T cells with enhanced Anti-tumor activity

Abstract

Provided herein is a T cell that expresses an A20 protein that lacks a functional ZnF7 domain or has a genome in which the endogenous A20 gene has been knocked out. In some cases, the A20 protein may have a C-terminal truncation that removes at least part of the ZnF7 domain, an amino acid substitution in the ZnF7 domain, a deletion that removes at least part of the ZnF7 domain, or an insertion that causes out-of-frame protein translation. Populations of the cells, methods of treatment using the cells, and a cell manufacturing method are also provided.

Description

[0001]T CELLS WITH ENHANCED ANTI-TUMOR ACTIVITY CROSS-REFERENCING This application claims the benefit of U.S. provisional application serial no 63 / 626,991, filed on January 30, 2024, which application is incorporated by reference for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCELISTINGXML FILEA Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF- 775WO_SEQLIST”, created on January 27, 2025, and having a size of 9,816 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. BACKGROUNDAdoptive cell therapy using T cells that have been genetically modified to express chimeric antigen receptors (i.e., “CAR-T cells”) show great promise for the treatment of cancer. However, its therapeutic benefits have been limited in many cases because even patients who respond to the immunotherapy remain at risk of relapse due to, among other things, the short- term persistence of the T cells. Therefore, a therapy that provides more robust and sustained anti-tumor immunity than current T cell therapies would be of great clinical value. This disclosure provides a solution to this problem. SUMMARY The A20 protein (otherwise known as Tumor Necrosis Factor α-Induced Protein 3 or “TNFAIP3”, for short) is encoded by the TNFAIP3 gene in humans. For ease of reference, this protein will be referred to herein as the “A20 protein” and the gene that encodes the A20 protein will be referred to herein as the “A20 gene”. It is understood that this protein and gene may be referred to by other names (e.g., TNFAIP3, etc.) in other publications. The names “A20” and “TNFAIP3” are interchangeable. It has been shown that the anti-tumor activity of CAR-T cells in vivo can be improved by knocking out the A20 gene or ablating the zinc finger 7 (ZnF7) domain of the A20 protein. In some embodiments, the functional persistence of the cells may be improved which, in turn, may allow the dosage of cells administered to a patient to be lowered. This lower dose may avoid some of the side effects of current ‘high dose’ CAR-T cell therapies that are currently being used, e.g., to treat some solid tumors. Following from the above, a T cell that expresses an A20 protein that lacks a functional ZnF7 domain (referred to as the “modified A20 protein” herein) is provided, as well as a T cell in which the endogenous A20 gene has been knocked out (which may be referred to as an “A20 knockout”). In some embodiments, the T cell may comprise: (i) a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain or a genome in which the endogenous A20 gene has been knocked out. In some embodiments, the T cell may additionally comprise: ii) a nucleic acid encoding a recombinant immune receptor such as a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR). A population of the subject T cells (which may have been grown ex vivo) is also provided. In embodiments that employ a modified A20 protein, the nucleic acid may be the endogenous A20 gene. In these embodiments, the endogenous A20 gene may contain one or more mutations that ablate the function of the ZnF7 domain where, in this context, the function may be ubiquitin binding or any other function of the ZnF7 domain of the A20 protein. Also provided is a method of treatment comprising administering a population of the present T cells to a patient in need thereof. As noted above, the dose of T cells used in the method may be lower than if the T cells do not contain the A20 knockout or modified A20 protein and, as such, in some embodiments, the number of T cells administered to the patient may be in the range of 100,000-50M, which is lower than the ‘normal’ dose of at least 50M cells. In other embodiments, a ‘normal’ dose of the cells (e.g., 50M-500M cells) may be administered to the patient. A T cell manufacturing process is also provided. In these embodiments, the method may comprise expanding T cells ex vivo to produce expanded T cells, wherein T cells comprise: a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain or a genome in which the endogenous A20 gene has been knocked out and a nucleic acid encoding a recombinant immune receptor, and harvesting the expanded T cells to produce a T cell population. In some embodiments, the expanded T cells may be harvested within 3-8 days of the initiation of cell expansion, thereby accelerating the manufacturing process (which is typically 10-14 days in length). In other embodiments, the expanded T cells may be harvested within 8-16 days of the initiation of cell expansion. Harvesting the cells within 8-16 days of the initiation of cell expansion may produce several doses of cells, which may be used in “off the shelf” embodiments in which allogeneic cells are made from a healthy person, genetically modified, stored, and then used only when they are needed. In some embodiments, the harvested T cell population may comprise 100,000-1 Bn of the T cells. In any embodiment, the harvested T cell population may be administered to a cancer patient in need thereof. Additional embodiments and other features, advantages and variations may become apparent in view of the description that follows below. BRIEF DESCRIPTION OF THE FIGURES The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIGS. 1A-1L. A genome-wide CRISPR perturbation screen identifies A20 / TNFAIP3 as a key regulator of T cell dysfunction in the setting of chronic antigen stimulation. (FIG. 1A) Graphic depiction of the genome-wide CRISPR screen to uncover genes regulating T cell dysfunction in the setting of chronic tumor antigen exposure. (FIG. 1B) Volcano plot portraying gene level scores for log fold change (x axis) and p-value (y axis) generated by MAgECK analysis for TFrep vs T0. Screen includes two human T cell donors. Highlighted genes include A20 / TNFAIP3 and genes with log FC > 1.8 and known roles in T cell fitness. (FIG. 1C) Representative normalized cancer growth curves of mKate+NY-ESO-1+A375 melanoma target cells. A20KOor AAVS1 control TCR-T cells either without prior repetitive stimulation (no rep stim) or after 5 prior rounds of repetitive stimulation (rep stim) were co-cultured with A375 cells at an E:T ratio of 1:2. Growth curves were determined by Incucyte live-cell imaging of mKate+cells and depict mean ± SEM of n = 3 technical replicates from one of two T cell donors. (FIG. 1D) Normalized supernatant concentrations (pg / ml) of secreted cytokine and cytotoxic proteins, as determined by multiplexed bead-based flow cytometric analysis (LEGENDplex). Cas9-edited A20KOor control AAVS1 TCR-T cells were analyzed before (pre) or after (post) 3 rounds of repetitive stimulation. Bar graphs depict mean ± SEM of n = 3 donors with n = 3 technical replicates per donor. Each technical replicate was normalized to mean of control AAVS1 group for corresponding donor and condition. Significance was assessed using paired t test. (FIG. 1E) Graphical timeline overview of the experimental workflow for the in vivo experiment depicted in (FIG. 1F) and (FIG. 1G). (FIG. 1F) Individual NALM6 growth curves in NSG mice treated with 1 x 105A20KO(sgRNA-1) or control AAVS1 CAR-T cells (n = 1 T cell donor, n = 5 mice per group). Leukemia burden was quantified via BLI. Units for average radiance = photons / s / cm2 / steradian. (FIG. 1G) Survival analysis for (FIG. 1F). (FIG. 1H) SPICE analyses of IR co-expression on Cas9-edited CD4+and CD8+CAR-T cells isolated from bone marrow of NALM6-bearing NSG mice 14 days after CAR-T cell injection, as determined by flow cytometry. Plots depict mean values of n = 3 mice per group per donor of n = 2 T cell donors. (FIG. 1I) Averaged MC38 growth curves in A20 haploinsufficient (A20+ / -) versus WT littermate (A20+ / +) mice. (FIG. 1J) Survival analysis for (FIG. 1I). (FIG. 1K) Averaged MC38 growth curves in bone marrow chimeric mice of the indicated donor and recipient genotypes. (FIG. 1L) Survival analysis for (FIG. 1K). For FIGS. 1G, 1J and 1L, Significance was assessed using a log- rank test. *p < 0.05; **p < 0.01. For FIGS. 1I and IK, each time point depicts mean + SEM of 10-19 mice per group combined from two or more independent experiments. Significance was assessed using two-way ANOVA or mixed-effects analysis. ****p < 0.0001. FIGS. 2A-2F. Inactivation of A20's zinc finger 7 domain enhances anti-tumor immunity. (FIG. 2A) (Top) Heatmap depicting ESM1b log likelihood ratio (LLR) score for all possible amino acid missense mutations (left) along the entire length of the A20 / TNFAIP3 gene, with more negative score indicating greater likelihood of pathogenicity, i.e. damaging to protein or domain function. (Middle) Average effects log2FC on human T cell TNF production by missense mutation base edits across the open reading frame of A20 / TNFAIP3, using either adenine (ABE; above) or cytosine (CBE; below) base editors. Red and blue indicate increased vs decreased TNF production over unedited cells, respectively. Dashed line = average log2FC of all terminating (knockout) guides within the ABE or CBE screen targeting the first 250 amino acid residues. Gray = amino acid residues not targeted by screen. (Bottom) Annotated domains of A20 protein as characterized on UniProt, with box colors indicating average effect of base edit screen guides targeting those domains. Annotation includes DUB catalytic triad of OTU domain (D100, C103, H256). Ub = ubiquitin. (FIG. 2B) Averaged MC38 growth curves in A20OTU(left), A20ZF4(middle), and A20ZF7(right) knock-in (KI) mice. (FIG. 2C) Survival analysis corresponding to mice from FIG. 2B. Significance was assessed using a log-rank test. *p < 0.05; ****p < 0.0001. (FIG. 2D) Horizontal bar graphs depicting proportion of mice from FIG. 2B that exhibited regressor phenotype (regression) versus those that did not (progression). Significance was assessed using Fisher's exact test. **p < 0.01. (FIG. 2E) Averaged MC38 growth curves from A20ZF7 / ZF7regressor mice that were rechallenged with MC38, compared to age-matched A20ZF7 / ZF7mice that were naive to tumor (primary challenge). (FIG. 2F) Averaged B16F10 (left) and CT26 (right) growth curves in A20ZF7KI mice. For FIGS. 2B, 2E and 2F, each time point depicts mean + SEM of 10-27 mice per group combined from two or more independent experiments. Significance was assessed using two-way ANOVA or mixed-effects analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. FIGS. 3A-3G. CD8+T cell-intrinsic A20ZF7inactivation invigorates anti-tumor immunity. (FIG. 3A) Averaged MC38 growth curves from A20ZF7KI mice with indicated genetic or antibody-mediated ablation of lymphocyte subsets. Isotype = isotype control antibody for anti-CD8α (rat IgG2b, κ). (FIG. 3B) Horizontal bar graphs depicting proportion of mice from FIGS. 3A and 3D that exhibited regressor phenotype (regression) versus those that did not (progression). Significance was assessed using Fisher's exact test. *p < 0.05; **p < 0.01. (FIG. 3C) Averaged MC38-OVA growth curves from CD45.1 mice that received adoptive transfer of either naive (one day prior to tumor inoculation) or pre-activated (day 7 post inoculation) A20+ / +or A20ZF7 / ZF7OT-I cells. (FIG. 3D) Averaged MC38 growth curves from E8Icremice interbred with mice carrying floxed A20 (A20lox) and / or A20ZF7KI alleles. (FIG. 3E) Survival analysis corresponding to mice from FIG. 3D. Significance was assessed using log-rank test. *p < 0.05. (FIG. 3F) Graphical timeline overview of the workflow for reconstitution of Cd8a- / -mice with CD8+T cells, related to FIG. 3G. XRT = sublethal irradiation. FSFC = full spectrum flow cytometry. (FIG. 3G) Averaged MC38 growth curves of Cd8a- / -mice reconstituted with CD8+T cells of the indicated A20ZF7KI genotype. For FIGS. 3A, 3C, 3D and 3G, each time point depicts mean + SEM of 10-34 mice per group combined from two or more independent experiments. Significance was assessed using two-way ANOVA or mixed-effects analysis. *p < 0.05; **p < 0.01; ****p < 0.0001 FIGS. 4A-4I. ZF7 inactivation alleviates terminal exhaustion of CD8+T cells. (FIG. 4A) Bar graphs depicting total intratumoral leukocyte density (CD45+cells; left), CD8+TIL density (middle) and CD8+TIL fraction of total leukocytes (right). (FIG. 4B) Horizontal bar graphs depicting CD8+TIL subset proportions. (FIG. 4C) Representative FSFC histograms showing IR, TOX and Ly108 expression on CD8+TIL subsets. Inset = proportion (%) of cells within ranged gate (bottom). (FIG. 4D) Bar graphs showing proportion of Ttex that express IRs, TOX and Ly108, related to FIG.4C. (FIG. 4E) Paired analysis of OT-I Ttex expression of IRs, TOX and Ly108 at 7 days following adoptive co-transfer into MC38-OVA-bearing mice, as determined by tumor FSFC. Each symbol / line pair represents A20+ / +vs A20ZF7 / ZF7OT-I cells retrieved from the same tumor (n = 7 samples combined from two independent experiments). Significance was assessed Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01. (FIG. 4F) Bar graphs depicting IR and Ly108 expression by OT-I cells following in vitro repetitive stimulation, as determined by FSFC. Significance was assessed using two-way ANOVA with Bonferroni’s multiple comparisons test. **p < 0.01; ****p < 0.0001. (FIG. 4G) Truncated violin plots depicting IR, TOX and Ly108 expression by each Ttex subset. Plots represents combined samples from A20+ / +, A20ZF7 / +and A20ZF7 / ZF7mice. (FIG. 4H) Representative contour plots showing Ttex expression of CX3CR1 vs KLRG1. Inset = proportion (%) of Ttex by quadrant. (FIG. 4I) Horizontal bar graphs depicting Ttex subset proportions, related to FIG. 4H. For FIGS. 4A-4D and 4G-4I, FSFC analyses of day 12-15 MC38 tumors. mean ± SEM, n = 5-15 samples (FIGS. 4A-4D, 4I) or 26-43 samples (FIG. 4G) per group combined from two or more independent experiments. Significance was assessed using one-way ANOVA (FIG. 4A), two- way ANOVA with Tukey’s multiple comparisons test (FIGS. 4B, 4D, and 4I), or Kruskal-Wallis test with Dunn's multiple comparisons test (FIG. 4G). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. In FIG. 4I, instead of asterisks, significance (p < 0.05) is depicted in the banner above the bar graphs as a bicolored line representing the two A20ZF7KI groups with significant differences for the indicated Ttex subset. For FIGS. 4C and 4H, each plot represents 3-5 concatenated samples of the same group from a single experiment. FIGS. 5A-5K. A20ZF7inactivation within Ttex unleashes perforin-dependent anti-tumor cytotoxicity. (FIG. 5A) Representative contour plots depicting IFNγ and TNF expression by Ttex following ex vivo stimulation with PMA and ionomycin. Plots show data from 2-4 concatenated samples per group from one experiment. Inset = proportion (%) of Ttex in that quadrant. (FIG. 5B) Bar graphs showing proportion of polyfunctional (IFNγ+cTNF+) cells by CD8+TIL subset. (FIG. 5C) Averaged MC38 growth curves from A20ZF7KI mice with indicated genetic ablation of IFNγ and / or TNF vs perforin (Prf1). Each time point depicts mean + SEM of 15-40 mice per group combined from three or more independent experiments. Significance was assessed using two-way ANOVA or mixed-effects analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (FIG. 5D) Horizontal bar graphs depicting proportion of mice from FIG. 5C that exhibited regressor phenotype (regression) versus those that did not (progression). Significance was assessed using Fisher's exact test. **p < 0.01. (FIG. 5E) Representative FSFC histograms showing perforin (top) and side scatter (SSC-A; bottom) expression by Ttex from A20+ / +(63, 8.8e5), A20ZF7 / +(62, 8.2e5) and A20ZF7 / ZF7(27, 6.3e5) tumors. Inset = proportion of perforin-expressing cells within ranged gate (top) or SSC-A MFI (bottom). Plots show data from 3-5 concatenated samples per group from one experiment. (FIG. 5F) Bar graphs showing proportion of perforin-expressing cells (left) or SSC-A MFI (right) among CD8+TIL subsets by A20ZF7KI genotype. (FIG. 5G) Scatter plot correlating Ttex SSC- A MFI with perforin expression, related to FIG. 5F. Statistics were calculated using simple linear regression (dashed line). (FIG. 5H) Bar graphs showing proportion of perforin-expressing cells among CD8+TIL subsets after ex vivo stimulation with PMA and ionomycin. (FIGS. 5I-5K) For FIGS. 5I, 5J and 5K, bar graphs depicting proportion of perforin- (FIG. 5I) or GzmB-expressing (FIG. 5J) CD8+TILs, or SSC-A MFI (FIG. 5K), in mice treated in vivo with either brefeldin or vehicle control (5% DMSO). For FIGS. 5A, 5B and 5E-5K, FSFC analyses of day 12-15 MC38 tumors. Bar graphs depict mean ± SEM of 7-15 samples per group combined from two or more separate experiments. Significance was assessed using two-way ANOVA with Tukey's multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. FIGS. 6A-6K. Base editing of A20's ZF7 motif enhances persistence and cytolytic function of human CAR-T cells in vitro and in vivo. (FIG. 6A) Individual NALM6 growth curves of NSG mice treated with 1 x 105Cas9-edited A20KO(sgRNA-2), A20ZF7or control AAVS1 CAR-T cells (n = 1 T cell donor, n = 5-11 mice per group). (FIG. 6B) Survival analysis for (FIG. 6A). (FIG. 6C) Individual NALM6 growth curves of NSG mice from FIG. 6A that controlled initial leukemia challenge and subsequently underwent multiple rechallenges with NALM6 cells (arrows). (FIG. 6D) Schematic illustration for the generation of B2Mi (control), A20KO, or A20ZF7CD19 TRAC-CAR-T cells using cytosine base editor (CBE) mRNA-mediated base editing. (FIG. 6E) Normalized cancer cell growth curves of mKate+A375-CD19 melanoma target cells. Base-edited A20KO, A20ZF7or control B2Mi CAR-T cells either without prior repetitive stimulation (no rep stim) or after 3 rounds of repetitive stimulation (rep stim) were co- cultured with A375-CD19 cells at an E:T ratio of 1:1. Growth curves were determined by Incucyte live-cell imaging of mKate+ cells and depict mean ± SEM of n = 3 technical replicates from n = 2 T cell donors (one representative donor shown). (FIG. 6F) Normalized supernatant concentrations of secreted cytokine and cytotoxic proteins, as determined by multiplexed bead- based flow cytometric analysis (LEGENDplex) of secreted cytokine and cytotoxic proteins. Base-edited (CBE) A20KO, A20ZF7or control B2Mi CAR-T cells were analyzed before (pre) or after (post) 3 rounds of repetitive stimulation. (FIG. 6G) Normalized IR expression by CD4+and CD8+base-edited control B2Mi control, A20KO, and A20ZF7CAR-T cells either before (pre) or after (post) 5 rounds of repetitive stimulation, as determined by flow cytometry. See legend from (FIG. 6F). (FIG. 6H) Individual NALM6 growth curves of NSG mice treated with 1 x 105base- edited A20KO, A20ZF7, or control B2Mi CAR-T cells, or with TRACKO(no CAR) T cells (n = 2 T cell donors, n = 6 mice per group per donor). (FIG. 6I) Survival analysis for (FIG. 6H). (FIG. 6J) Bar graphs depicting absolute numbers of CAR-T cells harvested from the bone marrow of Nalm6-bearing mice 14 days after CAR-T cell injection, as determined by flow cytometry (mean ± SEM, n = 2 T cell donors, n = 3 mice per group per donor). Significance was assessed using one-way ANOVA with Dunnett's T3 multiple comparisons test. (FIG. 6K) SPICE analyses of IR co-expression on base-edited CD4+and CD8+CAR-T cells isolated from bone marrow of NALM6-bearing NSG mice 14 days after CAR-T cell injection, as determined by flow cytometry. Plots depict mean values of n = 3 mice per group per donor of n = 2 T cell donors. For FIGS. 6A and 6H, leukemia burden was quantified via BLI. Units for average radiance = photons / s / cm2 / steradian. For FIGS. 6B and 6I, significance was assessed using log-rank test. *p < 0.05; **p < 0.01. Bar graphs depict mean ± SEM of n = 2 donors with n = 3 technical replicates per donor. Each technical replicate was normalized to mean of control B2Mi group for corresponding donor and condition. Significance was assessed using ***. