaling domain followed by a CD3 intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an 0X40 intracellular signaling domains, or a 4-1BB intracellular signaling domain followed by (c) a CD3^ intracellular signaling domain. In some cases, the intracellular signaling domain(s) of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No.2018 / 0000914 at paragraphs

[0164] -

[0167] ; and U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No. 2017 / 0183413 at paragraphs

[0112] -

[0115] ).In some cases, a CAR can be as set forth in Table 1.Table 1. Exemplary CARs for targeting tumor antigens.Any appropriate method can be used to express an antigen receptor on a T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide. For example, nucleic acid encoding an antigen receptor can be introduced into one or more T cells. In some cases, viral transduction can be used to introduce nucleic acid encoding an antigen receptor into a non-dividing a cell. Nucleic acid encoding an antigen receptor can be introduced in a T cell using any appropriate method. In some cases, nucleic acid encoding an antigen receptor can be introduced into a T cell by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection. In some cases, nucleic acid encoding an antigen receptor can be introduced ex vivo into one or more T cells. For example, ex vivo engineering of T cells expressing an antigen receptor can include transducing isolated T cells with a lentiviral vector encoding an antigen receptor. In cases where T cells are engineered ex vivo to express an antigen receptor, the T cells can be obtained from any appropriate source (e.g., a mammal such as the mammal to be treated or a donor mammal, or a cell line).In some cases, when a T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) anelevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide also expresses (e.g., is engineered to express) an antigen receptor, that T cell can be engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide and engineered to express an antigen receptor using any appropriate method. In some cases, a T cell can be engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide first and engineered to express an antigen receptor second, or vice versa. In some cases, a T cell can be simultaneously engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide and to express an antigen receptor. For example, (a) one or more nucleic acids used to reduce a level of a NEFM polypeptide, reduce a level of an UACA polypeptide, reduce a level of a TNFRSF9 polypeptide, reduce a level of an AKAP12 polypeptide, elevate a level of a GNLY polypeptide, elevate a level of a HLA-DRB5 polypeptide, and / or elevate a level of a CTDSP1 polypeptide, and (b) one or more nucleic acids encoding an antigen receptor (e.g., a CAR) can be simultaneously introduced into one or more T cells. One or more nucleic acids used to reduce a level of a NEFM polypeptide, reduce a level of an UACA polypeptide, reduce a level of a TNFRSF9 polypeptide, reduce a level of an AKAP12 polypeptide, elevate a level of a GNLY polypeptide, elevate a level of a HLA-DRB5 polypeptide, and / or elevate a level of a CTDSP1 polypeptide, and one or more nucleic acids encoding an antigen receptor can be introduced into one or more T cells on separate nucleic acid constructs or on a single nucleic acid construct. One or more nucleic acids used toreduce a level of a NEFM polypeptide, reduce a level of an UACA polypeptide, reduce a level of a TNFRSF9 polypeptide, reduce a level of an AKAP12 polypeptide, elevate a level of a GNLY polypeptide, elevate a level of a HLA-DRB5 polypeptide, and / or elevate a level of a CTDSP1 polypeptide, and one or more nucleic acids encoding an antigen receptor can be introduced ex vivo into one or more T cells. In cases where T cells are engineered ex vivo (a) to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide, and (b) to express an antigen receptor, the T cells can be obtained from any appropriate source (e.g., a mammal such as the mammal to be treated or a donor mammal, or a cell line).In some cases, a T cell having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be stimulated. A T cell can be stimulated at the same time as being engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide. For example, one or more T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy can be stimulated first, and can be engineered to have one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) areduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide second, or vice versa. A T cell can be stimulated using any appropriate method. For example, a T cell can be stimulated by contacting the T cell with one or more polypeptides. Examples of polypeptides that can be used to stimulate a T cell include, without limitation, CD3, CD28, inducible T cell co-stimulator (ICOS), CD137, CD2, 0X40, and CD27. In some cases, a T cell can be stimulated by contacting the T cell with two or more polypeptides.This document also provides methods and materials for treating cancer. For example, one or more T (e.g., CAR T) cells having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be administered (e.g., in an adoptive cell therapy such as a CAR T cell therapy) to a mammal (e.g., a human) having cancer to treat the mammal. In some cases, methods of treating a mammal having cancer as described herein can reduce the number of cancer cells (e.g., cancer cells expressing a tumor antigen) within a mammal. In some cases, methods of treating a mammal having cancer as described herein can reduce the size of one or more tumors (e.g., tumors expressing a tumor antigen) within a mammal.Any appropriate amount (e.g., number) of T (e.g., CAR T) cells having (e g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be administered (e.g., in an adoptive cell therapy such as a CAR T cell therapy) to a mammal (e.g., a human) having cancer. In some cases, from about 100,000 cells per kg body weight (cells / kg) to about 1,000,000,000 cells / kg (e.g., from about 100,000 cells / kg to about 750,000,000 cells / kg, from about 100,000 cells / kg to about 500,000,000 cells / kg, from about 100,000 cells / kg to about 250,000,000 cells / kg,from about 100,000 cells / kg to about 100,000,000 cells / kg, from about 100,000 cells / kg to about 750,000 cells / kg, from about 100,000 cells / kg to about 500,000 cells / kg, from about 100,000 cells / kg to about 250,000 cells / kg, from about 250,000 cells / kg to about 1,000,000,000 cells / kg, from about 500,000 cells / kg to about 1,000,000,000 cells / kg, from about 750,000 cells / kg to about 1,000,000,000 cells / kg, from about 1,000,000 cells / kg to about 1,000,000,000 cells / kg, from about 250,000,000 cells / kg to about 1,000,000,000 cells / kg, from about 500,000,000 cells / kg to about 1,000,000,000 cells / kg, from about 750,000,000 cells / kg to about 1,000,000,000 cells / kg, from about 250,000 cells / kg to about 750,000,000 cells / kg, from about 500,000 cells / kg to about 500,000,000 cells / kg, from about 750,000 cells / kg to about 250,000,000 cells / kg, from about 100,000 cells / kg to about 5,000,000 cells / kg, from about 250,000 cells / kg to about 750,000 cells / kg, from about 1,000,000 cells / kg to about 250,000,000 cells / kg, from about 250,000,000 cells / kg to about 500,000,000 cells / kg, or from about 500,000,000 cells / kg to about 750,000,000 cells / kg) of T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be administered to a mammal having cancer to treat the mammal.Any appropriate mammal (e.g., a human) having a cancer can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having a cancer can be treated with one or more T (e.g., CAR T) cells having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a ARAP 12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide in, for example, an adoptive T cell therapy such as a CAR T cell therapy using the methods and materials described herein.When treating a mammal (e.g., a human) having a cancer as described herein, the cancer can be any appropriate cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. In some cases, a cancer treated as described herein can express a tumor-associated antigen (e.g., an antigenic substance produced by a cancer cell). Examples of cancers that can be treated as described herein include, without limitation, mantle cell lymphomas (MCLs), diffuse large B cell lymphomas (DLBCLs), Hodgkin’s lymphomas, non-Hodgkin lymphomas, acute lymphoblastic leukemias (ALLs), chronic lymphocytic leukemias (CLLs), acute myeloid leukemias (AMLs), germ cell tumors, hepatocellular carcinomas, bowel cancers, lung cancers, breast cancers, ovarian cancers, melanomas, brain cancers, and multiple myelomas.In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having a cancer. Any appropriate method