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. FIG. 7 illustrates the domain structure of the human A20 protein, showing some mutations in the OTU, ZnF1, ZnF4 and ZnF7 domains. As shown, A20 can be divided into an N-terminal OTU (ovarian tumor) domain and seven C-terminal zinc finger (ZnF) domains. The catalytic cysteine (C103) in the OTU domain (green square) mediates A20’s deubiquitinating activity. A20’s ZnF4 domain (blue square) and ZnF7 domain (red square) mediate binding to K63- and M1-Ub chains, respectively. A20’s ZnF4 may also mediate E3 ubiquitin ligase activity to build K48 polyubiquitin chains. DETAILEDDESCRIPTIONBefore the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. T cells As summarized above, a T cell that has knocked out A20 gene or that expresses an A20 protein that lacks a functional ZnF7 domain (referred to as the “modified A20 protein” herein) is provided. The T cell may comprise: a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain; and / or a genome in which the endogenous A20 gene has been knocked out. In embodiments that involve modified A20 protein, the nucleic acid may be the endogenous A20 gene. In these embodiments, the gene may contain one or more mutations that ablate the function of the ZnF7 domain. If the endogenous gene has been altered, then the T cell may be homozygous or heterozygous for the alteration. A20 protein (otherwise known as Tumor Necrosis Factor α-Induced Protein 3 or “TNFAIP3”, for short) is encoded by the TNFAIP3 gene in humans (see Opipari et al J Biol Chem. 2020265: 14705–8, Genbank accession no KAI4020021.1 and Genbank Gene ID: 7128). The structure and function of A20 has been extensively reviewed (see, e.g., Ma and Malynn (Nat. Rev. Immunol. 201212: 774–785), Malynn and Ma (Cell. Immunol. 2019340: 103914), Razani et al. (Adv. Immunol. 2020148: 1–48), Martens et al (Cold Spring Harb. Perspect. Biol. 202012: a036418), Das et al (Front Immunol. 20189: 104), Wu et al (Arthritis Research & Therapy 202022: 220) (among many others). Structurally, the wild type human protein is 790 amino acids in length and, as illustrated in Fig. 1, has several domains, including an OTU (ovarian tumor) domain and seven zinc finger domains (designated ZnF1 to ZnF7). The OTU domain is defined by amino acids 41-261 and has a deubiquitinating enzyme (DUB) activity that relies on the catalytic residue Cys103. This domain has also been shown to bind to E2 enzymes Ubc13 and UbcH5c. The fourth zinc finger domain (ZnF4) is defined by amino acids 604-629 and has K63-linked polyubiquitin-binding affinity and possesses E3 ubiquitin (Ub) ligase activity. ZnF4 has also been shown to bind to E2 enzymes Ubc13 and UbcH5c. The seventh ZnF (ZnF7) domain (defined by amino acids 759-784) has strong binding affinity for linear (M1) ubiquitin chains (see Skaug et al., Mol Cell 201144: 559-571; Tokunaga et al., EMBO J. 2012 31: 3856-70). A20’s ZnF7 may play dominant roles in restraining inflammatory responses and preventing disease in vivo (Razani et al., Nat Immunol. 202021: 422–433). To the inventors’ knowledge, all of the prior work on A20’s ZnF7 domain has been done fibroblasts, myeloid cells, or intact mice. This prior work is not believed to be predictive of how T cells that express an A20 protein that lacks a functional ZnF7 domain (or A20 knockouts) would behave in vivo, particularly in a therapeutic setting. In some embodiments, the ZnF7 domain is either absent in the modified A20 protein or unable to bind to linear (M1) Ub chains. Relative to the wild type A20 protein, the modified A20 protein may have a C-terminal truncation that removes at least part of (including the entirety of) the ZnF7 domain, one or more amino acid substitutions in the ZnF7 domain (e.g., amino acid substitutes at C764 and / or C767, which are cysteine residues along with other amino acids that are responsible for coordinating the binding to zinc ions), a deletion that removes at least part of (including the entirety of) the ZnF7 domain, or an insertion that causes out-of-frame protein translation. As would be apparent, the changes can be effected by modifying the nucleic acid that encodes the protein, e.g., by introducing a point mutation, insertion, deletion, or substitution, etc. that changes the coding sequence for the protein. In any embodiment, the A20 protein may further comprise one or more amino acid alterations outside of the ZnF7 domain (e.g., amino acid substitutions in the in the DUB, ZnF1, ZnF2, ZnF3, ZnF4, ZnF5 or ZnF6 domains). In any embodiment, the modified A20 protein may be encoded by the endogenous A20 gene (i.e., the endogenous TNFAIP3 gene), modified to encode the modified A20 protein, i.e., by modifying the endogenous TNFAIP3 gene so that it contains one or more point mutations, insertions, deletions, or substitutions, etc. that result in an appropriate truncation, amino acid substitution or deletion that ablates the function of the ZnF7 domain in the encoded A20 protein. These changes can be readily implemented by tailored endonucleases such as but not limited to meganucleases, TAL effector nucleases, CRISPR / Cas9-based technologies or derivatives thereof including but not limited to base or prime editors. In some embodiments, the modified A20 protein may solely lack a functional ZnF7 domain. In these embodiments, the DUB and ZnF1-ZnF6 domains of the protein may be functional. In some embodiments, the first 758 amino acids of the protein may be wild type such that the DUB and ZnF1-ZnF6 domains have a wild type sequence). In these embodiments, the only change relative to wild type A20 will be a change in the ZnF7 domain. In other embodiments, the modified A20 protein may contain other changes. For example, in addition to lacking a functional ZnF7 domain, the modified A20 protein may lack: a functional DUB, ZnF1, ZnF2, ZnF3, ZnF4, ZnF5 or ZnF6 domain with the other domains functional. In some embodiments, the modified A20 protein may have a non-functional ZnF6 and ZnF7 with the remainder of the domains being functional, non-functional ZnF5, ZnF6 and ZnF7 domains with the remainder of the domains being functional, non-functional ZnF4, ZnF5, ZnF6 and ZnF7 with the remainder of the domains being functional, or non-functional ZnF3, ZnF4, ZnF5, ZnF6 and ZnF7 domains with the remainder of the domains being functional, etc. In addition to changes that ablate the ZnF7 domain, the modified A20 protein may also contain one or more amino acid substitutions (e.g., up to 10, up to 5 relative to the wild type sequence) at any position. For example, the alterations that inactivate ZnF7 may be combined with other alterations in A20 that effect A20’s activity, examples of which are shown in Fig.1 and 3A-C. For example, the alterations that inactivate ZnF7 may be combined with alterations that inactivate either A20’s deubiquitinase activity (e.g., C103A) or ZF4-mediated K63- binding / ligase activity (e.g., C609A, C612A). Examples of amino acid substitutes that abolish ZF7-mediated M1-Ub binding activity include (C764A, C767A) or (F755A, G756A). Some mutant A20 proteins may have alterations that effect both ZF4 and ZF7 functions (C609A, C612A, F755A, G756A), for example. As noted above, the cell may be a T cell, where the term “T cell” is intended to encompass all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg) and gamma-delta T cells, tumor infiltrating leukocytes (TILs), iPSC-derived T cells, natural killer T (NKT) cells etc. Cytotoxic T cells (i.e., CD8+T cells) are of particular interest, since they are the most common cell used for CAR- and TCR-based therapies. In theory, the present technology could be used in other types of therapeutic lymphocytes (e.g., natural killer (NK) cells) because those cells may have regulatory mechanisms. As such, in any embodiment of the present disclosure, the cell may be any type of lymphocyte, particularly an NK cell. In any embodiment, the T cell may be mammalian (e.g., mouse, human, primate). Human cells are often used, however. In any embodiment, the T cell may be a primary T cell, i.e., a cell obtained from peripheral blood, or a progenitor of the same (e.g., cell made by culturing primary T cells in proliferation or expansion medium) and in any embodiment, the T cell may be genetically modified to be allogeneic in a human host (e.g., by knocking out exons of the TCRα constant (TRAC) and / or TCRβ constant 1 (TRBC1) or 2 (TRBC2) loci and / or Beta 2 Microglobulin, CD52, etc.), methods for which are known (see, e.g., Bedoya et al (Front Immunol. 202112:640082) and Aparicio et al (Experimental Hematology & Oncology 2023 12: 73)). In some embodiments, the endogenous A20 gene of the T cell may encode an A20 protein that lacks a functional ZnF7 domain, the endogenous TCRα constant (TRAC) gene of the T cell may have been inactivated and the T cell may comprise a nucleic acid encoding a CAR. For example, in some cases the nucleic acid encoding the CAR may be inserted into the TRAC gene, thereby inactivating the TRAC gene in some cases. In some embodiments, the CAR may comprise an intracellular signaling domain from CD3ζ in which the second and third ITAM motifs have been altered to be non-functional, e.g., a CD3ζ signaling domain in which both tyrosine (Y) phosphorylation sites in the second and third ITAMs are be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated, as described in in Feucht et al (Nat Med. 201925: 82-88). T cells may be obtained from any suitable source. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population, or T cells may be obtained from a subject. T cells may be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is herein incorporated by reference in its entirety. In any embodiment, the cells that are expanded can be a primary T cell. T cells may be genetically modified, made from iPSCs, obtained from umbilical cord blood, central memory T cells, etc. As noted above, the cell may further a nucleic acid encoding a recombinant immune receptor, e.g., a chimeric antigen receptor (CAR), engineered T cell receptor (TCR) or HLA Independent TCR (HIT). In other embodiments (e.g., TILs), the T cell may comprise an endogenous immune receptor. In any embodiment, the recombinant immune receptor may be constitutively expressed in the cell. In either event, the immune receptor will be expressed on the surface of the cell and will be activated by binding to an antigen that is expressed by the cancerous cells, e.g., by the malignant cells, e.g., any of the antigens listed below, for example. Binding of the immune receptor to its cognate antigen activates the immune cell. In these embodiments, the receptor may be a chimeric antigen receptor (CAR), where the terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi- module molecules capable of triggering the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 A1 and US Patent Application No. 2015 / 0368342 A1, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151- 5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333- 47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19—4-1BB—CD3ζ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland). CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 201912: 145-156) and may include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region (which may be derived from CD4, CD8α, or CD28), a costimulatory signaling domain (which may be derived from the intracellular domains of the CD28 family (e.g., CD28 and ICOS) CD2 (US9783591B2) or the tumor necrosis factor receptor (TNFR) family of genes (e.g., 4-1BB, OX40, or CD27)), and an ITAM domain, e.g., the signaling domain from the zeta chain of the human CD3 complex (CD3zeta). In practice, any of these domains may be a variation of a wild type sequence. In practice, any of these sequences may be a variant of a wild type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example. For example, a CAR may have a signaling domain from CD3ζ in which two of the three ITAM motifs (the second and third ITAM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3ζ signaling domain is described in Feucht et al (Nat Med. 2019 25: 82-88). The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (α) and beta (β) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric αβ or γδ forms. The complete endogenous TCR complex in heterodimeric αβ form includes eight chains, namely an alpha chain (referred to herein as TCRα or TCR alpha), beta chain (referred to herein as TCRβ or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instance, a TCR is generally referred to by reference to only the TCRα and TCRβ chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate. Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRα or modified TCRβ chains as well as single chain TCRs that include modified and / or unmodified TCRα and TCRβ chains that are joined into a single polypeptide by way of a linking polypeptide Any engineered TCR having immune cell activation function can be induced using a method of the present disclosure. Such TCRs include, e.g., antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, High Affinity CDR2 Mutant TCRs, CD1-binding MTCRs, High Affinity NY-ESO TCRs, VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT Pub Nos. WO 2003 / 020763, WO 2004 / 033685, WO 2004 / 044004, WO 2005 / 114215, WO 2006 / 000830, WO 2008 / 038002, WO 2008 / 039818, WO 2004 / 074322, WO 2005 / 113595, WO 2006 / 125962; Strommes et al. Immunol Rev. 2014; 257(1):145-64; Schmitt et al. Blood. 2013; 122(3):348-56; Chapuls et al. Sci Transl Med. 2013; 5(174):174ra27; Thaxton et al. Hum Vaccin Immunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al. Immunol Rev. 2014; 257(1):237-49 (PMID:24329801); Hinrichs et al. Immunol Rev. 2014; 257(1):56-71 (PMID:24329789); Zoete et al. Front Immunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol. 2012; 167(2):216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev. 2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific World Journal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin Exp Immunol. 2005; 142(3):454-60 (PMID:16297157); Sami et al. Protein Eng Des Sel. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003; 16(9):707-11; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. Nat Biotechnol. 2005; 23(3):349-54; Dunn et al. Protein Sci. 2006; 15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. Nat Med. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891; McCormack, et al. Cancer Immunol Immunother. 2013 Apr;62(4):773-85; Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, et al. Mol Immunol. 2015 Oct;67(2 Pt A):67-74; the disclosures of which are incorporated herein by reference in their entirety. HLA-Independent TCRs (Eyquem et al. 2022 Feb;28(2):345-352) are also included in this definition. The immune receptor may be constitutively expressed (in which case its coding sequence will be operably linked to a constitutive promoter, i.e., a promoter that is always "on" in the cell) or induced, e.g., by activation of a proteolytic receptor or an inducible promoter. Depending on how the cells are going to be used, the a CAR may have an extracellular binding domain that recognizes, for example, CD19, CD20, BCMA, the folate receptor (FR-α), mesothelin (MSLN), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), MUC1, GD2, epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CAIX), L1-CAM, EGFRvIII, IL13Rα2, prostate- specisfic membrane antigen (PSMA), PSCA, fibroblast activation protein (FAP), CD133, c- MET, ephrin type-A receptor 2 (EphA2), Glypican-3 (GPC3), VEGFR-2, ROR1, MUC16, among others. Recombinant TCRs may recognize antigens such NYESO1, MAGE, mutated KRAS, mutated P53, MART-1, gp100, WT-1, CEA, and Tyrosinase, for example, although others exist. Other examples may exist below. A method comprising genetically modifying a T cell to have an A20 gene knockout or contain a nucleic acid encoding the modified A20 protein is provided. As would be apparent, this method may be done ex vivo (on primary T cells or their progenitors). In some embodiments, this method may involve modifying the endogenous A20 gene so that the encoded protein lacks a functional ZnF7 domain, as described above. For example, an A20 gene-targeted CRISPR / Cas9 gene modification system could be used, where the system either knocks out the endogenous A20 gene or introduces a point mutation, insertion, deletion, or substitution in region of the A20 gene that encodes the A20 ZnF7 domain Populations of T cells A population of the T cells is also provided. In some embodiments, these cells may be present in vitro and may be progenitors of primary T cells that have been genetically modified to have an A20 gene knockout or contain a nucleic acid encoding the modified A20. In some embodiments, the cell may contain a nucleic acid encoding an immune receptor, as discussed above. As noted above, in some embodiments T cells may be genetically modified to be allogeneic in a human host. In these embodiments, the cells may be frozen. The population may comprise any number of the CAR-T cells (e.g. 100,000-1 Bn cells). However, in some embodiments, the population may contain 100,000-50M of the CAR-T cells. In some embodiments, the harvested cells may be cryopreserved, where the term “cryopreserved” refers to cells that have been preserved or maintained by cooling to low sub- zero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. Useful methods of cryopreservation and thawing cryopreserved cells, as well as processes and reagents related thereto, include but are not limited to e.g., those described in U.S. Patent Nos. 10370638; 10159244; 9078430; 7604929; 6136525; and 5795711, the disclosures of which are incorporated herein by reference in their entirety. In contrast, the term “fresh”, as used herein with reference to cells, may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and / or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof. Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like. In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCl2, KCl, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. A population may include a therapeutically effective amount of the cells. By “therapeutically effective amount” it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a therapeutically effective amount of T cells may be 100,000-50M of the T cells. However, in other embodiments, a therapeutically effective amount of T cells may be 50M- 500M cells. The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like. Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration. The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation. A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM. In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v). Cell manufacture methods A cell manufacture method is also provided. In some embodiments, the method may comprise expanding the T cells ex vivo to produce expanded T cells, and harvesting the expanded T cells to produce a T cell population, where the term “harvesting” is intended to refer to a step in which the cells are removed from the container(s) / bioreactor(s) in which the cells were expanded. In some embodiments, the cells may be concentrated if desired, e.g., by centrifugation, a suitable cell separation technique (e.g., magnetic beads), and / or the like. In embodiments in which the T cells express a recombinant immune receptor, the cells may be made by any method, e.g., by introducing a transgene into T cells that have been genetically modified to encode a A20 protein that lacks a functional ZnF7 domain or to have an A20 gene knockout, by genetically modifying a population of T cells that comprise a transgene for the immune receptor to encode an A20 protein that lacks a functional ZnF7 domain or an A20 gene knockout, or by introducing a immune receptor transgene into T cells and, at the same time, genetically modifying the T cells to encode an A20 protein that lacks a functional ZnF7 domain or to knockout the A20 gene. As illustrated in the present examples section, these modifications can be implemented by CRISPR / Cas9 although other systems may be used. As shown, the transgene for the immune receptor may be inserted into the TRAC gene, thereby reducing the possibility of graft versus host disease. In other embodiments, expression of the transgene may be driven by a promoter that is endogenous to the cell. Other promoters, e.g., the EF1a, LTR, MND, SFFV, PGK could also be used. The general procedure for manufacturing CAR-T cells typically includes isolation of peripheral blood mononuclear cells (PBMCs). Next, PBMCs or T cells that have been further enriched from PBMCs are stimulated with antibody-coated beads (e.g. Dynabeads) or plate- bound antibodies to induce T cell activation and then genetically modified using lentiviral vectors, gamma-retroviral vectors, or other delivery methods, to express the cell surface CAR molecule. Subsequently, these engineered T cells are expanded in culture in the presence of one or more cytokines (e.g., IL-2, IL-15, IL-7 or any combination thereof (e.g., IL-2 and IL-15 or IL- 7 and IL-15)) for several days to reach the required cell numbers for either experimental testing or clinical treatment. In some embodiments, the T cells may be harvested within 3-8 days of the initiation of cell expansion (e.g., on day 3, day 4, day 5, day 6, day 7 or day 7), which is earlier than conventional procedures (which typically require 10-14 days of cell expansion). In these embodiments, the harvested T cell population may comprise 100,000-50M of the CAR-T cells, however more or less cells can be used. In other embodiments, the T cells may be harvested within 8-16 days of the initiation of cell expansion, in which case the population may comprise 50M-1 Bn cells. A cell population manufactured according to this method is also provided. Methods of treatment A method of treatment is also provided. This method may comprise administering an effective amount a population of the T cells to a patient in need thereof where, in some embodiments, an effective amount may be 100,000-50M cells or 50M to 1 Bn cells. In any embodiment, the T cells may be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human. Cells are typically infused into the patient, although routes of administration can be used. In any embodiment, the patient may be a cancer patient where the treatment may result in least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. The cancer being treated may be a blood cancer or a solid tumor, e.g., a carcinoma or a sarcoma. If the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. Cancers that can be treated with a method include any cancer that can be targeted by CAR-T cells, including, but not limited to carcinomas, sarcomas, melanoma, leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method include solid tumors, and cancers that begin in blood-forming tissue, i.e., hematological cancers such as leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method include metastatic cancers. Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. As would be apparent the choice of CAR may be determined by the type of cancer being treated. For example, the CAR may target CD19, CD20 or BCMA for B-cell malignancies, including acute myeloid leukemia (AML), multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), lymphoma, etc. Breast cancer antigens include MUC1, HER2, mesothelin, CEA, CAIX, FR-α, CD171, GD2, EGFRvIII, FAP, and vascular endothelial growth factor receptor2 (VEGF-R2). Ovarian cancer antigens include CA125, MUC16, HER2, hepatocyte growth factor receptor (c-Met), mesothelin, folate receptor alpha (FRα), and cancer / testis antigen 1B. Lung cancer antigens include mesothelin (MSLN), EGFR, PSCA, MUC1, CEA, CD80 / CD86, programmed death-ligand 1 (PD-L1), inactive tyrosine-protein kinase transmembrane receptor (ROR1), and HER2. Colorectal cancer antigens include CEA, EGFR, MUC1, NKG2DL, HER2, and CD133. Pancreatic cancer antigens include mesothelin, CD133, PSCA, claudin 6, claudin 18.2, EGFR, CEA, MUC1, and HER2. Glioblastoma antigens include interleukin-13 receptor alpha 2 (IL-13αR2), epidermal growth factor receptor variant III (EGFRvIII), and HER2. Neuroblastoma antigens include GD2 and L1-CAM (CD171). Melanoma antigens include HER2, ganglioside GD2 and c-MET. Hepatocellular carcinoma antigens include CEA, MUC-1, and GPC-3. Gastric cancer antigens include folate receptor 1 (FOLR1) and NKG2D. Prostate cancer antigens include prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), PSCA, T-cell receptor gamma alternate reading frame protein (TARP), transient receptor potential (trp)-p8, and PSMA. Renal cell carcinoma antigens include VEGFR2, CAIX, CCT301-38, CCT301-59, and CD70. Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, intraventricular, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. A method of treatment that involves gene editing in vivo is also provided. These embodiments may comprise administering an A20 gene-targeted CRISPR / Cas9-based therapeutic to a patient in need thereof, where the therapeutic either knocks out the endogenous A20 gene in the patient’s T cells or introduces a point mutation, insertion, deletion, or substitution in region of the A20 gene that encodes the A20 ZnF7 domain in the patient’s T cells. These embodiments may be performed using an AAV, VLP or LNP delivery system. Hamilton et al (Nat. Biotechnol. 2024 doi: 10.1038 / s41587-023-02085-z (epub ahead of print)) and Banskota et al (Cell 2022185: 250–265) describe how CRISPR / Cas9-based therapeutics can be delivered to target cells in vivo. Combination therapy In some cases, the T cells (e.g., CAR T or engineered TCR cells) may be administered along with at least one additional therapeutic agent or therapeutic treatment (together or sequentially). Suitable additional therapeutic agents include, but are not limited to, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor. Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like. A treatment method of the present disclosure can comprise co-administration of the CAR-T cells and at least one additional therapeutic agent. By “co-administration” it is meant that both the CAR-T cells and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the CAR-T cells and the at least one additional therapeutic agent. The administration of the CAR-T cells and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, the CAR-T cells of the present disclosure are administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the CAR-T cells can occur at different times and / or at different frequencies. In some cases, the subject may be additionally treated with an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA. Co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine,procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6-thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF- 06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinum-based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (S1)). In any embodiment, the present T cell therapy may be combined with FOLFOX therapy (i.e., a chemotherapy regimen that comprises administering folinic acid, fluorouracil and oxaliplatin (FOLFOX) to the patient), e.g., for the treatment of colorectal and other cancers. Also provided is a guide RNA that targets the ZnF7 domain of A20, which guide RNA may be packaged in a conjunction with an RNA-directed endonuclease and / or a base editor. Examples of such guide RNAs are set forth below. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. Abbreviations: CAR-T, chimeric antigen receptor-transduced T cells; IV, intravenous; IC, intracranial tumor models; SC, subcutaneous tumor models; MFI, Mean Fluorescence Intensity; Met, Metformin; Rap, Rapamycin; Met+Rap, combination therapy of Metformin and Rapamycin; 2DG, 2-Deoxy-D-glucose; DCA, Dichloroacetic acid; wt, wild type; LD, lymphodepletion; MS, median survival; DC, dendritic cells; UT, untransduced cells. Materials and methods Mice. Male and female mice between 6–12 weeks of age were used for all experiments, following a protocol approved by the UCSF Institutional Animal Care and Use Committee. All mice were housed with a 12 h / 12 h light / dark cycle, with food and water available ad libitum. Generation of A20lox / lox, A20OTU, A20ZF4and A20ZF7mice has been previously described. The following mouse strains were obtained from The Jackson Laboratory and interbred within the UCSF animal facility: C57BL / 6J (Strain #000664), BALB / cJ (#000651), NOD-scid IL2Rγnull(NSG) (#005557), CD45.1 (#002014), Cd8a- / -(#002665), E8Icre(#008766), Ifng- / -(#002287), Ighm- / -(#002288), OT-I (#003831), Prf1- / -(#002407), Tcrd- / -(#002120), and Tnf- / -(#0054400). NSG male mice were used for all in vivo CAR-T cell experiments. Human cell lines. A375 (ATCC), A375 mKate+, A375-CD19 and A375-CD19-mKate (generated by Alexander Marson’s lab at Gladstone Institutes / UCSF), were cultured in complete RPMI consisting of RPMI 1640 (Thermo Fisher) supplemented with 10% FBS (Corning), 1% glutamine (Life Technologies), 100U / ml penicillin (Thermo Fisher) and 0.1mg / ml streptomycin (Thermo Fisher). NALM6 (ATCC) expressing luciferase and eGFP (generated by Michel Sadelain’s lab at MSKCC) were cultured in complete RPMI supplemented with 1x MEM NEAA (Thermo Fisher), 1mM sodium pyruvate (Thermo Fisher), 55 µM 2-Mercaptoethanol (Thermo Fisher), and 1 mM HEPES (Thermo Fisher). HEK293T (ATCC) and Lenti X-HEK293T cells (Lenti-XTM 293T) were cultured in DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS; Corning), 1% glutamine (Life Technologies), 100U / ml penicillin (Thermo Fisher) and 0.1mg / ml streptomycin (Thermo Fisher). Flow cytometry was routinely performed on NALM6 and A375-CD19 cells to confirm CD19 antigen expression. NALM6, A375, A375- CD19 and 293T were routinely tested with the MycoAlert Detection kit (Lonza) and were found to be mycoplasma-free. Murine cell lines. MC38 (Kerafast), MC38-OVA (see below) and B16F10 (ATCC) tumor cells were cultured in complete DMEM (cDMEM; Corning), defined as DMEM supplemented with 10% fetal bovine serum (FBS; R&D Systems), 100 U / ml penicillin (Sigma- Aldrich), 0.1 mg / ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Thermo Fisher), 10 mM HEPES buffer (CellGro), 1x MEM nonessential amino acids (Corning), and 1 mM sodium pyruvate (CellGro). CT26 (ATCC) tumor cells were cultured in complete RPMI 1640 (cRPMI; Gibco), defined as RPMI with the aforementioned supplementation. MC38-OVA cells were generated by transfecting MC38 cells with pCI-neo-mOVA (Addgene #25099) by using Lipofectamine LTX Reagent with PLUS (Thermo Fisher Scientific) per the manufacturer's protocol, then selecting cells in the presence of 400 μg / ml G418. All cell lines were maintained in subconfluent conditions with minimal passage number. Primary human T cell isolation and culture. The isolation of primary human T cells from human PBMCs was conducted via negative selection using the EasySep Human T Cell Isolation Kit following the manufacturer’s instructions (STEMCELL). Leukopaks from deidentified healthy donors (STEMCELL) were used for this purpose and came with consent from donors and protocols approved by an Institutional Review Board. Following the isolation process, T cells were activated using CTS Dynabeads Human T-Cell Activator CD3 / CD28 (Thermo Fisher) at a cell to bead ratio of 1 to 1. The cells were cultured at a density of 1x 106cells / ml in complete human T cell growth medium defined as X-Vivo-15 media (Lonza) supplemented with 5% human serum (Gemini), 50 µM 2-mercaptoethanol (Thermo Fisher), and 10 mM N-acetyl-L-cysteine (MilliporeSigma). T cells were split every 48-72 hours. Human IL-7 and IL-15 (5 ng / ml each; Miltenyi) was added to this media, unless stated otherwise. Primary murine T cell isolation and culture. Naive mouse polyclonal CD8+T cells or OT-I cells were isolated by harvesting spleens from A20ZF7KI or OT-I mice and mechanically disaggregating through a 70 μm nylon cell strainer (Corning). Splenocytes were pelleted and erythrocytes were lysed in ACK buffer (Quality Biological) for 30 seconds, quenched in excess PBS (Gibco), filtered again, aliquoted for cell counting, and pelleted again. Splenocytes were resuspended in EasySep Buffer (STEMCELL) and naive polyclonal CD8+or OT-I cells were isolated with the Mouse Naive CD8+T cell Isolation Kit (STEMCELL) by following the manufacturer's protocol. Naive polyclonal CD8+T cells or OT-I cells were counted and then used directly for downstream in vitro or in vivo applications. In vivo NALM6 leukemia model. Male NSG mice aged 8-12 weeks were given an intravenous injection of 0.5 × 106effLuc-eGFP NALM6 cells. Four days later, mice were injected with a dose of 0.1 × 106CD19-specific CAR-T cells via tail vein injection. Tumor growth was monitored via bioluminescence imaging (BLI) using a Xenogen IVIS Spectrum Imaging System (PerkinElmer). Mice were administered 3 mg of D-luciferin (Gold Biotechnology) dissolved in DPBS via intraperitoneal injection and were imaged 10 min later. Randomization to CAR-T cell treatment groups was based on initial imaging results to maintain similar cancer burden across groups prior to CAR-T therapy. For the NALM6 rechallenge experiments, mice received 3 x106NALM6 cells via tail vein injection on days 44, 51, 58, and 65 following CAR-T cell injection. In vivo heterotopic tumor models. Recipient male and female mice had their right flank shaved two days prior to tumor inoculation. MC38 (5 x 105), MC38-OVA (5 x 105), B16F10 (1 x 105) or CT26 (5 x 105) tumor cells were injected subcutaneously (s.c.) in 100 μL PBS. For B16F10, tumor cells were mixed 1:1 in PBS:Matrigel (Corning). Bidirectional tumor size was measured every 3 days with digital calibers, and tumor volume was calculated as length x width2 / 2. Experimental endpoint was achieved when length or width reached greater than 2 cm, or when tumors demonstrated significant ulceration. For A20ZF7 / ZF7regressor rechallenge, 60-80 days after initial MC38 inoculation, left flanks of regressor mice or age-matched A20ZF7 / ZF7mice naive to tumor were shaved two days prior to inoculation. MC38 (2 x 106cells) were then injected s.c. CD8+T cell antibody-mediated depletion. For antibody-mediated CD8+T cell depletion in MC38-bearing A20ZF7KI mice, 500 μg anti-CD8α (Clone 2.43; BioXCell) was injected intraperitoneally (i.p.) two days prior to tumor inoculation. On day 5 after tumor inoculation, and every 7 days thereafter until experimental endpoint, mice received an additional 250 μg anti-CD8α i.p. In parallel, a separate group of control mice received isotype control antibody (Clone LTF-2; BioXCell) instead of anti-CD8α. CD8+T cell depletion in blood was confirmed by flow cytometry 14 days after tumor inoculation. CD8+T cell reconstitution of Cd8a- / -mice. For reconstitution of congenic Cd8a- / -(CD45.1 / 1) mice with CD8+T cells, Cd8a- / -recipient mice underwent sublethal X-ray irradiation (one dose of 450 cGy; X-Rad320; Precision X-Ray Irradiation). In parallel, naive polyclonal CD8+T cells were isolated from either A20+ / +, A20ZF7 / +or A20ZF7 / ZF7mice (CD45.2 / 2) as above. Three hours after irradiation, 3-8 x 106naive CD8+T cells were injected intravenously into recipients. After 12 days, and two days prior to tumor inoculation, engraftment was confirmed by flow cytometry on blood cells. In one experiment, recipient mice were euthanized 14 days after tumor inoculation, and tumors were analyzed by flow cytometry to confirm CD8+TIL reconstitution. OT-I cell adoptive transfer. For adoptive transfer of naive OT-I cells, isolation of OT-I cells was performed as above, and 1 x 106cells were injected intravenously into recipient mice one day prior to MC38-OVA inoculation. For adoptive transfer of pre-activated OT-I cells, total splenocytes harvested from OT-I mice were cultured at 2 x 106cells / ml in cRPMI containing 1 μg / ml OVA257-264peptide (Invivogen) for 48 h. Cells were then counted, washed and replated at 2 x 105cells / ml in fresh cRPMI containing 50 μM β-mercaptoethanol (Sigma-Aldrich) and 50 IU / ml human IL-2 (hIL-2; NCI BRB Preclinical Biologics Repository), and expanded for an additional 48 h. Finally, 2.5-10 x 106pre-activated A20+ / +or A20ZF7 / ZF7OT-I cells were injected intravenously into MC38-OVA-bearing mice on day 7 after tumor inoculation. For adoptive co- transfer experiments, recipient MC38-OVA-bearing WT mice (CD45.1 / 2) received equal numbers (2.5 x 106each) of A20+ / +(CD45.1 / 1) and A20ZF7 / ZF7(CD45.2 / 2) pre-activated OT-I cells on day 7 after tumor inoculation, and tumors were subsequently analyzed by FSFC on day 14 after tumor inoculation. Bone marrow chimera generation. Recipient mice (A20+ / +or A20+ / -; CD45.1 / 1) were treated with two doses of 450 cGy X-ray irradiation given three hours apart. In parallel, bone marrow was isolated from hindleg bones of congenic donor mice (A20+ / +or A20+ / -; CD45.2 / 2), lysed for 30 seconds in ACK lysis buffer (Quality Biological), quenched in excess PBS, filtered through 70 μm nylon mesh, and pelleted. Bone marrow from each mouse was resuspended in PBS and injected intravenously into recipients one hour after the last dose of irradiation at a donor:recipient ratio of 1:3. Six weeks after bone marrow transplant, chimerism was assessed by flow cytometry on blood cells, after which mice were inoculated with MC38 tumor cells. Lentiviral production and transduction. The day before transfection, Lenti-X 293T cells (Takara Bio) were seeded at 23 x 106cells per 225 cm² dish coated with poly-L-lysine (MilliporeSigma) in Opti-MEM (Thermo Fisher) supplemented with 5% FBS, 1X MEM Non- Essential Amino Acids (Thermo Fisher), and 1mM Sodium Pyruvate. The next day, 293T cells were transfected with transfer plasmids along with second-generation lentiviral packaging plasmids, pMD2.G (Addgene#12259) and psPAX2 (Addgene#12260), using the Lipofectamine 3000 reagent (Thermo Fisher), following the manufacturer's protocol. Six hours after transfection, the medium was replaced with fresh media plus ViralBoost Reagent (500X) (Alstem). Viral supernatants collected after 24 and 48 hours were centrifuged at 500xg at 4°C for 10 min to remove debris and concentrated using Lenti-X concentrator (Takara Bio) per manufacturer’s recommendations, then stored at 4°C overnight. The virus was further concentrated by centrifugation at 1500xg for 45 min at 4°C and resuspended in Opti-MEM with no additives at 100X the original volume, and then stored at −80°C. For T cell transduction, concentrated lentivirus was added 24 hours post-TCR activation to T cells at a 1:25 volume-to- volume ratio cultured in T cell media and gently mixed by tilting the culturing vessel. Adeno-associated virus (AAV) production and transduction. The AAV cargo plasmid containing an HDR template for a TRAC CAR was co-transfected with packaging plasmids into HEK293T cells using polyethylenimine (PEI) to produce AAV6 particles, which were subsequently purified using iodixanol gradient ultracentrifugation. Viral titers were quantified through quantitative PCR analysis performed on AAV samples treated with DNase I (NEB) and digested with proteinase K (Qiagen). HDR templates targeting TRAC were quantified using primers specific to the left homology arm of the HDR template. Quantitative PCR was conducted using the SsoFast EvaGreen Supermix (Bio-Rad) on a StepOnePlus Real-Time PCR System (Applied Biosystems). CBE mRNA production. An in vitro transcription (IVT) plasmid was constructed that contained evoCDA1-BE4max along with a mutated T7 promoter. IVT templates were generated through PCR amplification of evoCDA1-BE4max, utilizing a forward primer to correct the T7 mutation and a reverse primer to add a poly-A tail. The resulting PCR product included the wild- type (WT) T7 promoter, a 5′ untranslated region with a Kozak sequence, the codon-optimized evoCDA1-BE4max coding sequence, a 3′ untranslated region, and a 145-bp poly-A tail. After purification, the PCR product was stored at −20°C until use. IVT reactions were conducted using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs), substituting UTP with N1-Methylpseudouridine-5'-Triphosphate (Trilink Biotechnologies) and the addition of 4 mM CleanCap (TriLink Biotechnologies). The transcribed mRNA was purified using lithium chloride and eluted in RNA storage solution (Fisher Scientific). The mRNA product was analyzed using an Agilent 4200 Tapestation system and subsequently stored at −80°C. CRISPR editing of primary human T Cells. Lyophilized sgRNA (Synthego) was rehydrated in TE buffer (Synthego) to a concentration of 80 μM for Cas9 editors and 120 μM for Cas12a editors. 