can be used to identify a mammal having cancer. For example, imaging techniques and biopsy techniques can be used to identify mammals (e.g., humans) having cancer.In some cases, the methods described herein can include identifying a mammal (e.g., a human) as being in need of T cells (e.g., CAR T cells) having reduced susceptibility to T cell (e.g., CAR T cell) senescence. Any appropriate method can be used to identify a mammal as being in need of T cells (e.g., CAR T cells) having reduced susceptibility to T cell (e.g., CAR T cell) senescence.A mammal (e.g., a human) having a cancer can be administered one or more T (e.g., CAR T) cells having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA- DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide described herein. For example, one or more T cells having (e.g., engineered to have) one or more of (1) a reducedlevel of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be used in an adoptive T cell therapy (e.g., a CAR T cell therapy) to treat a mammal having a cancer. For example, one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be used in an adoptive T cell therapy (e.g., a CAR T cell therapy) targeting any appropriate antigen within a mammal (e.g., a mammal having cancer). In some cases, an antigen can be a tumor- associated antigen (e.g., an antigenic substance produced by a cancer cell). Examples of tumor-associated antigens that can be targeted by an adoptive T cell therapy provided herein include, without limitation, CD 19 (associated with DLBCL, ALL, and CLL), AFP (associated with germ cell tumors and / or hepatocellular carcinoma), CEA (associated with bowel cancer, lung cancer, and / or breast cancer), CA-125 (associated with ovarian cancer), MUC-1 (associated with breast cancer), ETA (associated with breast cancer), MAGE (associated with malignant melanoma), CD33 (associated with AML), CD123 (associated with AML), CLL-1 (associated with AML), E-Cadherin (associated with epithelial tumors), folate receptor alpha (associated with ovarian cancers), folate receptor feta (associated with ovarian cancers and AML), IL13R (associated with brain cancers), EGFRviii (associated with brain cancers), CD22 (associated with B cell cancers), CD20 (associated with B cell cancers), kappa light chain (associated with B cell cancers), lambda light chain (associated with B cell cancers), CD44v (associated with AML), CD45 (associated with hematological cancers), CD30 (associated with Hodgkin lymphomas and T cell lymphomas), CD5 (associated with T cell lymphomas), CD7 (associated with T cell lymphomas), CD2 (associated with T cell lymphomas), CD38 (associated with multiple myelomas and AML), BCMA (associated with multiple myelomas), CD 138 (associated with multiple myelomas and AML), FAP (associated with solid tumors), CS-1 (associated with multiple myeloma),and c-Met (associated with breast cancer). For example, one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a U AC A polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can be used in CAR T cell therapy targeting CD19 (e.g., a CART19 cell therapy) to treat cancer as described herein.In some cases, one or more T (e.g., CAR T) cells having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive T cell therapy (e.g., a CAR T cell therapy) can be administered to a mammal having a cancer as a combination therapy with one or more additional agents used to treat a cancer. For example, one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy can be administered to a mammal in combination with one or more anti-cancer treatments (e.g., surgery, radiation therapy, chemotherapy (e.g., alkylating agents such as busulfan), immunomodulating agents, checkpoint inhibitors, and / or targeted therapies small molecule targeted therapies). In cases where one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy are used with additional agents treat a cancer, the one or more additional agents can be administered at the same time or independently. In some cases, oneor more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA- DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy can be administered first, and the one or more additional agents administered second, or vice versa.In some cases, one or more T (e.g., CAR T) cells having (e.g., engineered to have) one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive T cell therapy (e.g., a CAR T cell therapy) can be administered to a mammal having a cancer as a combination therapy with one or more antisenescence treatments. For example, one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy can be administered to a mammal in combination with one or more senotherapeutic agents. Examples of senotherapeutic agents that can administered to a mammal (e.g., a human) having cancer together with one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide include, without limitation, IL-37 polypeptides, rapamycin, dasatinib, quercetin, and combinations thereof (e.g., dasatinib and quercetin). In cases where one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) areduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy are used with one or more senotherapeutic agents, the one or more senotherapeutic agents can be administered at the same time or independently. In some cases, one or more T (e.g., CAR T) cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide used in an adoptive cell therapy can be administered first, and the one or more senotherapeutic agents administered second, or vice versa.The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLESExample 1: CART cell susceptibility to senescence based on signalingCD19-directed chimeric antigen receptor (CART19) cells have emerged as a potentially curative immunotherapy in a subset of patients with hematological malignancies. While initial responses are impressive, the majority of responsive patients relapse within a year. Recent studies suggest that CART cells are susceptible to states of dysfunction.This Example evaluates the development of T cell senescence in CART cells using similar constructs to the FDA-approved therapies, CART19-BB^ and CART19-28^, to determine the impact of signaling domains on CART cell senescence. An in vitro model for repeated CART cell activation followed by rest was developed to study the development of senescence and its impact on effector T cell functions. CART cell immunophenotype, cell cycle regulators, and transcriptomic profile were examined on day 0 (DO; T cell), D8 (standard CART cell), DI 5 (after one activation cycle) and D22 (after two activation cycles).The immunophenotypic analyses suggested differential expression in inhibitory receptors between CART19-BB and CART 19-28C, following CART cell stimulation and rest. TIM-3 was upregulated in both CART19-BB(^ (DO vs D8 and D15, p=0.0289 and p=0.0090, respectively) and CART19-28q (DO vs D22 p=0.0394) while CTLA-4 was upregulated in only CARTT9-28^ (DO vs D8, D15, and D22; p=0.0002, p=0.0096, and p=O.O3O5, respectively). LAG-3 and PD-1 expression levels did not increase in either CARTT9-BB^ or CART 19-28 .Since increased TIM-3 expression was associated with T cell senescence, pl 6, p21 and p53 were further investigated by flow cytometry as the levels of these polypeptides is frequently increased in senescent cells, p 16 levels from D15 to D22 (p= 0.0165) and p53 levels from DO to D8, D15 and D22 were increased in CART19-BB^ (p= 0.0071, p=0.0281, and p= 0.0002, respectively) but not in CART19-28(^. p53 expression in CART19-28(^ increased at a later time point (DO vs D8 and D8 vs D22, p=0.4951, p=0.0257, respectively). The expression of p21 did not change upon serial activation for either CART19-BB^ or CART19-28^; however, p21 levels were higher in CART19-BB^ at D15 and D22 (p= 0.0056, p= 0.0113, respectively) compared to CARTT9-28^ (Figure 1). CD28 levels decreased from D8 to D15 and D22 in CART19-BB^ (p= 0.0327 and p= 0.0406, respectively) while CD28 levels did not change in CART 19-28^. Changes in the level of these markers suggested a more prominent senescent phenotype in CART19-BB^ compared to CART19-28^.Then, the transcriptomic signature of recurrently activated-rested cells was evaluated by performing RNA sequencing of CART19 cells at DO, D8, D15, and D22 in an in vitro repeated activation model. GSEA indicated enrichment of gene sets related to senescent T cells such as p53, DNA damage response, cell cycle checkpoint, glycolysis, and oxidative phosphorylation in CART19-BB(^ DI 5 compared to CART19-BB(^ D8 or CART 19-28^ DI 5. A senescence related gene set, senMayo, that defines senescing cell populations or tissue populations was used. This gene set is also significantly enriched in CART19-BB^ DI 5 compared to DO or CART19-28q D15 (p=O.OO53, p=0.16, respectively) suggesting a senescent fate in CARTT9-BB^ cells.Next, to