48 hours after activation, T cells were de-beaded using EasySep magnets (STEMCELL) and electroporation of Cas9–sgRNA–RNP was conducted using the Amaxa P3 Primary Cell 96-well 4D-Nucleofector Kit (Lonza). For Cas9-edited T cells, sgRNAs were used to target AAVS1 and TNFAIP3 (where guides were designed to either disrupt expression of the entire gene (A20KO), or to cause a truncation that eliminates the A20 ZF7 domain (the last exon) (A20ZF7). For targeted insertion of the CAR into the TRAC locus, sgRNA for Cas9 or crRNA sequences for Cas12a were used. These sgRNAs were mixed with Cas9 or Cas12a protein (stock concentration 40 μM, Q3 MacroLab / UC Berkeley) at a molar ratio of 2:1 (sgRNA:Cas) and incubated at a temperature of 37°C for 15 min. 3 μl of the resulting RNP was mixed with 2 x 106T cells that had been resuspended in 20 μL of P3 and transferred into a 96-well electroporation plate using the pulse code EH115. T cells were rapidly recovered by addition of 100 μL of T cell media and incubated at 37°C for 15 min. Cells were then resuspended in T cell media at a concentration of 2 x 106live T cells per ml with the addition of IL-7 and IL-15 and transferred to appropriate culture vessels. TRAC targeting of CD19 CAR construct. For AAV-mediated CRISPR knock-in of CD19 CAR into TRAC locus, cells were edited as described above. The TRAC locus was targeted utilizing sgRNAs. The recombinant AAV6 donor vector was added 30 min after electroporation at a multiplicity of infection of 5 x 104, with an overnight incubation in serum-free T cell growth medium. The following day, edited cells were resuspended in complete T cell growth medium, expanded first at a density of 2 x 106cells / ml, and maintained at 1.5 x 106cells / ml. Flow cytometry was used to evaluate the knock-in efficiency through CAR staining with an anti-G4S antibody (Cell Signaling Technologies). Base-editing. 48 hours after activation of human T cells with anti-CD3 / CD28 Dynabeads (ThermoFisher), cells were de-beaded and resuspended in P3 buffer plus supplements (Lonza) at 2 x 106cells per 20 μL. 2 µg of CBE mRNA along with 60 nmols of synthetic modified sgRNA (Synthego) targeting either an intronic region of beta-2 microglobulin , A20, or the A20ZF7motif (sgRNAs specified below). were then added to the resuspended cells. Cells were then electroporated and cultured as described in the Cas9-RNP electroporation section. Genome-wide CRISPR / Cas9 screening. T cells were isolated and stimulated as previously outlined, and 24 hours later were transduced with a lentiviral pool containing the genome-wide Brunello sgRNA library at a volume to volume (v / v) ratio of 1:500 to achieve a targeted transduction efficiency of 50%. The human Brunello CRISPR knockout pooled library was a gift from David Root and John Doench (Addgene #73178). Following this transduction, the T cells were washed with PBS and electroporated with Cas9-RNP containing a non-targeting sgRNA (sequence specified below) (Horizon Discovery), and subsequently cultured as described. On day 12, T cells were stained with Dextramer-HLAA*0201 / SLLMWITQV-APC (Immudex) and the percentage of TCR positive cells was determined by flow cytometry. These modified T cells were then co- cultured with A375 melanoma cells at an effector to target (E:T) ratio of 1:1 in complete T cell growth medium with the addition of 100 IU / ml IL-2 (R&D Systems). After 9 rounds of exposure to fresh tumor cells every 48 to 72 hours, cells were collected and genomic DNA was isolated using the NucleoSpin Blood XL kit (Macherey-Nagel). Genomic DNA concentration was quantified using Invitrogen 1X dsDNA High Sensitivity assay kit (Thermo Fisher) on a Qubit Fluorometer (Invitrogen). PCR of the amplicon containing the guide sequences was carried out using Ex Taq DNA Polymerase (Takara Bio) and P5 / P7 primers (IDT) (Ref: https: / / media.addgene.org / cms / filer_public / 61 / 16 / 611619f4-0926-4a07-b5c7- e286a8ecf7f5 / broadgpp-sequencing-protocol.pdf). The resulting amplicons were purified using SPRIselect Beads (Beckman-Coulter) and QC was conducted using a D1000 ScreenTape assay on a TapeStation (Agilent) prior to next-generation sequencing. Samples were pooled and sequenced on a NovaSeq X system at The Center for Advanced Technology (CAT) at UCSF. MAGeCK52 v0.5.9.5 was used to quantify guide counts in each sample and tested for guide enrichment. Paired robust rank aggregation (RRA) analysis of guide count data from two donors was performed using the default parameters. Samples of T cells collected before co- culture with tumor cells (Time-Zero [T0]) were compared to samples collected after 4 (Time- Intermediate [TI], 1 donor), and 9 rounds of co-culture (Time-Final [TF]) as well as an age- matched arm not exposed to tumor cells [TFctrl]. Guides with a read count of < 40 in the control samples were filtered out. Guide RNAs. The sequences of the guide RNAs used in the following work are shown below: TRAC gRNA sequence: 5’-CAGGGUUCUGGAUAUCUGU-3’ (SEQ ID NO:1) A20 KO gRNA sequence: 5’-UGAUGAAUGAUCCCAUUAGU-3’ (SEQ ID NO:2) A20 ZnF7 ablation gRNA sequence: 5’- GGCAUUGCCAAAAUGAUCAC-3’ (SEQ ID NO:3) Cas12a TRAC gRNA sequence 5’- GAGUCUCUCAGCUGGUACAC-3’ (SEQ ID NO:4) A20 KO CBE gRNA sequence 1 sgLRS009 5’- UUCCAGUUCUAAGGGGAGCG-3’ (SEQ ID NO:5) A20 KO CBE gRNA sequence 2 sgLRS010 5’- CGUCCAAGGCUGGGACCAUG-3’ (SEQ ID NO:6) A20 ZnF7 ablation CBE gRNA sequence 1 sgLRS005 5’- CCCGGCAACGCUGCUUGGGG-3’ (SEQ ID NO:7) A20 ZnF7 ablation CBE gRNA sequence 2 sgLRS007 5’- GUUGCAGUAGCCGUUGCACU-3’ (SEQ ID NO:8) Primers for gDNA amplification A20 Exon 4 Forward 5’- GTGCCTCACCATCCTCAGTC-3’ (SEQ ID NO:9) Reverse 5’- GGCTCCTGGAGAAAACCACA-3’ (SEQ ID NO:10) Human T cell repetitive stimulation assay. For repetitive stimulation of human TCR-T or CAR-T cells, the day before setting up the assay, A375 or A375-CD19 cells were seeded in cRPMI. The following day, the medium was replaced with complete T cell growth medium, and antigen-specific T cells were added on top of the tumor cells at a 1:1 effector-to-target (E:T) ratio, with IL-2 added at a concentration of 100 IU / ml. Subsequent co-cultures were conducted every 48 to 72 hours. For each co-culture, T cells were collected, counted with the Cellaca MX High-throughput Cell Counter (Revvity), and then replated onto fresh tumor cells at the same 1:1 E:T ratio. Murine T cell repetitive stimulation assay. Mouse OT-I repetitive stimulation assays were performed as previously described (Wu Wherry 2023 Sci Immunol). Briefly, splenocytes were harvested from WT mice by mechanically disaggregating spleens through a 70 μm nylon filter and pelleting. Erythrocytes were lysed in ACK Lysing Buffer for 30 seconds, after which excess PBS was added and cells were again filtered and pelleted. DCs were isolated via EasySep Mouse Pan-DC Enrichment Kit (STEMCELL) by following the manufacturer's protocol. DCs were counted, resuspended in cRPMI at 1 x 106cells / ml, plated in 96-well flat-bottom plates, and pulsed with 1 μg / ml OVA257-264peptide for 30 min at 37°C. Next, naive OT-I cells isolated as above were added at a 1:1 ratio. After 2, 4 and 6 days, remaining OT-I cells were counted and replated at 1 x105cells / ml in cRPMI containing 50 μM β-mercaptoethanol, 50 IU / ml hIL-2 and fresh 1 μg / ml OVA257-264peptide. On day 8, remaining OT-I T cells were collected for flow cytometry analysis. Cytokine withdrawal and antigen-dependence. CAR-T cells were cultured in complete T cell growth medium without the addition of cytokines and either stimulated once on day 0 with CD19+A375 melanoma cells at an effector-to-target (E:T) ratio of 1:1 or left unstimulated. For the stimulated arm, proliferation of CAR+cells was monitored over a 25-day period by automated cell counting with a Cellaca MX High-throughput Cell Counter (Revvity) using AO / PI staining solution (Revvity). CAR+cell percentages were evaluated alongside every count by flow cytometry using an anti-G4S antibody (Cell Signaling Technologies). The growth rates of CAR+cells were calculated, and the fold change relative to the initial count was determined. In the unstimulated arm, total T cell counts were used to assess proliferation and viability using AO / PI over a 15-day period and the fold change was calculated based on the initial count. Flow cytometry of human T cells in vitro. For in vitro analysis of cell surface activation markers, TCR-T and CAR-T cells (2 x 105to 5 x 105) were seeded into round-bottom 96-well plates (Corning). TCR-T cells were then stimulated with anti-CD3 / CD28 Dynabeads, and CD19 CAR-T cells were stimulated with CD19+A375 melanoma cells at a bead / cell to cell ratio of 1 to 1 at 37°C for 6 hours. Cells were then centrifuged at 300g for 5 min, washed once with 200 μL of FACS buffer, and stained with the appropriate antibodies (1 μL per 100 μL staining buffer) for 20 min at 4°C in the dark. Cells were then washed twice with FACS buffer. For exhaustion and differentiation markers, cells were stained without prior stimulation following the same approach. Samples were analyzed using the Attune NXT Cytometer (Invitrogen) or LSRFortessa X-50 (BD) and data were processed and analyzed using FlowJo 10.9.1 (FlowJo, Inc.). For compensation controls, UltraComp eBeads compensation beads (ThermoFisher) were used. Antibodies used for staining are specified in key reagents table. Flow cytometry of human CAR-T cells ex vivo. For analysis of bone marrow, NALM6- bearing mice were euthanized 14 days after CAR-T cell injection. For bone marrow extraction, the femur and tibia from each leg were dissected and crushed in MACS buffer consisting of PBS (Gibco), 2% FBS and 1mM EDTA using a mortar and pestle. The resulting cell suspension was filtered through a 70 μm cell strainer (Corning), centrifuged at 300xg for 5 min, and red blood cells were lysed using ACK lysing buffer (Quality Biological) for 2 min, with the reaction subsequently quenched using MACS buffer. Each sample was first resuspended in 500 μL of MACS buffer. 250 μL of each sample were used for analysis, centrifuged, and resuspended in 100 μL of FACS buffer. Cells were first incubated with 10 μL of mouse Fc block (Miltenyi Biotec) per sample and incubated for 20 min at room temperature. The cells were then stained with the appropriate antibody mix (see key reagents table) and incubated for 45 min at room temperature (RT). After staining, the cells were washed, resuspended in 150 μL of FACS buffer with 50 μL of counting beads per sample (Thermo Fisher), and analyzed on an Aurora spectral flow cytometer (Cytek). Data were processed and analyzed in FlowJo. SPICE plots were generated using SPICE 6.1. Multiplex immunoassay. Before and after several rounds of co-culture with tumor cells, 2 x 105TCR T or CAR-T cells were resuspended in 200 μL fresh complete T cell growth medium without cytokines. TCR-T cells were seeded on top of A375 melanoma cells at an effector to target ratio of 1 to 1 or stimulated with CTS Dynabeads Human T-Cell Activator CD3 / CD28 at a cell to bead ratio of 1 to 1. CAR-T were seeded on top of CD19+A375 melanoma cells at an effector to target ratio of 1 to 1. 24 hours later, the supernatant from the co- cultures was harvested and spun for 5 min at 300g at 4 °C to remove cell debris. The collected supernatant was then stored at -80 °C and subsequently analyzed using the LEGENDplex Human CD8 / NK Panel (13-plex) (Biolegend) as per the manufacturer’s guidelines. Results were analyzed using the LEGENDplex analysis tool (Biolegend). When necessary, the supernatant was diluted, and the resulting concentration was multiplied by the dilution factor to obtain the absolute concentration in pg / ml. Full spectrum flow cytometry of murine TILs. For FSFC analysis of heterotopic tumors in immunocompetent mice, tumor-bearing mice were euthanized and tumors were harvested. Tumors were digested for 30 min at 37°C in RPMI 1640 containing 0.13 Wunsch units / ml Liberase TM (Roche) and 72 μg / ml DNase I (Roche), with intermittent agitation. Tumors were pelleted and resuspended in FSFC buffer (PBS + 2% FBS + 5 mM EDTA) and incubated for an additional 15 min at 37°C, after which samples were filtered through 70 μm nylon cell strainer to create a single cell suspension. Samples were then stained for 15 min at room temperature (RT) in PBS containing Zombie NIR live / dead stain (BioLegend), then washed in FSFC buffer. Samples were then treated with Fc receptor blocking reagent (10 μg / ml anti-CD16 / CD32; BioXCell) in FSFC buffer containing a 1:10 dilution of Brilliant Stain Buffer (Thermo Fisher) for 10 min at RT, after which surface antibody staining cocktail was added and samples were incubated for an additional 20 min at 37°C. Samples were washed in FSFC buffer, then fixed and permeabilized for 40 min at RT using eBioscience Foxp3 Transcription Factor Staining Kit (Thermo Fisher). Samples were then blocked for 10 min at RT in Foxp3 kit buffer containing 1:10 dilution of Brilliant Buffer and 2% rat serum (Jackson ImmunoResearch), after which intracellular antibody staining cocktail was added and samples were incubated an additional 30 min at RT. Samples were washed, resuspended in FSFC buffer, and acquired on the Cytek Aurora instrument. Quality control of data was performed using SpectroFlo software (Cytek) before subsequent analysis using FlowJo. SPICE plots were generated using SPICE 6.1. Flow cytometry of murine blood leukocytes. Mice were anesthetized with isoflurane and underwent retro-orbital phlebotomy with heparanized capillary tubes (Thermo Fisher). Collected blood cells were pelleted and lysed for 15 min in ACK buffer, quenched in excess PBS, filtered via 70 μm cell strainer, and pelleted before performing live / dead and surface stain as above. Intracellular cytokine staining of murine TILs. For analysis of cytokine production in mouse CD8+TILs, tumors were harvested and processed into single cell suspensions as above, then plated at 1x106cells / ml in cRPMI containing 50 ng / ml PMA (Sigma-Aldrich) and 500 ng / ml ionomycin (Sigma-Aldrich) and incubated at 37°C. After 1 hour, 5 μg / ml brefeldin A (BioLegend) was added, and cells were incubated an additional 4 h at 37°C. Cells were subsequently pelleted and submitted to live / dead and surface stain as above. Cells were then fixed, permeabilized, and stained for cytokines and perforin using the Cytofix / Cytoperm kit (BD) per manufacturer's protocol. In vivo brefeldin treatment. Six hours prior to euthanization, day 12-15 MC38-bearing mice were given 500 μL i.p. of PBS containing 250 μg brefeldin (Sigma-Aldrich) or 5% DMSO (vehicle control). Mice were closely monitored for six hours and showed no visible signs of distress. In vitro killing assay. Killing assays were performed using TCR-T and CAR-T cells, where antigen-specific T cells were co-cultured with pre-plated mKate+A375 melanoma cells in a 96-well flat-bottom plate (Corning). T cells were seeded in various effector-to-target (E:T) ratios. For the TCR-T assays, mKate+A375 melanoma cells (ATCC) were utilized, whereas for the CAR-T assays, CD19-expressing mKate+A375 cells were used (generated by the Marson lab at Gladstone Institutes). Plates were imaged every 4-6 hours for at least 72 hours using the IncuCyte S3 live-cell imaging system (Essen Bioscience). The red object counts of mKate+ objects per well were recorded over time. Cancer cell growth was calculated at each time point for each replicate by normalization to the red object count at time zero. Proliferation assay. TCR-T or CAR-T cells were resuspended in PBS (Gibco) and stained with the fluorescent dye CellTrace Violet (Thermo Fisher Scientific) at a working concentration of 5 µM, following the manufacturer's protocol. After staining, TCR-T cells were resuspended in 200 µL complete T cell growth medium and stimulated with 3.125 µL / mL of ImmunoCult Human CD3 / CD28 T Cell Activator (STEMCELL) for 72 hours. For stimulation of CAR-T cells, CD19+A375 melanoma cells were used. Flow cytometry was used to measure cell division percentages across conditions and analyzed with the proliferation modeling tool in FlowJo 10.9.1. Immunoblotting. For activated conditions only, A20 modified and control T cells were stimulated for 24 hours with CTS Dynabeads Human T-Cell Activator CD3 / CD28 (Thermo Fisher). Frozen cell pellets were resuspended in Pierce RIPA buffer (Thermo Fisher) plus complete protease inhibitor (Roche) and incubated on a rotator at 4 °C for 30 min and spun down at >16,000 g for 20 min at 4 °C. The cell lysates were stored at -80 °C if not used immediately. Protein concentrations were measured using the Pierce BCA Protein Assay (Thermo Fisher). 20 µg of protein from each sample were loaded onto 4–20% tris-glycine SDS gels (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad) using the Bio-Rad Trans-Blot Transfer System. The membrane was blocked with AdvanBlock-Chemi solution (Advansta) for 1 hour at RT and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were utilized: A20 / TNFAIP3 antibody (1:1000 dilution, Santa Cruz), β-actin rabbit monoclonal antibody (1:5000 dilution, Abclonal). For secondary antibodies, anti-rabbit IgG HRP-linked antibody (1:2000 dilution, Cell Signaling) and anti-mouse IgG HRP-linked antibody (1:2000 dilution, Cell Signaling) were used. Membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and imaged on a ChemiDoc Touch Imaging System (Bio-Rad). Sanger sequencing. Frozen cell pellets were resuspended in QuickExtract DNA Extraction Solution (Biosearch Technologies) at a concentration of 100 x 105cells per 50 µL of solution. gDNA was extracted following instructions according to manufacturer’s protocol. 5 µL of resulting solution was used as input template for a PCR reaction using Phusion High-Fidelity DNA Polymerase (NEB) to survey Exon 2, 5, 9 at the TNFAIP3 locus, and AAVS1. Resulting amplicons were purified using SPRI selection beads (Beckman Coulter) at 2X concentration following kit instructions. Purified amplicons were submitted to Quintara Biosciences for sanger sequencing using the forward and reverse primer used for the PCR amplification. Indel % and knock-out (KO) score (proportion of indels that are at least 21 bp long or indicate a frameshift) were analyzed with the Synthego ICE analysis tool. Next-generation sequencing. Cell pellets and gDNA extraction were prepared as above. 5uL of extraction solution was used as input for the PCR reaction to survey TNFAIP3 Exon 2, TNFAIP3 Exon 9 (A20ZF7domain), and B2Mi loci. The PCR reaction is comprised of Q5 High- Fidelity 2X Master Mix (New England Biolabs), loading 5 µL of the gDNA extraction in a reaction volume of 50 µL, and following recommended cycling conditions from manufacturer for 32 cycles. Resulting amplicons were purified using SPRI selection beads (Beckman Coulter) at 2X concentration following the kit instructions. Amplicons were analyzed with Agilent High Sensitivity D5000 ScreenTape (Agilent) on a TapeStation (Agilent) to quantify expected product sizes. Purified amplicons were submitted to Quintara Biosciences for next-generation sequencing. Human cancer transcriptomic dataset analyses. For analysis of TNFAIP3 expression in human tumor microenvironment cell subsets (data not shown), single cell RNA-sequencing datasets were collected, normalized, curated and annotated by TISCH2. All raw data was converted to transcripts per million (TPM) and depicted in a heatmap as the mean expression values of TNFAIP3 in different cell types of different datasets. All datasets that included, at a minimum, values for both malignant cells and immune cells were selected. For correlation of TNFAIP3 expression with exhaustion markers in human tumor infiltrating lymphocytes (data not shown), bulk RNA sequencing data from sorted intratumoral conventional T cells (CD45+CD3+CD8+and CD45+CD3+CD4+CD25-) was analyzed from the UCSF Immunoprofiler database as previously described. Briefly, datasets were downloaded from NCBI GEO (GSE184398). Exhaustion gene signature scores for all samples across all cancer subtypes were determined from an 11-gene set (AFAP1L2, AKAP5, CBLB, FUT8, HAVCR2 [TIM-3], LAG3, NUSAP1, SYT11, TNFSF4 [OX40L], TNFRSF9 [4-1BB], and TOX) after gene expression was converted to percentile ranks across samples, and exhaustion score for each samples was calculated as the average of percentiles across the 11 genes. Quantifications and statistical analyses. Details for the statistical analyses for all experiments are provided in the figure legends. Plots were generated in GraphPad Prism. Paired t tests of in vitro data from 2-3 human donors do not account for correlation among the technical replicates and do not include corrections for multiple comparison. The survival outcomes for mice are depicted through Kaplan-Meier curves. Screen analysis was done with MAGeCK in R and visualization with the package ggplot2, with editing to enhance clarity. Illustrations were created in Adobe Illustrator. Results A genome-wide CRISPR perturbation screen identifies A20 / TNFAIP3 as a key regulator of T cell dysfunction after chronic antigen stimulation. The ability to repeatedly eradicate tumor cells during sustained tumor antigen exposure is an indispensable characteristic of therapeutic T cells. To reveal genes that have the potential to enhance T cell persistence and deter dysfunction, an in vitro system that models chronic exposure to tumor antigen was employed. Human T cells were engineered to express the NY- ESO-1 specific TCR (1G4), and then half of these 1G4 TCR-T cells were exposed to repeated co-culture with fresh tumor cells (data not shown), while the other half were not exposed to tumor cells but otherwise cultured similarly for the same duration. After multiple rounds of tumor exposure, the ability of TCR-T cells to eradicate tumor cells was assessed. Notably, TCR- T cells matched for time in culture, without prior exposure to antigen, exhibited robust tumor- killing capability, while TCR-T cells which were continuously exposed to tumor antigen displayed a substantial decline in their capacity to control tumor cell growth after each successive round of tumor stimulation (data not shown). Cell surface inhibitory receptors (IRs) whose expression are associated with T cell exhaustion (LAG-3, TIM-3, PD-1, and CD39) increased as TCR-T cells were repeatedly exposed to tumor cells, with the difference between tumor-exposed and unexposed TCR-T cells growing progressively over successive rounds of stimulation (data not shown). The capacity of these TCR-T cells to secrete effector cytokines (IL-2, IFNγ, TNF) and cytotoxic mediators (granzyme B (GzmB), soluble Fas ligand (sFasL), and perforin) decreased after several rounds of exposure to tumor cells (data not shown), providing further evidence of dysfunction from chronic antigen stimulation. Altogether, these observations provide evidence that the chronic antigen stimulation models key aspects of T cell dysfunction and has the potential to nominate gene modifications that overcome these functional limitations. To identify genes with the potential to enhance T cell persistence and long-term effector function, a genome-wide CRISPR knockout screen using this repetitive tumor stimulation system was performed. Primary human T cells from two human donors were transduced with the 1G4 TCR along with a genome-wide single-guide RNA (sgRNA) library, and Cas9 protein was introduced by electroporation. These T cells were then exposed to tumor cells over nine successive rounds (FIG. 1A), maintaining an effector to target (E:T) ratio of 1:1 each time until the bulk TCR-T cells could no longer effectively eliminate the tumor cells, indicating a reduction in their functional capacity. TCR-T cells were collected before initial exposure to tumor cells (Time-Zero [T0]), after 4 four rounds of stimulation (Time-Intermediate [TI]), and after 9 rounds of stimulation (Time-Final [TF]) for next-generation sequencing to identify sgRNAs enriched in TCR-T cells after repeated tumor exposure. A parallel set of pooled edited TCR-T cells was kept in culture for the duration of the screen but not exposed to tumor cells [TFctrl]. Using MAGeCK analysis, numerous gene targets were identified with sgRNAs that were enriched in the TF compared to T0 screen conditions, many of which have been previously targeted to enhance T cell fitness and / or counter T cell exhaustion. For instance, RASA2, CISH, DNMT3A, MAPK14, and members of the Mediator, Cullin-5, and SWI / SNF complexes were found to be enriched in the TF compared to T0 screen conditions (FIG. 1B). TNFAIP3, which encodes the ubiquitin- binding adaptor and modifying enzyme A20, displayed one of the highest enrichments of its associated sgRNAs in comparison to the baseline count (rank 4) (FIG. 1B), and exhibited high correlation between both T cell donors (data not shown). When the TFctrl was compared to the baseline T0 sample, A20 / TNFAIP3 (hereafter referred to as A20) was ranked 4718th (Fi data not shown), suggesting that the high rank in TF compared to T0 baseline was specific to the repeated tumor stimulation. A sample sequenced at an earlier timepoint in the screen [TI] and compared to T0 showed A20 was ranked 70th at that time point (data not shown), suggesting that there is a relative increase in the A20 persistence advantage with increasing chronic stimulation pressure. The potential of targeted A20 ablation to enhance the functional persistence and anti- tumor activity of human T cell therapies was further investigated. Primary human T cells were engineered to express the 1G4 TCR and edited using RNP-Cas9 electroporation with a sgRNA targeting A20 (a new sgRNA not included in the screen library, sgRNA-1, targeting exon 2) or a safe harbor locus (AAVS1) as a control. Sequencing analyses of edited T cells from multiple donors confirmed the high editing efficiency (indel ~88-93%) of the sgRNA targeting A20, as well as successful editing of the AAVS1 locus (data not shown). Upon initial re-stimulation, A20-targeted (A20KO) cells demonstrated a higher percentage of dividing cells when compared to control AAVS1-edited cells, suggesting increased proliferative capacity (data not shown). While both the A20KOand control AAVS1 TCR-T cells demonstrated robust tumor-killing activity upon initial co-culture, the A20KOTCR-T cells demonstrated substantially improved recursive killing of cancer cells compared to control cells, the latter of which failed to control cancer cell growth after multiple rounds of tumor exposure (FIG. 1C and data not shown). Additionally, repetitively stimulated A20KOcells secreted significantly higher levels of effector cytokines (IL-2, IFNγ, TNF) and effector proteins (perforin, GzmB, sFasL) when compared to control cells (FIG. 1D). Overall, these findings suggest that A20KOT cells resist functional decline induced by chronic antigenic exposure, thereby maintaining enhanced tumoricidal ability and production of cytotoxic proteins. To investigate whether the functional enhancements of A20KOT cells observed in vitro translate to improved in vivo performance, therapeutically relevant CD19-specific CAR-T cells were used. The ability of A20KOCD19-CAR-T and control CD19-CAR-T cells to control human NALM6 B cell leukemia growth in immunodeficient NOD-scid IL2Rγnull(NSG) mice was tested. TRAC-CAR-T cells were generated, where a CD19 CAR with a CD28 costimulatory domain is integrated into the TRAC locus, which has previously shown to be an optimized best- in-class CAR-T cell therapy, vastly outperforming conventionally generated CAR-T cells. These cells were additionally edited with CRISPR / Cas9 RNPs using either A20KOor control AAVS1 sgRNAs. Sequencing analyses of edited CAR-T cells confirmed a high frequency (indel % ~95%) of A20 editing as well as successful editing of the AAVS1 locus (indel % ~62) (F data not shown). A20KOor control AAVS1 CD19 TRAC-CAR-T cells were injected into NSG mice four days after they were engrafted with 5 x 105luciferase-expressing GFP+NALM6 leukemia cells (FIG. 1E). The CAR-T cells were introduced at a low dose (1 x 105CAR+cells / mouse) designed as a “stress-test” to evaluate CAR-T cell efficacy at controlling a high tumor challenge. Bioluminescence imaging (BLI) was then used to serially monitor NALM6 growth in these mice. BLI analyses revealed that most mice receiving the control AAVS1 CAR-T cells succumbed to NALM6 within three weeks (FIGS. 1F and 1G). Conversely, mice treated with A20KOCAR-T cells at the same dose showed greatly improved tumor control as well as prolonged survival when compared to the control group (FIGS. 1F and 1G). These in vivo findings were validated using multiple T cell donors and a different sgRNA (sgRNA-2, targeting exon 5) to ablate A20 expression (data not shown). To understand how A20 regulates the behavior of these CAR-T cells, A20KOand control AAVS1 CAR-T cells were isolated from the bone marrow of NALM6- engrafted mice and these cells were profiled via flow cytometry. Less than 30% of A20KOCAR- T cells co-expressed two or more IRs across donors, compared to more than 40% of control AAVS1 CAR-T cells (FIG. 1H). These findings suggest that A20KOCAR-T cells persist in more functional states than control CAR-T cells. A trend toward increased numbers of A20KOCAR-T cells compared to control cells was observed, suggesting increased proliferation or survival of these cells (data not shown). A20 ablation did not substantially alter CAR-T cell memory phenotypes (data not shown). Overall, A20 ablation in human CAR-T cells potently enhances their ability to suppress leukemia growth in vivo. To gain insight into the potential clinical implications of A20 expression in human cancer patients, A20 / TNFAIP3 expression was analyzed in available transcriptomic profiling datasets of human cancers. It was found that A20 expression is selectively elevated in immune cell subsets, including T cells, within the tumor microenvironment (TME) of multiple human cancer subtypes compared to non-immune cells (data not shown). As A20 restrains TCR induced NF-kB signaling, the increased levels of A20 in TME immune cells may suppress their activation and thereby inhibit anti-tumor immune responses. To test whether modulating A20 expression levels can limit anti-tumor immunity in an immunocompetent model, the growth of MC38 – a widely used colon adenocarcinoma derived from C57BL / 6 inbred mice and considered to be a “hot” tumor that responds well to immunotherapy – was compared in heterozygous A20+ / -mice and littermate control wild-type (WT; A20+ / +) mice. Of note, A20+ / -mice appear grossly normal without spontaneous inflammation, but have reduced A20 expression and increased susceptibility to experimentally-induced inflammation; homozygous A20- / -mice die perinatally and are hence not suitable for testing anti-tumor responses. Interestingly, A20+ / -mice consistently controlled MC38 growth better than A20+ / +littermates (FIGS. 1I-1J and data not shown). Thus, A20 haploinsufficiency enhances anti-tumor immunity. Next, the anti-tumor efficacy of A20+ / -hematopoietic cells was tested by inoculating MC38 cells into radiation chimera reconstituted with either A20+ / -or A20+ / +bone marrow cells. Chimeras bearing A20+ / - hematopoietic cells suppressed MC38 tumor growth better than chimeras containing A20+ / +hematopoietic cells (FIGS. 1K-1L and data not shown). Together, these findings suggest that A20 expression is elevated within TME immune cells, and that reduction of A20 expression in radiation sensitive hematopoietic cells enhances anti-tumor immunity. Inactivation of the zinc finger 7 domain of A20 invigorates anti-tumor immunity. Complete loss of A20 from cells can perturb both cellular activation and cell death responses in distinct cell types and differentiation states. These disparate outcomes reflect the ability of A20 to regulate several distinct ubiquitinated signaling complexes. The A20 protein contains several structurally defined biochemical motifs that perform distinct biochemical functions (FIG. 2A). Selective mutation of individual A20 motifs may thus differentially impact T cell functions such as activation, differentiation, cytokine secretion, and / or survival. Accordingly, it was desired to investigate the domains and associated biochemical functions of A20 that regulate acute T cell responses. To carry this out, a deep protein language model – ESM1b – was used and a modified workflow capable of predicting the phenotypic consequences of missense variants throughout the genome was utilized. Specific interrogation of the A20 open reading frame highlighted several hotspot regions within A20 where missense variants were predicted to have a high likelihood of functional impact on the A20 protein (FIG. 2A). To complement this approach with more defined functional outcomes, data from a recent large-scale base editor (BE) screen in primary human T cells was analyzed. This screen utilized a massive sgRNA library tiled across the coding sequences of known regulators of T cell activity, including A20 / TNFAIP3, to probe for missense mutations capable of enhancing T cell effector functions. Briefly, T cells were activated, transduced with BEs and sgRNAs, expanded in cytokines, and then restimulated. T cells with the highest level of activation (e.g., TNF production) upon restimulation were then flow-sorted and sequenced to uncover enriched sgRNAs and their associated missense mutations. Analysis of this BE mutagenesis screen revealed sgRNAs that linked A20 missense mutations with enhanced TNF production by activated T cells (FIG. 2A). These missense mutations were again clustered in several hotspot regions. Aligning the results from the ESM1b analysis and BE screen altogether nominated three distinct A20 domains as potentially critical for T cell functionality: the ovarian tumor (A20OTU) domain, the seventh zinc finger motif (A20ZF7), and to a lesser extent the fourth zinc finger motif (A20ZF4) (FIG. 2A). The A20OTUand A20ZF4domains carry out deubiquitinase (DUB) and E3 Ub ligase enzymatic activity, respectively, and A20ZF4can also bind to K63 Ub chains. The A20ZF7motif binds to M1 linked Ub chains, but has no known enzymatic activity. To better understand how these A20 motifs regulate anti-tumor immunity in vivo, the unique series of A20 knock-in (KI) mice that express structurally defined point mutations that abrogate distinct biochemical functions within the A20OTU, A20ZF4, or A20ZF7motifs, while leaving the remainder of the A20 protein intact, was leveraged. Notably, while A20- / -mice die shortly after birth, A20OTU / OTU, A20ZF4 / ZF4, and A20ZF7 / ZF7mice live over 8 months, thereby allowing for the analysis of their responses to implanted tumors. First, the panel of A20 KI mice and their WT (A20+ / +) littermates with MC38 were challenged. Tumor growth and survival curves in mice either heterozygous or homozygous for the A20OTUor A20ZF4mutations matched those of A20+ / +littermates (FIGS. 2B and 2C), arguing against a dominant role for either the A20OTUor the A20ZF4domain in regulating anti-tumor immunity. In striking contrast, both heterozygous A20ZF7 / +and homozygous A20ZF7 / ZF7mice exhibited significantly slower tumor growth and prolonged survival compared to A20+ / +littermates (FIGS. 2B and 2C). Intriguingly, tumor growth did not diverge between A20+ / +and A20ZF7 / +or A20ZF7 / ZF7mice until days 9-12 following tumor inoculation (FIG. 2B). This timing hinted at an important role for the A20ZF7domain in regulating tumor-specific T cell responses, which typically require 7-10 days for naïve antigen-specific lymphocytes to become activated, proliferate, and differentiate into effector cells. Moreover, in a remarkable 43% of homozygous A20ZF7 / ZF7mice, tumors grew for 9-12 days and then regressed, ultimately becoming undetectable for the remainder of the experiment (through day 60; FIG. 2D and data not shown). Mice exhibiting this pattern of tumor growth were defined as “regressors.” A smaller fraction (7%) of heterozygous A20ZF7 / +mice also exhibited this regressor phenotype (FIG. 2D and data not shown), suggesting