study the antitumor activity of recurrently activated and rested CART 19 in vivo, xenograft models were generated through the engraftment of NOD-SQD-y" ' (NSG)mice with the luciferase+CD19+cell line JeKo-1. Engrafted mice were randomized based on bioluminescence to treatment with UTD (untransduced T cell control), D8, DI 5, and D22 CART19-BB^ or CART19-28i (n>5 mice / group). Mice treated with CART19-BB^ D8, CART19-BBi D15, CART19-28i; D8, and CART19-28J D15 had significantly less tumor burden than UTD mice (p= 0.0005, p= 0.0013, p= 0.0004 and p= 0.0012, respectively). However, mice treated with CART19-BB^ D22 or CART19-28< D22 CART cells had similar tumor burden to UTD mice. CART19-BB(^ D22 mice had a significantly lower tumor burden compared to CART19-28(^ D22 mice (p= 0.0140). The survival of the mice correlated well with the tumor burden (Figure 2). Collectively, these results indicate that CART19-BB^ cells were more susceptible to developing a senescent phenotype than CART19-28g cells, as indicated by persistent expression of TIM-3, elevated cell cycle regulators, and a predominant transcriptional signature of senescence.Example 2: Prevention of senescence improves CAR T cell functionsCART cell functions decrease upon repeated stimulationSummary of CART cell production (D0-D8) and activation protocol (D8-D15 and D15-D22) is shown in Figure 3 A. T cells from ND were activated with anti-CD3 / CD28 Dynabeads for 24 hours and divided into three groups for UTD (untransduced control), BBz (transduced with CAR19-4-1BBQ and 28z (transduced with CAR19-CD28 Q. The CART cells were fed until D6 when the magnetic beads were removed to rest the CART cell for two days. The cells were co-cultured twice in a week with resting intervals to generate DI 5 and D22 CART cells. CART cells at different activation cycles were cocultured with Luc+ JeKo- 1 cell line with varying E:T ratios (Figure 3B). The viability of the Luc+ JeKo-1 was measured by bioluminescence after 24 hours of co-culture (E:T, 0.32: 1) (*p < 0.05, Mixed- effects analysis; Tukey's multiple comparisons test, error bars, SEM; up to seven biological and three technical replicates). Proliferation assays are shown in Figure 3C. Same cells from Figure 3B were co-cultured with JeKo-1 at 1: 1 E:T ratio. The co-cultures were fed at D3 with fresh media and CART cells were counted by volumetric assays with flow cytometer ad D5. E (*p < 0.05, two-way ANOVA; Tukey's multiple comparisons test, error bars, SEM; seven biological and three technical replicates).CART cell exhaustion is not responsible for impaired T cell function upon recurrent activation-resting cycleBBz and 28z cells at indicated days were stained with indicated T cell exhaustion markers for flow analysis. Expression of CTLA-4 in 28z increased significantly but the change was less than 3% (Figure 4A) which brings questions about the biological significance of this changes. LAG-3 expression didn’t change over time in either of the CART groups tested (Figure 4B) PD-1 expression stayed similar in BBz but decreased in 28z (Figure 4C). TIM-3, whose expression is associated with T cell senescence, was the only exhaustion marker whose expression in both CARTs was tested Figure 4D).Recurrent activation induces senescence-like immunophenotypical changes in CART cells, more prominent in BBzThe same cells in Figure 4 were stained for CD28 (Figure 5 A) and CD27 (Figure 5B) for flow analysis. Senescent T cells had decreased expressions of CD28 and CD27. The expression levels for both markers decreased more prominently in BBz. SA-Bgal activity, which is increased in senescent cells, of CART cells at indicated time point were measured by CellEvent™ Senescence Green Detection Kit according to the manufacturers protocol. Both CART cells had increased SA-Bgal activity (Figure 5C). The amount of cycling cells decreased in senescent cells (Figure 5D). The levels of cycling cells were measured with EDU assay. CART cells were cocultured with lethally irradiated JeKo-1 in the presence of 2 pM EDU and CART cell portions that were positive for EDU were plotted. The Click-iT EdU Flow Cytometry Cell Proliferation Assay was used.Recurrent activation induces impaired CART cell activity in vivoLuc+JeKo-1 cells were transplanted to NSG mice. The mice were randomized based on the tumor load which was measured by bioluminescence. In this experiment, mice were randomized while the tumor burden was low. Mice groups received indicated CART cells. The tumor burden 16 days after the CART cell infusion was plotted. Tumor levels was lower in D8 and DI 5 CART cell mice than UTD mice while the tumor levels in D22 CART cell mice was not lower than UTD mice for both BBz and 28z (Figure 6A). Tumor levels in BBzD22 mice were lower than 28z D22 mice. Blood samples from mice in Figure 6A were collected from each mouse to count CART cell in circulating blood as a measurement of CART cell persistency. The mice in BBz D8 group had detectable levels of T cell while mice in BBz DI 5 or D22 groups did not, showing the impairing effect of recurrent activation on CART cells (Figure 6B). Similar to the experiment shown in Figure 6A, CART cells were infused while the tumor burden in mice was high. In this experiment only mice from D8 BBz and D8 28z mice had significantly less tumor compared to UTD indicating impaired CART cell activity in DI 5 CART cells as well (Figure 6C). Blood samples from mice in Figure 6C were collected from each mouse to count CART cell in circulating blood. The mice in BBz D8 group had more T cell compared to blood from mice in BBz DI 5 and D22 groups (Figure 6D). The 28z D8 mice had detectable levels of T cells. E & F. Peripheral blood samples were collected from the mice 21 days after CART cell infusion. The cytokine analysis from the blood samples indicated increase TNF-a and IL-10 levels for recurrently activated BBz cells while only IL-10 levels were increased in recurrently activated 28z cells. Increased TNF-a and IL- 10 levels are reported in senescent T cells (Figures 6E and 6F).Recurrent activation-resting results in extensive changes in gene expressions according to RNAseq analysisCART cell RNAs from 3 normal donors were sequenced. The table shows the number of genes whose expression changed significantly (Figure 7A). Gene set enrichment analysis (GSEA) indicated that senMayo gene set was enriched in BBz DI 5 cells compared to both DO T cells and 28z DI 5 (Figure 7B). The senMayo gene set was recently reported to be enriched in senescing cells populations. Some of the senescence associated gene sets were also enriched in BBz compared to other groups (Figure 7C). DNA damage marker levels changed extensively based on the CSD or on the activation cycle (Figure 7D). p-H2AX (Serl39), p-ATM (Ser 1981 ) and p-Chk2 (Thr68) levels were higher in BBz D15 compared to BBz D8, but their levels were mostly similar in 28z D8 and D 15. Overall level of p-BRCAl (Seri 524) was higher in BBz while levels of p-p53 (Seri 5) and p-Chk2 (Thr68) levels were higher in 28z indicating preferential DNA damage response based on the CSD present in the CAR cells. Recurrently activated CART cells were immunoblotted against OS responseproteins Catalase and SOD1 (Figure 7E). The level of both proteins increased consistently upon recurrent activation. The increase was independent of the CSD present in the CART cells.Senescence induced with irradiation have different impact on BBz and 28z efficacyA schema of CART cell production protocol is shown in Figure 8A. Senescence was induced in CART cells by 3 Gy IR exposure at D2. Expression levels of indicated senescence marker were plotted by qPCR (Untreated vs. IR; Figures 8B-8E). IR and untreated CART cells were cocultured with JeKo-1 cell lines. CART cell proliferation (Figure 8F) and killing (Figure 8G) were plotted. 