a dose dependence to A20ZF7activity. By contrast, no regressors were detected in A20+ / +, A20OTU / OTU, or A20ZF4 / ZF4mice (FIG. 2D and data not shown). Thus, inactivation of the A20ZF7motif leads to a robust primary anti-tumor immune response capable of outright tumor rejection. Augmented primary anti-tumor immune responses can lead to enhanced memory responses. On the other hand, some genetic enhancements of acute CAR-T cell responses can compromise the durability of these responses, resulting in tumor relapse. It was investigated whether regressor A20ZF7 / ZF7mice develop memory responses by rechallenging them with four times the number of MC38 cells used in the primary challenge (data not shown). Strikingly, regressor A20ZF7 / ZF7mice nearly completely resisted this larger secondary challenge, with significantly less tumor growth and prolonged survival when compared to age-matched A20ZF7 / ZF7mice receiving their primary tumor challenge (FIG. 2E and data not shown). Therefore, abrogation of the A20ZF7motif also confers a strong memory anti-tumor immune response. The vigor of anti-tumor immune responses varies between tumor types and between host immune backgrounds. The responses of A20+ / +, A20ZF7 / +and A20ZF7 / ZF7littermates to challenges was tested with two additional heterotopic tumor models. First, B16F10 melanoma, a C57BL / 6 “cold” tumor that responds poorly to immunotherapy, was used. As with MC38, both A20ZF7 / +and A20ZF7 / ZF7mice suppressed B16F10 tumor growth better than A20+ / +littermates, accompanied by prolonged survival (FIG. 2F and data not shown). Next, CT26 colon carcinoma, a “hot” tumor derived from BALB / c mice, was used. BALB / c mice exhibit more robust type 2 immune responses characterized by IL-4, IL-5, and IL-13 expression, and less robust type 1 responses typified by IFNγ and TNF expression. A20ZF7KI mice were backcrossed to BALB / c mice for seven generations and, upon challenge with CT26, again observed that both A20ZF7 / +and A20ZF7 / ZF7mice suppressed tumor growth better than A20+ / +littermates (FIG. 2F and data not shown). Hence, the A20ZF7domain restrains anti-tumor immunity against multiple tumor types and across divergent immune backgrounds. CD8 T cell-intrinsic A20ZF7inactivation enhances anti-tumor immunity. A20 is a potent regulator of myeloid cells and innate immunity25,31,32, and myeloid cells greatly outnumber lymphocytes in the TMEs of MC38 tumors grown in A20+ / +, A20ZF7 / +and A20ZF7 / ZF7mice alike (data not shown). By contrast, the findings in human T cells (FIG. 1) suggest A20 plays a T cell-intrinsic role in regulating anti-tumor immunity. Therefore, it was desired to determine which immune cells are critical for the superior anti-tumor immunity of A20ZF7 / +and A20ZF7 / ZF7mice. A20ZF7KI mice were interbred with Rag1- / -mice to remove B and T lymphocytes. RAG-1 deficiency abolished the advantages in MC38 tumor suppression and survival of both A20ZF7 / +and A20ZF7 / ZF7mice (FIG. 3A and data not shown), while also eliminating the regressor phenotype (FIG. 3B and data not shown). These findings could not, however, be attributed to B or gd T cells, as both A20ZF7 / +and A20ZF7 / ZF7mice with genetic ablation of either B cells (Ighm- / -a.k.a. mMT) or gd T cells (Tcrd- / -) retained tumor suppression advantages over A20+ / +mMT and A20+ / +Tcrd- / -controls, respectively (FIGS. 3A-3B and data not shown). Next, CD8+T cells were investigated. Strikingly, both genetic (Cd8a- / -) and antibody- mediated (anti-CD8a) depletion of CD8+T cells from A20ZF7 / +and A20ZF7 / ZF7mice abolished their tumor suppressive advantage (FIG. 3A and data not shown) and phenotype (FIG. 3B and data not shown). Altogether, these results highlight the necessity for CD8+T cells in driving superior anti-tumor immunity in A20ZF7mice. Next, the degree to which A20ZF7inactivation within CD8+T cells is sufficient for driving enhanced anti-tumor immunity was investigated. A20ZF7mice were interbred with OT-I transgenic mice expressing a TCR specific for the ovalbumin peptide OVA. A20ZF7 / ZF7OT-I or control A20+ / +OT-I cells were then adoptively transferred into congenic (CD45.1) WT hosts. After challenging these host mice with OVA-expressing MC38 cells (MC38-OVA), it was observed that recipients of either naive or pre-activated A20ZF7 / ZF7OT-I cells both suppressed tumor growth and prolonged survival better than recipients of A20+ / +OT-I cells (FIG. 3C and data not shown). Next, to test the ability of A20ZF7CD8+T cells to kill MC38 tumors via the recognition of endogenous tumor antigens, two separate approaches were taken. First, Cd8acre(a.k.a. E8Icre) mice were utilized to direct either CD8+T cell-restricted A20 deficiency (E8IcreA20loxP / loxPmice) or A20ZF7hemizygosity (E8IcreA20ZF7 / loxPmice). E8IcreA20loxP / loxPmice outperformed E8IcreA20+ / +littermates in tumor growth suppression (FIGS. 3D-3E and data not shown) and achieved regressor rates comparable to A20ZF7 / ZF7mice (FIG. 3B), confirming that A20KOCD8+T cells boost anti-tumor immunity. Remarkably, E8IcreA20ZF7 / loxPmice also demonstrated regressor rates equivalent to E8IcreA20loxP / loxPmice (FIG. 3B), and prolonged mouse survival to a greater extent than E8IcreA20ZF7 / +or E8IcreA20loxP / +mice (FIGS. 3D-3E and data not shown), indicating that A20ZF7hemizygosity within CD8+T cells promotes anti-tumor immunity. Second, Cd8a- / -mice (CD45.1) were reconstituted with congenic (CD45.2) polyclonal CD8+T cells (FIG. 3F). Using sublethal irradiation prior to adoptive transfer, durable CD8+T cell engraftment was achieved (data not shown). Importantly, Cd8a- / -mice reconstituted with either A20ZF7 / +or A20ZF7 / ZF7CD8+T cells suppressed MC38 growth significantly better than Cd8a- / -mice reconstituted with control A20+ / +CD8+T cells (FIG. 3G and data not shown). Taken together, these data show that abrogating the A20ZF7motif specifically within CD8+T cells enhances anti-tumor immunity. A20ZF7inactivation inhibits terminal exhaustion of CD8+T cells. To better understand how A20ZF7regulates tumoricidal CD8+T cells, CD8+TILs from MC38 tumors in A20+ / +, A20ZF7 / +and A20ZF7 / ZF7mice were analyzed by full spectrum flow cytometry (FSFC) 12-15 days after tumor injection. This time point was chosen to allow enough time for mice to mount an adaptive immune response, while still maintaining similar tumor sizes across genotypes. No significant difference in total CD8+TIL numbers between A20+ / +, A20ZF7 / +and A20ZF7 / ZF7mice was observed, whether evaluating by fraction of total leukocytes or by total density (FIG. 4A). Next, CD8+TIL subsets were investigated, as differences in CD8+T cell differentiation can impact anti-tumor immunity. CD8+TILs can be broadly defined as Tex (PD- 1+) or PD-1–cells, and Tex can be further divided into progenitor exhausted (Tpex) and terminally exhausted (Ttex) subsets. Tpex have stem-like features but possess minimal cytotoxic potential, and are broadly defined by expression of TCF-1. Tpex ultimately differentiate into Ttex, characterized by loss of TCF-1 expression, upregulation of the IR TIM-3, and increased expression of TOX. Ttex increase their expression of cytotoxic molecules but remain hypofunctional, as TOX orchestrates upregulation of various additional IRs and epigenetic silencing of T cell effector genes. Within CD8+TILs, PD-1–cells, Tpex (TCF-1+TIM-3–) and Ttex (TCF-1–TIM-3lo / +) were identified in A20+ / +and A20ZF7KI mice alike (data not shown). No differences in the distribution of these CD8+TIL subsets across genotypes were observed (FIG. 4B). Thus, quantitative differences in CD8+TILs or their subsets did not appear responsible for superior anti-tumor immunity in A20ZF7KI mice. Phenotypes of CD8+TIL subsets were interrogated by FSFC, focusing on surface markers and transcription factors connected to memory and exhaustion. While A20ZF7 / ZF7mice exhibited a profound memory response to secondary tumor challenge (FIG. 2E), no differences in expression of the canonical surface markers denoting central memory (CD62L+; Tcm) or tissue resident memory (CD69+CD103+; Trm) CD8+T cells were observed (data not shown). Thus, A20ZF7inactivation did not preferentially drive CD8+TILs toward a memory phenotype at this early time point. Next, the expression of IRs linked to exhaustion (2B4, CD39, ICOS, LAG- 3, and TIGIT) as well as the intracellular expression of TOX were profiled. Additionally, the expression of Ly108 (a.k.a. SLAMF6), a stemness marker implicated in T cell activation which is often used as a cell surface surrogate for TCF-1, was assessed. As expected, Tpex across genotypes collectively expressed the highest level of Ly108, while Ttex consistently expressed the highest levels of IRs and TOX (data not shown). With few exceptions, the fraction of PD-1– CD8+TILs and Tpex that expressed these markers was similar in A20+ / +, A20ZF7 / +and A20ZF7 / ZF7mice (data not shown). However, striking differences between A20ZF7 / ZF7CD8+Ttex were uncovered when compared to A20+ / +and A20ZF7 / +Ttex. While Ttex across genotypes appropriately lost expression of TCF-1 and gained expression of TIM-3 (data not shown), a significantly greater percentage of A20ZF7 / ZF7Ttex retained expression of Ly108, while a significantly lower percentage expressed TOX or additional IRs when compared to A20+ / +and A20ZF7 / +Ttex (FIGS. 4C and 4D). Indeed, while greater than 70% of A20+ / +and A20ZF7 / +Ttex co-expressed 4 or more IRs in addition to PD-1, fewer than 25% of A20ZF7 / ZF7Ttex did the same (data not shown). These differences in the A20ZF7 / ZF7Ttex phenotype persisted in more advanced MC38 tumors (day 24; data not shown), and were also present in A20ZF7 / ZF7mice inoculated with B16F10 and CT26 tumors (data not shown). Furthermore, this altered phenotype was observed solely in A20ZF7 / ZF7Ttex, and not in A20OTU / OTUor A20ZF4 / ZF4Ttex (data not shown). Together these results show that inactivation of the A20ZF7motif alters the CD8+Ttex compartment such that they appear less exhausted. Intriguingly, analysis of transcriptomic data from the UCSF Immunoprofiler database revealed a positive correlation between A20 / TNFAIP3 expression and markers of exhaustion in TILs from various human cancers (data not shown), pointing to a TIL-intrinsic role for A20 in orchestrating exhaustion. Hence, to test whether A20ZF7inactivation combats CD8+TIL exhaustion in a truly cell autonomous manner, congenically marked pre-activated A20+ / +OT-I (CD45.1 / 1) and A20ZF7 / ZF7OT-I (CD45.2 / 2) cells were adoptively co-transferred into WT CD45.1 / 2 recipient mice bearing MC38-OVA tumors, and tumors were analyzed by FSFC. Both A20+ / +and A20ZF7 / ZF7OT-I TILs were present at similar densities and differentiated into Ttex (PD-1+TCF-1–TIM-3lo / +) in similar proportions (adoptively co-transferred). However, A20ZF7 / ZF7OT-I Ttex displayed increased Ly108 expression and decreased levels of IRs and TOX when compared with A20+ / +OT-I Ttex within the same TME (FIG. 4E), confirming the CD8+T cell- intrinsic role of A20ZF7inactivation in relieving the exhaustion phenotype. Finally, to gain insight into whether this effect may be driven by repetitive or exhaustive TCR engagement, A20+ / +and A20ZF7 / ZF7OT-I cells were submitted to an in vitro repetitive stimulation assay benchmarked as a surrogate for promoting exhaustion. This assay revealed increased Ly108 and decreased IR expression in repetitively stimulated A20ZF7 / ZF7OT-I T cells when compared to A20+ / +OT-I cells (FIG. 4F), mirroring the in vivo findings and confirming that A20ZF7 inactivation acts downstream of repetitive TCR engagement to prevent or relieve terminal exhaustion of CD8+T cells. Further characterization of Ttex from day 12-15 MC38 tumors revealed an intriguing dichotomy in differentiation based on expression of CX3CR1 and KLRG1. CX3CR1 has been described as a marker of an intermediate state between Tpex and Ttex in a mouse model of chronic viral infection, though whether it demarcates a similar Tex population in the TME remains to be addressed. Meanwhile, KLRG1 commonly denotes short-term effector CD8+T cells, and is not typically seen expressed on Ttex within the TME. Within Ttex, four distinct subpopulations were identified based on CX3CR1 and KLRG1 expression. Curiously, broad characterization of Ttex across genotypes revealed that, among these subpopulations, KLRG1+CX3CR1–Ttex had the highest proportion of Ly108-expressing cells and the lowest proportion of IR- and TOX-expressing cells, while the exact opposite was true for KLRG1–CX3CR1+Ttex (FIG. 4G). Remarkably, while KLRG1–CX3CR1+Ttex dominated in A20+ / +and A20ZF7 / +tumors, A20ZF7 / ZF7tumors showed a substantial increase in KLRG1+CX3CR1–Ttex (FIGS. 4H and 4I). This pattern of increased KLRG1+CX3CR1–Ttex was similarly observed in later stage MC38 tumors, in B16F10 and CT26 tumors (adoptively co-transferred), and in adoptively transferred A20ZF7 / ZF7OT-I cells within MC38-OVA tumors (adoptively co- transferred), but was absent in Ttex from A20OTU / OTUor A20ZF4 / ZF4mice (adoptively co- transferred). Thus, A20ZF7inactivation promotes the differentiation of a unique KLRG1+CX3CR1–Ttex subset that appears less exhausted and may provide enhanced tumoricidal activity. Perforin, but not IFNγ or TNF, drives enhanced anti-tumor immunity in A20ZF7 / ZF7mice. The reduced apparent exhaustion in A20ZF7 / ZF7Ttex may align with enhanced tumoricidal effector function, explaining the superior anti-tumor response of A20ZF7 / ZF7mice. To define the relevant effector mechanism, cytokines were first investigated, as most studies of T cell exhaustion have highlighted diminished polyfunctionality, i.e., co-production of cytokines including IFNγ and TNF, as a key indicator of dysfunction. A significant increase in polyfunctional IFNγ+TNF+Ttex in both A20ZF7 / +and A20ZF7 / ZF7mice was observed upon ex vivo stimulation (FIGS. 5A-5B and data not shown). Notably, A20ZF7 / +Ttex displayed increased cytokine production when compared to A20+ / +Ttex despite similar expression of IRs and TOX (FIG. 4D), suggesting that even monoallelic A20ZF7inactivation in Ttex can uncouple inhibitory signals from suppression of effector functions. In contrast, A20+ / +, A20ZF7 / +and A20ZF7 / ZF7Ttex all showed minimal IL-2 production (data not shown), suggesting A20ZF7inactivation does not universally restore effector functions in Ttex within this tumor model. The findings above suggest that increased cytokine production by A20ZF7-deficient CD8+TILs might contribute to their enhanced anti-tumor activity. To test this, either IFNγ or TNF expression in A20ZF7KI mice was genetically ablated by crossing A20ZF7KI mice with Ifng- / -or Tnf- / -mice. Surprisingly, neither IFNγ or TNF deficiency alone were able to reverse the superior MC38 growth control and prolonged survival of A20ZF7 / +and A20ZF7 / ZF7mice when compared to A20+ / +littermates (FIG. 5C and data not shown). In addition, A20ZF7 / ZF7Ifng- / -and A20ZF7 / ZF7Tnf- / -mice both showed regressor rates similar to A20ZF7 / ZF7mice (FIG. 5D), altogether indicating that neither of these cytokines individually are required for the anti-tumor phenotype in A20ZF7KI mice. To account for potential redundancies or compensatory actions between the anti-tumor activities of IFNγ and TNF, A20ZF7mice were interbred with Ifng- / -Tnf- / -mice that lack both cytokines. Interestingly, A20ZF7 / +Ifng- / -Tnf- / -mice were no longer able to control tumor growth or prolong survival better than their A20+ / +Ifng- / -Tnf- / -mice counterparts (FIG. 5C and data not shown), indicating that these cytokines drive the superior tumor suppression observed in heterozygous A20ZF7 / +mice. By contrast, homozygous A20ZF7 / ZF7Ifng- / -Tnf- / -mice retained significant advantages in controlling tumor growth and prolonging survival when compared to A20+ / +Ifng- / -Tnf- / -mice, while maintaining a 45% regressor rate (FIGS. 5C-5D and data not shown). These findings suggested the presence of an additional IFNγ / TNF-independent tumor growth suppression mechanism unlocked in A20ZF7 / ZF7but not A20ZF7 / +mice. CD8+T cells can also kill target cells via exocytosis of cytotoxic granules (i.e. degranulation) that contain perforin and granzymes. Perforin disrupts target cell membranes to facilitate granzyme entry, and granzymes in turn trigger cell death pathways. The perforin- granzyme signaling axis was evaluated for its role in A20ZF7anti-tumor immunity by crossing A20ZF7KI mice with perforin knockout (Prf1- / -) mice. Strikingly, perforin deficiency almost entirely abolished the tumor suppression and survival advantages, as well as the regressor phenotype, of A20ZF7 / ZF7mice (FIGS. 5C-5D and data not shown). Importantly, perforin deficiency did not change CD8+TIL numbers, CD8+TIL subset distribution, or markers of T cell exhaustion in A20+ / +, A20ZF7 / +or A20ZF7 / ZF7mice (data not shown). Therefore, perforin mediates enhanced anti-tumor immunity in A20ZF7 / ZF7mice without perturbing CD8+T cell differentiation. A20ZF7inactivation unleashes perforin degranulation by exhausted CD8+T cells. The superior perforin-dependent cytotoxicity shown by A20ZF7 / ZF7CD8+TILs could result from increased numbers of perforin-expressing Ttex. Therefore, CD8+TIL perforin expression was assessed by FSFC which, unlike most cytokines, can be detected without ex vivo stimulation. Regardless of genotype, few PD-1–TILs or Tpex expressed perforin, while a substantial proportion of Ttex had detectable perforin expression (FIGS. 5E and 5F). This finding aligns with prior reports of enhanced perforin and granzyme levels in Ttex. Surprisingly, while A20+ / +and A20ZF7 / +Ttex exhibited similarly high proportions of perforin-expressing cells, an unexpected reduction in the numbers of perforin-expressing A20ZF7 / ZF7Ttex was observed when compared to A20+ / +Ttex (FIGS. 5E and 5F). This result seemingly contradicted the findings of enhanced perforin-dependent tumoricidal activity in A20ZF7 / ZF7mice and enhanced perforin secretion by A20KOTCR-T cells (FIG. 1E). It was also observed that side scatter (SSC) median fluorescence intensity (MFI) was greatly increased in Ttex when compared to PD-1–and Tpex subsets (FIGS. 5E and 5F). SSC indicates cellular complexity, and in the case of CD8+T cells, likely reflects granule content. Indeed, a strong positive correlation between Ttex perforin expression and SSC was observed (FIG. 5G). Remarkably, as with perforin, SSC MFI was significantly reduced in A20ZF7 / ZF7Ttex when compared to A20+ / +and A20ZF7 / +Ttex (FIGS. 5E and 5F). This phenomenon of decreased Ttex perforin expression was unique to A20ZF7 / ZF7Ttex, as it was not observed in A20OTU / OTUor A20ZF4 / ZF4Ttex (data not shown). Furthermore, similar reductions in perforin-expressing A20ZF7 / ZF7Ttex from later stage MC38 tumors as well as B16F10 and CT26 tumors were observed (data not shown), arguing against tumor size- or model-specific effects. Adoptively co-transferred A20ZF7 / ZF7OT-I Ttex also showed reduced proportions of perforin-expressing cells compared to A20+ / +OT-I Ttex (data not shown), as did A20ZF7 / ZF7OT-I cells upon in vitro repetitive stimulation (data not shown). Altogether, these findings highlight a paradox wherein CD8+T cell-intrinsic A20ZF7inactivation unleashes perforin-dependent anti-tumor cytotoxicity by Ttex, yet engenders fewer perforin-expressing Ttex. Diminished perforin expression