28z-IR cells killed JeKo-1 more successfully while no increase detected in BBz-IR. Proliferation of CART cells upon IR did not increase as expected of senescent cells.Differentially expressed genes in recurrently rested-activated BBz cellsDifferential gene expression (DGE) analysis, which considers expression levels of thousands of genes, was performed on recurrently rested-activated BBz cells. NEFM, UACA, TNFRSF9, AKAP12, GNLY, HLA-DRB5, and CTDSP1 were determined to be differentially expressed upon recurrent activation resting cycles.Change in RPKM of UACA (Figure 9A) and NEFM (Figure 9B) in BBz at indicated time points was determined. UACA was knocked out by CRISPR / Cas9 technology (Figure 9C). CART cells were transduced with sgControl and sgUACA. The figure shows the cleavage assay indicating presence of mutations in intended region in sgUACA transduced cells but not sgControl. CART cells with knocked out UACA were cocultured with Luc+Jeko cell lines similar to Figure 3B (Figure 9D). UACA KO cells killed target cells more efficiently compared to control cells.Knocking out UACA and NEFM in T cell based malignant cell line Jurkat cellsCART cells were transduced with sgControl, sgUACA, sgNEFM or both (dual) lentiviral particles (Figure 10). A cleavage assay was done showing the presence of mutations in intended locations.Jurkat cells from Figure 10 were stained for indicated T cell exhaustion markers for flow analysis (Figures 11 A-l ID). Exhaustion markers were low in sgUACA while higher in sgNEFM transduced cells. CD57 levels were low in sgUACA transduced cells (Figure 1 IE) KLRG1 was low in sgUACA transduced cells (Figure 1 IF). CD57 and KLRG1 are senescence markers for T cells. This data suggest UACA KO may ameliorate T cell senescence.Jurkat cells with indicated transduction from Figure 11 were cultured and counted daily (Figure 12). The fold change in the cell number was higher in sgUACA group while lower in sgNEFM and intermediate with dual sgRNA transduced group. This indicated UACA and NEFM may have different impact on CART cell activity.Jurkat-Lucia™ NFAT-CD28 (JNFAT) cells were initially transduced with either CAR19-4-1BB (JNFAT-BBz) or CAR19-CD28 (JNFAT-28z). These cells were similar to BBz and 28z respectively except for made out of Jurkat-Lucia™ NFAT-CD28 instead of normal T cells collected from healthy donors. JNFAT-BBz cells were transduced with sgControl, sgUACA, sgNEFM or both (dual) lentiviral particles (Figure 13 A). A cleavage assay to indicate mutations in intended areas was done. JNFAT-28z cells were transduced with sgControl, sgUACA, sgNEFM or both (dual) lentiviral particles (Figure 13B). A cleavage assay to indicate mutations in intended areas was done. JNFAT-BBz and JNFAT- 28z cells were cultured in normal media and counted daily with flow cytometer. Absolute number (Figure 13C) and fold change (Figure 13D) of sgUACA transduced cells were increased compared to control.JNFAT reporter activity increased with sgUACA compared to sgControl transduced cells. Activation of JNFAT cells correlated with luciferase activity which was measured by a luminometer after providing luciferase substrate. JNFAT-BBz and JNFAT-28z cells were cultured and activated with anti-CD3 / CD28 Dynabeads at 1: 1 E:T ratio. The bioluminescence levels after 24 hours (Figure 14A) and 48 hours (Figure 14B) were plotted. sgUACA transduced cells had increased activation response compared to control.Example 3: Exemplary SequencesGNLY polypeptide (SEQ ID NO: 13)MATWALLLLAAMLLGNPGLVFSRLSPEYYDLARAHLRDEEKSCPCLAQEGPQGDLLTKTQELGRDYRTCLTIVQKLKKMVDKPTQRSVSNAATRVCRTGRSRWRDVCRNFMRRYQSRVTQGLVAGETAQQICEDLRLCIPSTGPLNucleic acid encoding a GNLY polypeptide (SEQ ID NO: 16)ATGGCTACCTGGGCCCTCCTGCTCCTTGCAGCCATGCTCCTGGGCAACCCAGGTCTGGTCTTCTCTCGTCTGAGCCCTGAGTACTACGACCTGGCAAGAGCCCACCTGCGTGATGAGGAGAAATCCTGCCCGTGCCTGGCCCAGGAGGGCCCCCAGGGTGACCTGTTGACCAAAACACAGGAGCTGGGCCGTGACTACAGGACCTGTCTGACGATAGTCCAAAAACTGAAGAAGATGGTGGATAAGCCCACCCAGAGAAGTGTTTCCAATGCTGCGACCCGGGTGTGTAGGACGGGGAGGTCACGATGGCGCGACGTCTGCAGAAATTTCATGAGGAGGTATCAGTCTAGAGTTACCCAGGGCCTCGTGGCCGGAGAAACTGCCCAGCAGATCTGTGAGGACCTCAGGTTGTGTATACCTTCTACAGGTCCCCTC TGAHLA-DRB5 polypeptide (SEQ ID NO: 14)MVCLKLPGGSYMAKLTVTLMVLSSPLALAGDTRPRFLQQDKYECHFFNGTERVRFLHRDIYNQEEDLRFDSDVGEYRAVTELGRPDAEYWNSQKDFLEDRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPARTQTLQHHNLLVCSVNGFYPGSIEVRWFRNSQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRAQSESAQSKMLSGVGGFVLGLLFLGAGLFIYFKNQKGHSGLHPTGLVSNucleic acid encoding a HLA-DRB5 polypeptide (SEQ ID NO: 17)ATAGTTCTCCCTGAGTGAGACTTGCCTGCTCCTCTGGCCCCTGGTCCTGTCCTGTTCTCCAGCATGGTGTGTCTGAAGCTCCCTGGAGGTTCCTACATGGCAAAGCTGACAGTGACACTGATGGTGCTGAGCTCCCCACTGGCTTTGGCTGGGGACACCCGACCACGTTTCTTGCAGCAGGATAAGTATGAGTGTCATTTCTTCAACGGGACGGAGCGGGTGCGGTTCCTGCACAGAGACATCTATAACCAAGAGGAGGACTTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTGACGCTGAGTACTGGAACAGCCAGAAGGACTTCCTGGAAGACAGGCGCGCCGCGGTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAGTTGAGCCTAAGGTGACTGTGTATCCTGCAAGGACCCAGACCCTGCAGCACCACAACCTCCTGGTCTGCTCTGTGAATGGTTTCTATCCAGGCAGCATTGAAGTCAGGTGGTTCCGGAACAGCCAGGAAGAGAAGGCTGGGGTGGTGTCCACAGGCCTGATTCAGAATGGAGACTGGACCTTCCAGACCCTGGTGATGCTGGAAACAGTTCCTCGAAGTGGAGAGGTTTACACCTGCCAAGTGGAGCACCCAAGCGTGACGAGCCCTCTCACAGTGGAATGGAGAGCACAGTCTGAATCTGCACAGAGCAAGATGCTGAGTGGAGTCGGGGGCTTTGTGCTGGGCCTGCTCTTCCTTGGGGCCGGGCTATTCATCTACTTCAAGAATCAGAAAGGGCACTCTGGACTTCACCCAACAGGACTCGTGAGCTGAAGTGCAGATGACCACATTCAAGGGGGAACCTTCTGCCCCAGCTTTGCATGATGAAAAGCTTTCCTGCTTGGCTCTTATTCTTCCACAAGAGAGGACTTTCTCAGGCCCTGGTTGCTACCGGTTCAGCAACTCTGCAGAAAATGTCCATCCTTGTGGCTTCCTCAGCTCCTGCCCTTGGCCTGAAGTCCCAGCATTGATGGCAGTGCCTCATCTTCAACTTTAGTGCTCCCCTTTACCTAACCCTACGGCCTCCCATGCATCTGTACTCCCCCTGTGTGCCACAAATGCACTACGTTATTAAATTTTTCTGAAGCCCAGAGTTAAAAATCATCTGTCCACCTGGCTCCAAAGACAAAAAATAAAAAGAAAAGAAAAAGGGAAGATTACTDSP1 polypeptide (SEQ ID NO: 15)MVAAPWATQEQEEGRGIQPGDRGDQKSAASQKPRSRGILHSLFCCVCRDDGEALPAHSGAPLLVEENGAIPKTPVQYLLPEAKAQDSDKICVVIDLDETLVHSSFKPVNNADFIIPVEIDGVVHQVYVLKRPHVDEFLQRMGELFECVLFTASLAKYADPVADLLDKWGAFRARLFRESCVFHRGNYVKDLSRLGRDLRRVLILDNSPASYVFHPDNAVPVASWFDNMSDTELHDLLPFFEQLSRVDDVYSVLRQPRPGSNucleic acid encoding a CTDSP1 polypeptide (SEQ ID NO: 18)GCCTTTGCCCTTGCCGGTAGACCCGAAGCACGTCGCTTCCTTTCTTGAAACTCAGTTTCCTCATCTGTGAAATGGGTTAAGAAGGAGGCCGTTCTAAGGGGTAAATGAGATTAGGGGGCGCAGCCAGAGCAGGCCTAGGAAAACGCCAGCAGCGTGTGGCGATCACGGTGATGAAGCGCAATGGTGGCCGCCCCGTGGGCTACCCAGGAGCAGGAGGAGGGCCGAGGGATCCAGCCCGGGGACCGGGGTGACCAGAAGTCAGCAGCTTCCCAGAAGCCCCGAAGCCGGGGCATCCTCCACTCACTCTTCTGCTGTGTCTGCCGGGATGATGGGGAGGCCCTGCCTGCTCACAGCGGGGCGCCCCTGCTTGTGGAGGAGAATGGCGCCATCCCTAAGACCCCAGTCCAATACCTGCTCCCTGAGGCCAAGGCCCAGGACTCAGACAAGATCTGCGTGGTCATCGACCTGGACGAGACCCTGGTGCACAGCTCCTTCAAGCCAGTGAACAACGCGGACTTCATCATCCCTGTGGAGATTGATGGGGTGGTCCACCAGGTCTACGTGTTGAAGCGTCCTCACGTGGATGAGTTCCTGCAGCGAATGGGCGAGCTCTTTGAATGTGTGCTGTTCACTGCTAGCCTCGCCAAGTACGCAGACCCAGTAGCTGACCTGCTGGACAAATGGGGGGCCTTCCGGGCCCGGCTGTTTCGAGAGTCCTGCGTCTTCCACCGGGGGAACTACGTGAAGGACCTGAGCCGGTTGGGTCGAGACCTGCGGCGGGTGCTCATCCTGGACAATTCACCTGCCTCCTATGTCTTCCATCCAGACAATGCTGTACCGGTGGCCTCGTGGTTTGACAACATGAGTGACACAGAGCTCCACGACCTCCTCCCCTTCTTCGAGCAACTCAGCCGTGTGGACGACGTGTACTCAGTGCTCAGGCAGCCACGGCCAGGGAGCTAGTGAGGGTGATGGGGCCAGGACCTGCCCCTGACCAATGATACCCACACCTCCTCCCAGGAAGACTGCCCAGGCCTTTGTTAGGAAAACCCATGGGCCGCCGCCACACTCAGTGCCATGGGGAAGCGGGCGTCTCCCCCACCAGCCCCACCAGGCGGTGTAGGGGCAGCAGGCTGCACTGAGGACCGTGAGCTCCAGGCCCCGTGTCAGTGCCTTCAAACCTCCTCCCCTATTCTCAGGGGACCTGGGGGGCCCTGCCTGCTGCTCCCTTTTTCTGTCTCTGTCCATGCTGCCATGTTTCTCTGCTGCCAAATTGGGCCCCTTGGCCCCTTCCGGTTCTGCTTCCTGGGGGCAGGGTTCCTGCCTTGGACCCCCAGTCTGGGAACGGTGGACATCAAGTGCCTTGCATAGAGCCCCCTCTTCCCCGCCCAGCTTTCCCAGGGGCACAGCTCTAGGCTGGGAGGGGAGAACCAGCCCCTCCCCCTGCCCCACCTCCTCCCTTGGGACTGAGAGGGCCCCTACCAACCTTTGCCTCTGCCTTGGAGGGAGGGGAGGTCTGTTACCACTGGGGAAGGCAGCAGGAGTCTGTCCTTCAGGCCCCACAGTGCAGCTTCTCCAGGGCCGACAGCTGAGGGCTGCTCCCTGCATCATCCAAGCAATGACCTCAGACTTCTGCCTTAACCAGCCCCGGGGCTTGGCTCCCCCAGCTCTGAGCGTGGGGGCATAGGCAGGACCCCCCTTGTGGTGCCATATAAATATGTACATGTGTATATAGATTTTTAGGGGAAGGAGAGAGGGAAGGGTCAGGGTAGAGACACCCCTCCCTTGCCCCTTT CCTGGGCCCAGAAGTTGGGGGGAGGGAGGGAAAGGATTTTTACATTTTTTAAAC TGCTATTTTCTGAATGGAACAAGCTGGGCCAAGGGGCCCAGGCCCTGTCCTCTGT CCCTCACACCCCTTTGCTCCGTTCATTCATTCAAAAAAACATTTCTTGAGCACCTT CTGTGCCCAGCATATGCTAGGCCCACCAGCTAAGTGTGTGTGGGGGGTCTCTACG CCAGCTCATCAGTGCCTCCTTGCCCATCCTTCACCGGTGCCTTTGGGGGATCTGT AGGAGGTGGGACCTTCTGTGGGGTTTGGGGATCTCCAGGAAGCCCGACCAAGCT GTCCCCTTCCCCTGTGCCAACCCATCTCCTACAGCCCCCTGCCTGATCCCCTGCTG GCTGGGGGCAGCTCCCAGGATATCCTGCCTTCCAACTGTTTCTGAAGCCCCTCCT CCTAACATGGCGATTCCGGAGGTCAAGGCCTTGGGCTCTCCCCAGGGTCTAACG GTTAAGGGGACCCACATACCAGTGCCAAGGGGGATGTCAAGTGGTGATGTCGTT GTGCTCCCCTCCCCCAGAGCGGGTGGGCGGGGGGTGAATATGGTTGGCCTGCAT CAGGTGGCCTTCCCATTTAAGTGCCTTCTCTGTGACTGAGAGCCCTAGTGTGATGAGAACTAAAGAGAAAGCCAGACCCCTAExample 4: Differential susceptibility and role for senescence in CART cells based on costimulatory domainsThe results in this Example re-present and expand on at least some of the results provided in other Examples.This Example identifies senescence as a potential mechanism of failure in CART cells containing a CAR that includes 4-1BB intracellular signaling domain.ResultsA model to study irreversible CART cell dysfunction using recurrent activationAn in vitro model was developed that incorporates serial CART cell activation to mimic cancer relapse along with resting periods between stimulations to study senescence. Unlike exhaustion, senescence is an irreversible cell fate and does not recover after rest (Blackburn et al., Nature Immunology 10(l):29-348 37 (2008); and Zhao et al., Cell Mol Immunol. 17(l):27-35 (2020)), BB(^, and 28(^ cells were generated from healthy donor T cellsfrom Day 0 (DO) to Day 8 (D8) (Figure 20A). D8 CART cells served as a baseline timepoint for manufactured CART cell function and phenotype. Both BB(^ and 28^ were activated through repeated cocultures with lethally irradiated CD 19+ mantle cell lymphoma cell line JeKo-1, followed by a period of rest (Figure 20A, D8 to D22). During rest, CART cells were cultured in T cell medium without additional target cells for four days prior to analysis.CART cells after one or two activation / rest cycles are referred to as Day 15 (DI 5) or Day 22 (D22) CART cells, respectively.In this model, it was found that the expression of multiple exhaustion markers — lymphocyte activation gene 3 (LAG-3), cytotoxic T lymphocyte-associated protein 4 (CTLA- 4), and programmed cell death 1 (PD-1) — increased significantly three days after activation with anti-CD3 / CD28 Dynabeads (used during CART cell production, Figure 20A) but returned to baseline by D8. T cell immunoglobulin and mucin domain 3 (TIM-3), however, remained elevated (Figure 20B). Exhaustion marker expression was also measured at DO and after each resting period at D8, D15, and D22. LAG-3, CTLA-4, and PD-1 all returned to near basal levels at the end of each activation / rest cycle (Figures 20C-20E), but TIM-3 remained persistently elevated even after resting periods Figure 20F).Recurrent activation impairs CART cell activity and cytokine profile in vivoThe effect of recurrent CART cell activation on CART cell efficacy was tested in vivo. 1 x 106luciferase+ JeKo-1 cells were intravenously engrafted to NOD-SCID-IL2rY- / - (NSG) mice. Mice then underwent serial bioluminescence imaging (BLI) to monitor tumor engraftment. After 10 days, when bioluminescence intensity reached ~107photons / second (p / s), mice were randomized by tumor burden and intravenously treated with 1 x 106BB^ or 28^ intravenously which were previously cryopreserved at D8, D15, or D22 (Figure 21A).Mice treated with BBi^ or 28(^ D8 and D15 had significantly less tumor burden compared to UTD-treated mice (Figures 15A and 15B). On the other hand, mice treated with BB(^ or 28^ D22 had similar tumor burden to mice treated with UTD, indicating severely impaired CART activity in vivo for both BB^ and 28(^ after two cycles of recurrent activation (Figures 15A and 15B). In fact, the probability of survival for mice receiving 28(^ D22 was statistically decreased compared to mice treated with UTD, D8, and DI 5 (Figure 15C). Thesefindings highlight the vast impact of repeated antigen-specific activation of CART cells on their function and ultimate therapeutic outcome.To further assess this phenomenon, the recurrently activated CART 19 cells were also tested in a stress tumor model where CART cells were administered 14 days after JeKo-1 administration when bioluminescence intensity reached ~108p / s. Mice treated with BB^ or 28C, D8 again had significantly lowered tumor burden compared to UTD-treated mice (Figure 15D, Figure 21B). However, in both BBc D15 and 28C, D15 treated mice, tumor burdens were not significantly different from UTD-treated mice, indicating pronounced CART dysfunction which emerged at earlier timepoints in this more aggressive stress tumor model (Figure 15D, Figure 2 IB). BBi^ D8-treated mice exhibited an enhanced CART cell proliferation compared to all other BB(^ treatment groups (Figure 21C).Secretion of inhibitory cytokines, including TNF-a and IL-10, have been reported to be elevated in senescent T cells30. Therefore, inhibitory cytokine levels from the serum of mice from the in vivo stress model were measured. As such, serum levels of tumor necrosis factor (TNF)-a were significantly elevated in BB^ D I 5- and BBc D22- treated mice compared to BB(^ D8-treated mice, but these trends were not observed in 28^-treated mice (Figure 15E). Serum IL-10 levels increased in mice treated with D22 CART cells compared to D8 for both BB^ and 28^ (Figure 15F).Senescence-like immimophenotype in CART cells differs based on costimulatory domainTo further characterize the development of a senescence-like phenotype in CART19 cells, expression of cell cycle regulators was assessed in the recurrently activated CART cells. Specifically, the levels of senescence markers CDKN2A (p 16) and TP53 (p53) were measured in CART cells via intracellular staining and flow cytometry at the indicated activation cycles. The percentage of p 16+CART cells increased significantly in repeatedly activated BB(^ but not 28^ (Figure 16A). p53 levels increased in both BB(^ and 28^, but p53 upregulation started at an earlier time point in BB(^ upon recurrent activation cycles (Figure 16B).Levels of the oxidative stress (OS) markers, catalase and superoxide dismutase 1 (SOD1) were measured upon repeated activation / rest cycles. Catalase and SOD1 levels wereincreased in both BB(^ and 28c upon recurrent activation (Figure 16C). CD27 and CD28 expression on the CART cells was monitored at different time points. Upon recurrent activation, expression of both CD27 and CD28 were significantly downregulated in BBc but remained similar in 28^ Figures 16D-16E).Finally, recurrently activated CART cells were assessed for upregulation of well- established DNA damage markers by immunoblotting. The results indicated that levels of DNA damage markers p-H2AX (Seri 39), p-ATM (Seri 981), and p-Chk2 (Thr68) were elevated in BB(^ D15 compared to BB(^ D8, but their levels was similar in 28^ DI 5 compared to 28(^ D8 (Figure 16F), indicating differing DNA damage response based on the costimulatory domain.A senescent transcriptional signature is differentially enriched upon recurrent activation / rest cycles in BBC vs. 28<CNext, the transcriptome of recurrently activated CART cells from three healthy donors was assessed by performing RNA sequencing on T cells at DO, BB(^, and 28^ on D8, DI 5, and D22 as illustrated in Figure 20A. Notably, D8 CART cells performed well in both low and high (stress) tumor burden models, whereas D22 CART cells failed in both models in vivo (Figures 15A-15D, Figures 21B-21C). However, D15 CART cells exhibited antitumor activity only in low tumor burden model. Therefore, DI 5 CART cells represent a sensitive timepoint to study early signs of T cell impairments.Samples clustered based on the costimulatory domain rather than clustering by donor or activation cycle according to principal component analysis (PCA) and unsupervised hierarchical clustering (Figures 22A-22B). Normalized gene counts data from T cell DO and D15 CART cells to were used perform gene set enrichment analysis (GSEA) with senMayo (Saul et al., Nat Commun. 13(1) (2022)). It was found that the senMayo gene set was significantly enriched in BBq D15 compared to T cell DO or 28C, D15 (Figure 17A). Next, the normalized gene counts in hallmark gene sets were run and significantly enriched gene sets were noticed in BBq DI 5 related to senescent T cell phenotypes, including DNA damage, oxidative stress, and glycolysis (Figure 22C).Collectively, the data suggest that both BB(^ and 28C, exhibit signs of senescence as evident by impaired effector functions, persistent elevation of TIM-3, increased secretion of inflammatory cytokines, and increased cell cycle regulator expression upon repeated stimulation followed by rest. However, the experiments strongly indicated that BB(^ were more susceptible to senescence as indicated by their significant loss of CD27 and CD28 expression, accumulated DNA damage markers, and enrichment of senescence gene signature.MYC activation due to recurrent activation / re sting cycles has differential impact on BBC and 28CGSEA revealed that numerous MYC-related gene sets were enriched in BB while the same genes were depleted in 28^ following repeated activation / rest cycles (Figure 17B). To test whether MYC activation differentially impact BB(^ and 28^, oncogenic MYC T58A (Figure 17C) and wild-type MYC (Figure 18) was overexpressed (MYC OE) in BB(^ and 28 . MYC T58A activation upregulated known T cell senescence markers, such as p21 , p53, and p-H2AX, in both BB^ and 28c as determined by immunoblotting or flow cytometric analysis (Figures 17C-17D). MYC activation did not induce exhaustion marker expression (Figures 22D-22F). Antigen-specific stimulation of MYC OE BB^, but not MYC OE 28^ resulted in decreased cell cycle, measured with EDU, compared to control BB(^ and 28^ (Figure 17E). MYC OE BB(^ but not MYC OE 28^ displayed decreased antigen-specific killing and proliferation (Figures 17F-17G). Ectopic MYC expression had no impact on OS (Figure 22G). These data suggest that MYC is activated in recurrently activated BB7, and MYC- induced senescence may have a detrimental impact on BB(^ functions, but not on 28^.Wild-type MYC overexpressing BB(^ killed target cells less efficiently while 28^ killing was not impacted in vitro (Figures 18B and 18C). NSG mice bearing luciferase positive JeKo-1 tumor received control or wild-type MYC overexpressing CART cells. The wild-type MYC overexpressing CART cell mice had decreased number of CART cells in peripheral blood (Figure 18D). BB^ mice that had MYC overexpression had higher tumor burden (Figures 18E G) and had significantly shorter survival compared to control (Figure 18H). These affects were not observed in MYC overexpressing 28^ (Figure 18).Induction of senescence through irradiation impairs BBC but enhances 28 'To further validate the effect of senescence on CART efficacy in BB(^ and 28Q senescence was induced in CART cells through sublethal irradiation on Day 2 of CART manufacturing (Figure 23 A). Irradiated CART cells showed increased senescence marker expression by qPCR (Figures 19A-19D) and flow cytometry (Figures 23B-23E), but no upregulation of exhaustion makers LAG-3, CTLA-4, and PD-1 (Figures 23F-23H). It should be noted that the yield of irradiated CART cells was ~10 times lower than non-irradiated CART cells. Upon co-culture with target cells, irradiated 28^, but not irradiated BBQ killed target cells and entered the cell cycle more efficiently as measured by EDU assay (Figures 19E-19G). These phenomena occurred in irradiated 28L, without a corresponding increase in proliferation.Since the experiments demonstrated that induction of a senescence-like state in CART cells impaired BB^ activity, but improved 28^ activity, it was next inquired how senescence impacted CART activity in the clinic (see “Analysis of scRNAseq data” section in Methods for detailed explanation). Analysis of publicly available RNA sequencing datasets from patients treated with CART cells indicated that the senMayo gene set was enriched in non-responders vs. responders to tisagenlecleucel (tisa-cel, equivalent to BBQ, but not in non-responders vs. responders to axicabtagene ciloleucel (axi-cel, equivalent to 28Q, providing additional support for the role of senescence in the failure of 4- IBB- but not CD28-costimulated CART cells (Figure 19H). A scoring system was generated based on each patient’s senMayo gene expression signature. Interestingly, axi-cel -treated but not tisa- cel-treated patients with high senMayo scores had longer progression-free survival (see “Analysis of scRNAseq data” section in Methods for detailed explanation) (Figure 19H). Similar to recurrently activated / rested BBQ numerous MYC-related pathways were enriched in non-responders vs. responders to tisa-cel but not in non-responders vs. responders to axi- cel (Figure 17B, Figure 19J). Altogether, these data added support for the role of senescence in the failure of 41BB- but not CD28-costimulated CART cells.MethodsCell LinesThe mantle cell lymphoma cell line JeKo-1 was purchased from ATCC (Manassas, VA, USA) and cultured in R20 medium, which contained RPMI 1640 (cat# 21870092, Gibco, Gaithersburg, MD, USA), 20% fetal bovine serum (cat# 250517, FBS, Sigma, St. Louis, MO, USA), and 1% penicillin-streptomycin-glutamine (PSG) (cat# 221675, Gibco). JeKo-1 was lentivirally transduced with luciferase-GFP (pBMN (CMV-copGFP-Luc2-Puro), cat# 80389, Addgene, Cambridge, MA, USA) and sorted to 100% purity.CART Cell GenerationCART cells were generated. In summary, second-generation CAR constructs were synthesized de novo (Integrated DNA Technologies, Coralville, IA, USA) containing an anti- CD19 single chain variable fragment (clone FMC63) and either CD28-CD3^ or 41BB-CD3^ signaling domains in a third-generation lentiviral backbone. Lentiviral particles were generated through transfection of plasmids into HEK-293T cells (ATCC) using Lipofectamine 3000 (cat# L3000075, Invitrogen, Carlsbad, CA, USA). T cells were isolated from de-identified healthy donors on Day 0 (DO) using EasySep Human T Cell Isolation Kits (cat# 17951, STEMCELL Technologies, Vancouver, BC, Canada) and stimulated with CD3 / CD28 Dynabeads (cat# 40203D, LifeTechnologies, Oslo, Norway) at a 3: 1 beads-to- cell ratio. On Day 1, T cells were lentivirally transduced with the CAR constructs at a multiplicity of infection of 3. T cells were cultured in T cell medium containing X-VIVO™ 15 (cat# 123696, Lonza, Walkersville, MD, USA), 10% human serum albumin (cat# 12667- 50ML-M, Coming, NY, USA), and 1% PSG (cat# 221675, Gibco). Beads were removed and CAR expression was assessed on Day 6. CART cells were used in the repeated activation cycle model beginning on Day 8. CAR expression was detected with anti-FMC63 (cat# FM3- AY54P1, ACROBiosystems, Newark, DE, USA).Repeated Activation Cycle ModelAfter generating untransduced control T cells (UTD), CART 19-41BB-CD3(^ (BBQ cells, and CART19-CD28-CD3(^ (28Q cells through the eight-day protocol described above,Day 8 CART cells (UTD D8, BB(^ D8 and 28C, D8) were co-cultured with JeKo-1 cells that had been irradiated at 120 Gy (JeKo-1 IR) at a 1 : 1 effector-to-target ratio. A second round of 1 : 1 JeKo-1 IR was added to the co-culture after three days. The media was replaced with fresh media two days later and CART cells were rested for two additional days before concluding the activation cycle and performing functional assays. Each subsequent activation cycle took an additional 7 days. As such, CART cells after one activation cycle were referred as UTD D15, BB(^ D15, and 28^ D15; and after 2 cycles of activation were referred to as UTD D22, BBi D22, and 28 D22.T Cell Functional AssaysCART cell proliferation and cytotoxicity assays were performed as described elsewhere (Sterner et al., Blood. 133(7):697-335 709 (2019)). In summary, for killing assays, effector cells were cocultured with luciferase+JeKo-1 cells at various ratios. Target killing was assessed at the indicated timepoints by adding 1 pL of 30 pg / mL D-luciferin (cat# LUCK-1G, Gold Biotechnology, St. Louis, MO, USA) per 100 pL media to the cocultures and performing bioluminescence imaging (BLI) (Promega GloMax Explorer, Madison, WI, USA) to measure the remaining live cells. For proliferation assays, effector cells and JeKo-1 cells were cocultured at a 1 : 1 ratio. After five days, absolute counts of CD3 cells were assessed via flow cytometry using anti-human CD3 (cat# 344818, BioLegend, San Diego, CA, USA) and LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (cat# L34966, Invitrogen).In Vivo ExperimentsNOD-SCID-IL2rY- / - (NSG) mice at 6- to 8-week were purchased from Jackson Laboratories. Mice were intravenously engrafted with luciferase+ 1x106 JeKo-1 cells, and tumor burden was serially assessed via BLI with a Xenogen IVIS-200 Spectrum camera (PerkinElmer, Hopkinton, MA, USA). When tumor burden reached ~lxl07-108photons / second, mice were randomized to treatment with IxlO6effector cells through tail vein injection as indicated in the specific experiment. Mice were monitored for tumor burden and overall survival.Blood CART cell count and cytokine analysisCytokine profile and CART cell expansion were followed by peripheral blood sampling. To determine CART cell counts, ~70 pL of blood was lysed of red blood cells with FACSTM Lysing Solution (cat# 349202, BD Biosciences, San Diego, CA, USA). Antibody staining was done with anti-human CD3, anti-human CD45 (cat# 317308, BioLegend), antimouse CD45 (cat# 103116, BioLegend) and CD20 (cat# 980208, BioLegend). Flow cytometry was then performed to determine the absolute CD3+T cell count and the expression of inhibitory receptors. Absolute cell counts were calculated by determining amount of cell per unit volume on flow cytometer.Cytokine concentration in the plasma of mice was determined with MILLIPLEX MAP Human High Sensitivity T Cell Panel Premixed 13-plex (cat# HSTCMAG28PMX13BK, Millipore Sigma, Ontario, Canada) according to the manufacturer’s instructions with 25 pL of either serum per sample. Analysis was completed with Belysa software after running the samples on a Luminex 200 (Millipore Sigma).Intracellular Staining p 16 INK4A (cat# CST 82548) and p53 (cat# CST 5429) antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). The cells were stained according to the manufacturer’s recommendations. In summary, the cells were washed with PBS and fixed with 4% formaldehyde for 15 minutes at room temperature. Fixed cells were permeabilized with 90% methanol for 10 minutes on ice. The cells were stained with the antibodies for 1 hour at room temperature for flow analysis. All measurements were performed in technical triplicates. Fluorescence minus one (FMO) was used as a negative control to provide the positive and negative gates.EdU AssayClick-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit from Life Technologies (cat# Cl 0635) was purchased from ThermoFisher Scientific. UTD and CART cells were co-cultured with JeKo-1 IR at a 1 : 1 ratio in the presence of 2 pM EdU for 24 hours and stained according to the manufacturer’s protocol. All measurements were performed intechnical triplicates. Cells without EdU served as a negative control to gate the positive and negative EdU populations.RNA isolation, RNAseq and qPCRCells used for RNA isolation were cultured in serum-free media for 24 hours before being flash frozen and stored at -80°C until RNA isolation. RNA was isolated from ~1 x 107cells in one batch with RNeasy Plus Mini Kit (cat# 74134, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA quality was initially assessed using Qubit fluorometry (Invitrogen, Carlsbad, CA) and the Agilent Fragment Analyzer (Santa Clara, CA, USA). cDNA libraries were prepared using 100 ng of total RNA according to the manufacturer's instructions for the QuantSeq 3' mRNA-Seq Library Kit (Lexogen, Greenland, NH, USA). The concentration and size distribution of the completed libraries were determined using an Agilent TapeStation DNA 1000 chip (Santa Clara, CA, USA) and Qubit fluorometry (Invitrogen). Libraries were sequenced following Illumina’s standard protocol for the NovaSeq 6000 (San Diego, CA, USA). The flow cell was sequenced as 100 bp single end reads using the NovaSeq SP sequencing kit and NovaSeq Control Software vl.7.5. Base-calling was performed using Illumina’s RTA version 3.4.4. cDNA for qPCR was produced with iScript Advanced cDNA Kit for RT-qPCR (cat# 1725038, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s suggestions. Real Time-Quantitative Polymerase Chain Reaction (RT-qPCR) was performed according to manufacturer’s instructions with RT-qPCR SsoAdvanced Universal SYBR Green Supermix (cat# S7563, Bio-Rad).Primers used for qPCR were TBP28 (F: GCCAGCTTCGGAGAGTTCTGGGATT (SEQ ID NO: 19), R: CGGGCACGAAGTGCAATGGTCTTTA (SEQ ID NO:20)), p2128 (F: GCCATTAGCGCATCACAGT (SEQ ID NO:21), R: ACCGAGGCACTCAGAGGAG (SEQ ID NO:22)), p5328 (F: GAGGTTGGCTCTGACTGTACC (SEQ ID NO:23), TCCGTCCCAGTAGATTACCAC (SEQ ID NO:24)), MMP12 28 (F: CTGAGGACATAGCAAATATGCAATAAA (SEQ ID NO:25), R: TGGTTTGGTTGTTAGAAATGGTGTA (SEQ ID NO:26)), and PIM129 (F:CGACATCAAGGACGAAAACATC (SEQ ID NO:27), R: ACTC- TGGAGGGCTATACACTC (SEQ ID NO:28)).RNA-seq analysisRNA-seq analysis was used to compare 3 untransduced control T cell (UTD) samples, 3 CART19-41BB-CD31 (BBQ samples both for day 8 (BB D8), day 15 (BB D15), and day 22 (BB D22), and 3 CART19-CD28-CD31 (28Q samples both for day 8 (28^ D8), day 15 (28^ D15), and day 22 (28^ D22). Differentially expressed genes in UTD versus BB(^ D8, D15, D22 and 28C, D8, D15, D22 was performed using the “limma” (v3.58. 1) R package after removing lowly expressed genes and voom transformation. Expression of SenMayo genes were visualized in heatmaps after scaling each gene and hierarchical clustering where distance metric was set to “correlation” and the clustering method was set to “complete” using “pheatmap” (vl.0.12) R package. Principal component analysis (PCA) was performed using the voom-normalized gene expression matrix via the “factoextra” R package (vl.0.7). The entire set of genes were used for this analysis and individual samples were visualized along the first two principal component axes. Lastly, GSEA between select pairs of samples was performed where the genome-wide log fold-change values were provided as a pre-ranked input to the fgsea algorithm (vl.28.0).Analysis of scRNAseq dataScRNAseq data of B-cell lymphoma patients treated with CD 19 CAR-T therapy were downloaded from the GEO database (GSE197268) (Haradhvala et al., Nat. Med., 28(9): 1848- 1859 (2022)). This data set consists of time-course analyses of PBMCs before and during treatment with axicabtagene ciloleucel (axi-cel, containing CD28 signaling domain) or tisagenlecleucel (tisa-cel, containing 4-1BB signaling domain). Raw barcode-feature matrices were processed using Seurat R package (v5.0.2) and low-quality cells containing <200 or >6000 mapped features and >10% mitochondrial transcripts were removed from the analyses. Gene expression data were normalized using cpm method of Seurat and data were log2 -transformed subsequently. 28 out of 32 patients with scRNAseq data on day 0 (infusion timepoint) and day 7 (on-treatment timepoint) were selected for further analysis. Out of these 27 patients, 15 patients were treated with axi-cel (9 with complete response [Axi_R], 6 withprogressive disease [Axi_NR]) and 13 patients were treated with tisa-cel (5 with complete response [Tisa R], 9 patients with progressive disease [Tisa NR]). For gene set enrichment analyses (GSEA), single-cell data were converted to pseudobulk data by first “gating” cells based on CAR expression (non-zero counts of the annotated Yescarta and Kymriah transcripts) and calculating an average gene expression value for each gene. Pseudobulk data were aggregated across patients and GSEA was performed using fgsea R package (vl.28.0) following ranking genes based on their signal-to-noise ratio (Subramanian et al., Proc. Natl. Acad. Sci. USA, 102(43): 15545-50 (2005)). Survival analyses were performed using pseudobulk data from this cohort. To that end, progression-free survival duration for each patient was extracted by matching the FDG-PET scan timepoint and the classification of response at the time of the scan. Survival analyses were performed using the “survival” (v3.5-8) and “survminer” (vO.4.9) R packages after categorizing the patients as high or low at the median value of the averaged SenMayo gene set.Western BlotWestern blot was performed as described elsewhere (Ye et al., Blood, 120(10):2021 - 2031 (2012)). CART cells pellets were washed once with ice cold PBS. Then, the cell pellet was resuspended for 15 minutes on ice with cold cell lysis buffer (cat# R0278, RIPA buffer, Thermo Fisher, Waltham, MA, USA) with freshly added protease and phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher, Waltham, MA, USA). Protein concentration was measured by BCA assay (Thermo Fisher, Waltham, MA, USA). 20 pg of protein lysate was run on an SDS-PAGE gel (Mini-PROTEAN TGX Precast Protein Gels, cat# 4561094, Bio-Rad), followed by protein transfer on a PVDF membrane. The antibodies used were Oxidative Stress Defense (Catalase, SOD1, TRX, smooth muscle Actin) (cat# abl79843, Abeam, Waltham, MA, USA), -Actin (cat# 4967S), p-Chk2 (Thr68) (cat# 2197), p-ATM (Serl981) (cat# 5883), p-Histone H2A.X (Serl39) (cat# 9718), c-Myc (cat# 13987S) (CST), p53 (cat# sc-126), and p21 (cat# sc-53870) (Santa Cruz, Dallas, Texas US).MYC T58A OverexpressionPIG-MycT58A (cat# 177648) was purchased from Addgene to amplify the oncogenic MYC and clone it into pLenti CMV Blast empty (w263-l) (cat# 17486) to generate pLenti CMV Blast MYC T58A. Lentiviruses were produced from pLenti CMV Blast empty (control) and pLenti CMV Blast MYC T58A following the same protocol for CAR19 virus production. MYC T58A-expressing CART cells were selected with blasticidin (cat# Al 113903, Thermo Fisher) at 10 pg / mL concentration between days 3-8.SoftwareAll statistics were performed using GraphPad Prism version 10.2.0 for Windows (GraphPad Software, La Jolla, CA, USA). Statistical tests are described in detail in the respective figure legends. Flow cytometry data were analyzed by FlowJo (Ashland, Oregon, US). Mouse bioluminescence images and measurements were done with Living Image version 4.7.2. Illustrations were generated by Biorender (Toronto, Ontario, Canada).Example 5: Treating CancerA human having cancer is administered CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide.The administered CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (6) an elevated level of a HLA- DRB5 polypeptide, and (7) an elevated level of a CTDSP1 polypeptide can target (e.g., target and destroy) cancer cells (e.g., cancer cells expressing a tumor antigen targeted by the CAR T cells) within a mammal.Example 6: Treating CancerT cells are obtained from a mammal having cancer and are engineered to be CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide.The CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA-DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide are administered back to the human.The administered CAR T cells having one or more of (1) a reduced level of a NEFM polypeptide, (2) a reduced level of a UACA polypeptide, (3) a reduced level of a TNFRSF9 polypeptide, (4) a reduced level of a AKAP12 polypeptide, (5) a reduced level of a MYC polypeptide, (6) an elevated level of a GNLY polypeptide, (7) an elevated level of a HLA- DRB5 polypeptide, and (8) an elevated level of a CTDSP1 polypeptide can target (e.g., target and destroy) cancer cells (e.g., cancer cells expressing a tumor antigen targeted by the CAR T cells) within a mammal.OTHER EMBODIMENTSIt is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A T cell having reduced susceptibility to T cell senescence, wherein said T cell comprises (a) a reduced level of a NEFM polypeptide, a reduced level of a UACA polypeptide, a reduced level of a TNFRSF9 polypeptide, a reduced level of a AKAP12 polypeptide, a reduced level of a MYC polypeptide, an elevated level of a GNLY polypeptide, an elevated level of a HLA-DRB5 polypeptide, or an elevated level of a CTDSP1 polypeptide, and (b) nucleic acid encoding a chimeric antigen receptor, and wherein said T cell expresses said chimeric antigen receptor.

2. The T cell of claim 1, wherein said T cell comprises said reduced level of said NEFM polypeptide.

3. The T cell of any one of claims 1-2, wherein said T cell comprises said reduced level of said UACA polypeptide.

4. The T cell of any one of claims 1-3, wherein said T cell comprises said reduced level of said TNFRSF9 polypeptide.

5. The T cell of any one of claims 1-4, wherein said T cell comprises said reduced level of said AKAP12 polypeptide.

6. The T cell of any one of claims 1-5, wherein said T cell comprises said reduced level of said MYC polypeptide.

7. The T cell of any one of claims 1-6, wherein said T cell comprises said elevated level of said GNLY polypeptide.

8. The T cell of any one of claims 1-7, wherein said T cell comprises said elevated level of said HLA-DRB5 polypeptide.

9. The T cell of any one of claims 1-8, wherein said T cell comprises said elevated level of said CTDSP1 polypeptide.

10. The T cell of claim 1, wherein said T cell comprises said reduced level of said NEFM polypeptide, said reduced level of said UACA polypeptide, said reduced level of said TNFRSF9 polypeptide, said reduced level of said AKAP12 polypeptide, said reduced level of said MYC polypeptide, said elevated level of said GNLY polypeptide, said elevated level of said HLA-DRB5 polypeptide, and said elevated level of said CTDSP1 polypeptide.

11. The T cell of any one of claims 1-10, wherein said chimeric antigen receptor targets a tumor-associated antigen.

12. The T cell of claim 11, wherein said tumor-associated antigen is CD19.

13. The T cell of any one of claims 1-12, wherein said chimeric antigen receptor comprises a BB^ signaling domain or a 28C, signaling domain.

14. The T cell of any one of claims 1-13, wherein said T cell is obtained from a human.

15. The T cell of any one of claims 1-14, wherein said T cell does not exhibit T cell senescence as rapidly as a comparable T cell lacking said reduced level of said NEFM polypeptide, said reduced level of said UACA polypeptide, said reduced level of said TNFRSF9 polypeptide, said reduced level of said AKAP12 polypeptide, said reduced level of said MYC polypeptide, said elevated level of said GNLY polypeptide, said elevated level of said HLA-DRB5 polypeptide, and said elevated level of said CTDSP1 polypeptide.

16. A method for treating a mammal having cancer, wherein said method comprises administering, to said mammal, a composition comprising the T cell of any one of claims 1-17. The method of claim 16, wherein said mammal is identified as being in need of T cells having reduced susceptibility to CAR T cell senescence.

18. The method of any one of claims 16-17, wherein said mammal is a human.

19. The method of any one of claims 16-18, wherein said composition comprises from about 100,000 to about 1,000,000,000 of said T cells.

20. The method of any one of claims 16-19, wherein said cancer is selected from the group consisting of a mantle cell lymphoma (MCL), a diffuse large B cell lymphoma (DLBCL), a Hodgkin’s lymphoma, a non-Hodgkin lymphoma, an acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a germ cell tumor, a hepatocellular carcinoma, a bowel cancer, a lung cancer, a breast cancer, an ovarian cancer, a melanoma, a brain cancers, and a multiple myeloma.

21. The method of any one of claims 16-20, wherein said chimeric antigen receptor targets a tumor-associated antigen expressed by a cancer cell of said cancer.

22. The method of claim 21, wherein said tumor-associated antigen is CD19.

23. The use of a composition comprising the T cell of any one of claims 1-15 to treat a mammal having cancer.

24. A composition comprising the T cell of any one of claims 1-15 for use in the preparation of a medicament to treat a mammal having cancer.

25. A composition comprising the T cell of any one of claims 1-15 for use in the treatment of cancer.

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