by A20ZF7 / ZF7Ttex could either result from reduced production or from enhanced degranulation. To distinguish between these possibilities, the capacity of Ttex to manufacture perforin protein was measured. First, dissociated MC38 tumors were stimulated ex vivo with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin to block intracellular vesicle transport and, by extension, inhibit degranulation. Using this approach, it was discovered that a similar fraction of A20ZF7 / ZF7Ttex expressed perforin as compared to A20+ / +and A20ZF7 / +Ttex (FIG. 5H). Thus, A20ZF7 / ZF7Ttex are capable of equivalent perforin production, at least in these pharmacologically stimulated cells. Next, to quantify Ttex granule content in their native environment without additional stimulation, MC38-bearing mice were treated for 6 hours in vivo with brefeldin or vehicle control, then analyzed tumors by FSFC. This experiment revealed several important facets of perforin degranulation by A20+ / +and A20ZF7-deficient CD8+TILs. Most strikingly, the fraction of A20ZF7 / ZF7Ttex expressing perforin increased markedly upon brefeldin treatment (FIG. 5I), indicating that many A20ZF7 / ZF7Ttex are likely degranulating perforin in the absence of brefeldin. Meanwhile, the fraction of perforin-expressing Ttex remained similar in brefeldin-treated versus control A20+ / +and A20ZF7 / +mice (FIG. 5I), suggesting minimal degranulation by these Ttex. Parallel findings of increased SSC and increased percentage of GzmB-expressing Ttex were observed in brefeldin-treated A20ZF7 / ZF7mice, but not in A20+ / +or A20ZF7 / +mice (FIGS. 5J and 5K). Notably, few perforin-expressing PD-1–TILs or Tpex were detected in any genotype even after brefeldin treatment (FIG. 5I), indicating that perforin production is highly restricted to Ttex. Finally, among Ttex subsets, it was observed that KLRG1+CX3CR1–A20ZF7 / ZF7Ttex had the lowest proportion of perforin-expressing cells, while KLRG1–CX3CR1+Ttex had the highest proportion (data not shown). These fractions became relatively normalized upon brefeldin treatment (data not shown) suggesting that the least exhausted appearing KLRG1+CX3CR1–Ttex subset was more actively secreting perforin in vivo. Overall, these data highlight an unappreciated defect in Ttex degranulation that is regulated by A20 and can be sharply reversed upon A20ZF7inactivation. Deletion of A20 or mutation of A20ZF7in human CAR-T cells enhances anti-tumor immunity in vivo. Given the potent ability of A20ZF7to regulate anti-tumor immunity, it was tested whether specific mutation of the A20ZF7motif in human CD19-specific TRAC-CAR-T cells would enhance leukemia control in preclinical models. A Cas9 sgRNA targeting a DSB at Asp768 in the A20ZF7motif was designed to inactivate the domain via indel frameshift. This strategy removes the two most C-terminal zinc coordinating cysteines in the A20ZF7domain. Sanger sequencing analysis confirmed efficient indel generation (91%) and relative KO score (84%) (data not shown). Immunoblotting for A20 in activated T cells revealed efficient deletion of A20 protein expression in A20KOT cells, while A20ZF7targeted T cells retained A20 protein expression (data not shown). Next, A20KOor A20ZF7CD19 CAR-T cells were injected into NALM6-bearing NSG mice and monitored leukemia growth. A large fraction of mice treated with either A20KOor A20ZF7CAR-T cells potently suppressed NALM6 leukemia growth and demonstrated increased survival compared to mice treated with control AAVS1 CAR-T cells (FIGS. 6A and 6B). In addition, the majority of recipients of A20KOor A20ZF7CAR-T cells that eradicated NALM6 remained leukemia-free for several months without relapse, and showed no evidence of splenic enlargement or decline in health indices for underlying lymphoproliferative disease. To assess whether these leukemia-free mice had developed durable anti-tumor immunity, the mice were rechallenged with 3 x 106NALM6 cells on days 44, 51, 58, and 65 after the original NALM6 injection. Despite repeated NALM6 rechallenge, the majority of these mice remained resistant to leukemic engraftment (FIG. 6C). Thus, as with A20KOCAR-T cells, Cas9-directed abrogation of the A20ZF7domain in CAR-T cells dramatically enhances the anti- tumor efficacy and functional persistence of these cells. Base-editing of A20ZF7enhances persistence and cytolytic function of human CAR-T Cells in vitro and in vivo. Cas9-based CRISPR editing relies upon DNA DSBs that lead to deletions or frameshifts. By contrast, CRISPR BEs generate targeted base pair edits without causing DSBs. Hence, BE- directed mutagenesis usually mutates a single amino acid and avoids triggering DNA DSB repair pathways, which altogether confers a more defined and clinically safer approach to genomic engineering. To test the therapeutic potential of this strategy, BEs were used to introduce a targeted A20ZF7domain mutation into CAR-T cells. From the ESM1b analysis and large-scale BE screen28(FIG. 2A), a specific amino acid residue within the A20ZF7motif (C779), and a corresponding sgRNA targeting a missense mutation (C779Y), together were predicted to disrupt A20 function and enhance T cell activation (data not shown). Notably, C779 represents the third (of four) Zn++coordinating cysteines in the A20ZF7motif, and is likely important for proper coordination of the zinc ion and M1 Ub binding. For comparison, base-edited A20KOCAR-T cells were generated using an sgRNA that introduces a premature stop codon in exon 2 of the A20 gene (R90*), and elimination of the A20 protein by was confirmed immunoblot (data not shown). Control T cells from the same donors were edited with an sgRNA targeting an intronic sequence of the B2M gene encoding beta-2 microglobulin (β2m). This intronic mutation (hereafter referred to as B2Mi) does not disrupt β2m surface protein expression (data not shown). Next-generation sequencing analysis of T cells collected 7 days post editing confirmed that both the A20KOand A20ZF7sgRNAs edited A20 with 90% efficiency, generating the expected R90* or C779Y mutations, while the B2Mi sgRNA edited with 97% efficiency (data not shown). Next, CD19-specific CAR-T cells were engineered that were base-edited with either A20KO, A20ZF7or control B2Mi sgRNAs. Cas12a RNP carrying TRAC-targeting sgRNAs, cytosine base editor (CBE)-encoding mRNA, and a CBE sgRNA targeting either A20KO, A20ZF7or B2Mi were introduced into T cells via electroporation (FIG. 6D). As CBE is coupled to a nicking Cas9 (nCas9) for targeting, Cas12a-mediated CAR knock-in was utilized here to mutually exclude TRAC KO sgRNAs and CBE sgRNAs from complexing with the opposing CRISPR effector. Next, these CAR-T cells were tested in in vitro assays. Base-edited CAR-T cells were repeatedly stimulated with fresh CD19+tumor cells, then evaluated for expression of activation and exhaustion markers, production of cytokines and cytotoxic proteins, and tumor cell killing capacity. Remarkably, A20KOand A20ZF7base-edited CAR-T cells maintained robust recursive killing compared to control B2Mi CAR-T cells after three rounds of tumor stimulation (FIG. 6E and data not shown). Furthermore, compared to control B2Mi CAR-T cells, base-edited A20KOand A20ZF7CAR-T cells produced more effector cytokines (IFNγ, TNF) and secreted substantially more cytotoxic proteins (perforin, GzmB and sFasL) after repetitive stimulation (FIG. 6F), consistent with the findings of increased secretion by Cas9-edited A20KOTCR-T cells (FIG. 1D) and increased degranulation by A20ZF7 / ZF7Ttex (FIGS. 5H-5J). A20KOand A20ZF7CAR-T cells also exhibited enhanced proliferation when compared to control B2Mi CAR-T cells (data not shown). In addition, repetitively stimulated A20KOand A20ZF7CAR-T cells expressed lower levels of exhaustion markers (PD-1 and LAG-3) (FIG. 6G) and higher levels of activation markers (CD25, CD137, CD69 and CD154) (data not shown). Importantly, by all metrics, A20ZF7CAR-T cells performed similarly to A20KOCAR-T cells. Next, it was tested whether A20 disruption by base-editing enables human CAR-T cells to grow in a cytokine- or antigen-independent fashion, which could indicate a safety challenge for cell therapy applications. First, the growth of A20KOand A20ZF7CAR-T cells was evaluated after the withdrawal of cytokines, where consistent decreases in cell numbers and viability of both A20KOand A20ZF7CAR-T cells were observed, similar to control B2Mi CAR-T cells (data not shown). Further, the numbers of A20KOand A20ZF7CAR-T cells contracted as expected after initial exposure to tumor cells without cytokines (data not shown). The level of dependence on antigen of A20KOand A20ZF7CAR-T cells for tumor cell killing was also assessed. Here, all CAR-T cell conditions were exposed to CD19-negative tumor cells and their ability to eradicate tumor cells was assessed over several days. A small degree of allogenic killing was observed, which was similar across all conditions (data not shown). Therefore, targeting A20 or its ZF7 motif does not confer cytokine-independent growth or antigen-independent killing. These safety metrics suggest A20 targeted CAR-T cells could potentially be safely utilized as clinical CAR-T cell products. To test the ability of base-edited A20 mutant CAR-T cells to suppress tumor growth in vivo, NALM6-engrafted mice were treated with either base-edited A20KO, A20ZF7, or control B2Mi CD19-CAR-T cells and growth of the luminescent NALM6 tumors was serially measured via BLI. Mice receiving B2Mi CAR-T cells failed to control tumor progression, leading to rapid mouse mortality (FIGS. 6H and 6I). In striking contrast, the majority of mice treated with A20KOor A20ZF7base-edited CAR-T cells exhibited robust tumor control 20 days after T cell injections, while also demonstrating significantly improved survival (FIGS. 6H and 6I). Importantly, in all treatment groups, there were no gross differences in mice based upon visual inspection or body weight (data not shown). To understand why A20KOand A20ZF7engineered CAR-T cells suppress leukemia growth better than control B2Mi CAR-T cells, bone marrow samples from tumor bearing mice were evaluated 14 days after CAR-T cell adoptive transfer by flow cytometry. At this early time point, leukemia burden remained relatively even across treatment groups. The findings showed a trend toward higher numbers of CAR-T cells in the bone marrow of mice treated with A20KOor A20ZF7CAR-T cells when compared to those treated with control B2Mi CAR-T cells (FIG. 6J). Notably, editing of A20 did not substantially change the distribution of memory phenotypes of the isolated CAR-T cells (data not shown). The functional state of these cells was further investigated by analyzing IR surface expression. Importantly, the frequency of CD4+or CD8+cells co-expressing 2 or more IRs was markedly lower in A20KO(24-30%) and A20ZF7(34-46%) CAR-T cells when compared to B2Mi control (51-65%), suggesting a superior functional state of A20-mutated CAR-T cells (FIG. 6K). Altogether, these data highlight that ablation of A20 or the A20ZF7motif via base editing can lead to remarkable improvements in vivo using an already optimized best-in-class CAR-T cell therapy, accompanied by reduced CAR-T cell exhaustion, without any ostensible safety risk. In above study, the entire protein-coding genome was filtered through a CRISPR knockout screen, a base-editing mutagenesis screen, a protein language model, and transgenic knock-in mice to pinpoint a single protein domain — A20ZF7— as a critical negative regulator of T cell activity within the TME. This precise molecular lever operated both in murine and human T cells, in liquid and solid tumors, and in immunodeficient and immunocompetent mice. Base- editing was incorporated into the human CAR-T cell production workflow, allowing for introduction of an A20ZF7missense mutation at high editing efficiency that dramatically enhanced CAR-T cell anti-tumor activity both in vitro and in vivo. This study is believe to set a new standard for engineering CAR-T cell clinical products using base-editing. CRISPR BE-directed gene ablation offers a distinct advantage over Cas9 by generating precise nonsense mutations or interrupted splice sites, without introducing DNA DSBs and their accompanying chromosomal rearrangements. This approach was taken one step further by introducing a strategic missense mutation. For multifunctional proteins like A20, a targeted missense mutation that otherwise preserves the remaining protein and its functions could prove functionally superior with fewer undesirable side effects, as compared to gene ablation. Recent large-scale base-editing mutagenesis screens have begun to pinpoint key amino acid residues that impact T cell functions in vitro. This approach is herein translated to in vivo CAR-T cell enhancement by targeting A20ZF7. BE-guided gene ablation can also be multiplexed, thus setting the stage for multiplexing missense mutations to develop safer and more efficacious next- generation T cell therapies. Indeed, multiplexed editing within A20 itself might lead to further enhancements, as synergistic interactions between the A20ZF4and A20ZF7motifs has been described. The aforementioned base-editing screens converged on mutations found in hereditary syndromes characterized by autoimmunity. Also of note, naturally occurring T cell lymphoma oncogenic mutations were shown to improve CAR-T cell therapy, particularly those that augmented NF-κB signaling. Likewise, inherited mutations in A20 can lead to haploinsufficiency of A20 (HA20), a syndrome with autoimmune manifestations, while somatic A20 mutations have been described in B cell lymphomas and rarely in T cell lymphomas. Most mutations in HA20 patients and in A20-deficient lymphomas occur throughout A20 and result in loss or truncation of A20 expression, which would be consistent with a role for the C-terminal A20ZF7motif. Altogether, these findings highlight that the optimal enhancement of T cell therapies may already be written in the genetics of existing pathophysiologic processes, including those tied to A20 dysfunction. Importantly, it is emphasized that studies using A20KOor A20ZF7CAR-T cells showed no evidence of cytokine- or antigen-independent proliferation, or of CAR-T cell malignant transformation. The findings herein show that A20ZF7orchestrates T cell exhaustion. These results align with multiomics analyses wherein Ttex differentiation correlated with increased A20 / Tnfaip3 chromatin accessibility and decreased NF-κB transcriptional signatures. The human CRISPR screen also extends the findings of similar genome-scale by incorporating more rounds of T cell repetitive tumor stimulation. Notably, these screens with shorter duration of repetitive antigenic exposure did not find enrichment of A20, indicating that A20’s role likely emerges in later stages of T cell exhaustion. Accordingly, A20 / TNFAIP3 became progressively enriched in the screen with increasing rounds of repetitive stimulation, and A20ZF7inactivation in murine TILs specifically impacted Ttex but not Tpex phenotypes. The mechanism by which A20ZF7reinforces Ttex differentiation has yet to be elucidated, though findings in A20ZF7 / ZF7Ttex suggest that A20ZF7supports TOX expression. Indeed, the predominance of KLRG1+A20ZF7 / ZF7Ttex mirrors the skewing of chronically stimulated TOX-deficient CD8+T cells toward KLRG1+effector-like cells. Additional factors that connect A20ZF7with exhaustion might involve restraint of NF-κB signaling. A20ZF7binds M1 ubiquitin dimers and chains on IKKγ to impede the NF-κB pathway. In acutely activated T cells, A20 can disrupt NF-κB activity following recruitment to the CARD11-BCL10-MALT1 (CBM) signalosome, possibly via the A20ZF7motif. Repeated TCR stimulation might thus engage a similar mechanism that ultimately connects downstream to induction of TOX, thereby silencing effector genes and increasing IR expression. Two layers of anti-tumor effector mechanisms unlocked by targeting A20ZF7in human and murine T cells alike are revealed. The first layer includes enhanced production of IFNγ and TNF, consistent with prior studies of A20KOCD8+TILs. These studies are extended by showing that the A20ZF7motif regulates IFNγ and TNF production, and that neither cytokine was individually required for enhanced tumor suppression in A20ZF7KI mice. Instead, an unexpected redundancy was discovered between IFNγ and TNF activity required for tumor suppression in A20ZF7 / +mice. Notably, A20ZF7 / +Ttex express more IFNγ and TNF than A20+ / +Ttex despite similar levels of TOX expression, indicative of additional TOX-independent pathways by which A20ZF7regulates Ttex functions. The second layer of anti-tumor immunity involves A20’s restraint of the perforin- granzyme axis. This regulatory function of A20 likely occurs independently of Tox, since Tox deficient CD8 T cells are relieved of exhaustion markers but remain dysfunctional. Through the lens of A20ZF7deficiency, an unappreciated impairment in Ttex is revealed, not at the level of perforin or granzyme expression, but at the ultimate step of degranulation. Indeed, while Ttex differentiation leads to gene silencing of effector cytokines, these cells consistently express perforin and granzymes. However, their ability to degranulate has not been addressed, and would furthermore not be revealed by transcriptomics, nor by intracellular staining in the absence of a protein trafficking inhibitor. Here, in vivo brefeldin treatment was used to demonstrate that A20ZF7 / ZF7Ttex were rapidly secreting perforin and GzmB, while A20+ / +and A20ZF7 / +Ttex were not. Moving forward, the use of brefeldin could better discriminate the functional state of T cells in the TME and help resolve the complex nature of perforin and granzyme flux in T cells that are labeled “terminally exhausted.” Furthermore, the signaling mediators that connect TCR engagement to degranulation remain incompletely understood. Genome-scale CRISPR screens have begun to discover regulators of degranulation in acutely activated CD8+T cells, and thus future studies in chronically stimulated T cells might be expected to find regulators of Ttex degranulation aside from A20ZF7. This study definitively shows a requirement for perforin degranulation to achieve MC38 rejection in A20ZF7 / ZF7mice. These findings parallel prior studies using immunotherapy to treat MC38 tumor-bearing mice wherein perforin was required not to slow tumor growth but to reject tumors outright. Indeed, heterozygous A20ZF7 / +mice slowed MC38 growth via increased production of IFNγ and TNF, but could not reject MC38 given their ongoing impairment in Ttex degranulation. Conversely, while homozygous A20ZF7 / ZF7Ttex also increased their production of IFNγ and TNF, this was not required for tumor rejection. These findings reflect differential A20ZF7dosage requirements for regulation of cytokine production versus granule exocytosis. In other tumor models, the requirements for cytokine versus granzyme-mediated cytotoxicity can be dramatically different. Altogether, this study emphasizes the various layers of anti-tumor immunity available to TILs, and the importance of finding targets such as A20ZF7that enhance T cell activity in multiple ways. Here, these multiple advantages of A20 deficiency are harnessed in a single base edit to engineer CAR-T cell therapeutics that easily outperform the current clinical standard. Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein

Claims

CLAIMS What is claimed is:

1. A human T cell that comprises: a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain; and / or a genome in which the endogenous A20 gene has been knocked out.

2. The human T cell of claim 1, wherein the T cell comprises a genome in which the endogenous A20 gene has been knocked out.

3. The human T cell of claim 1, wherein T cell comprises a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain.

4. The human T cell of any prior claim, wherein the T cell further comprises: a nucleic acid encoding a recombinant immune receptor.

5. The human T cell of claim 4, wherein the recombinant immune receptor is a chimeric antigen receptor (CAR), and HLA-Independent TCR (HIT) or engineered T cell receptor (TCR).

6. The human T cell of any prior claim, wherein the human T cell is a primary T cell or a progenitor thereof.

7. The human T cell of any prior claim, wherein the human T cell is genetically modified to be allogeneic in a human host.

8. The human T cell of any prior claim, wherein the ZnF7 domain is either absent in the A20 protein or unable to bind to linear (M1) Ub chains.

9. The human T cell of any prior claim, wherein the A20 protein comprises: a C-terminal truncation that removes at least part of the ZnF7 domain,an amino acid substitution in the ZnF7 domain, a deletion that removes at least part of the ZnF7 domain, or an insertion that causes out-of-frame protein translation.

10. The human T cell of any prior claim, wherein the A20 protein further comprises one or more amino acid alterations outside of the ZnF7 domain.

11. The human T cell of any prior claim, wherein the nucleic acid is the endogenous A20 gene and the gene contains one or more mutations that ablate the function of the ZnF7 domain.

12. The human T cell of any prior claim, wherein: the endogenous A20 gene encodes an A20 protein that lacks a functional ZnF7 domain; the endogenous TCRα constant (TRAC) gene has been inactivated; and the T cell comprises a nucleic acid encoding a CAR.

13. The human T cell of claim 12, wherein: the nucleic acid encoding the CAR is inserted into the TRAC gene.

14. The human T cell locus of claims 4-13, wherein the CAR comprises an intracellular signaling domain from CD3ζ in which the second and third ITAM motifs have been altered to be non-functional.

15. A population of the human T cells of any of claims 1-14.

16. The population of T cells of claim 15, wherein the population comprises 100,000-1 Bn of the T cells.

17. A method of treatment comprising: administering a therapeutic amount of the population of T cells of claim 15 or 16 to a patient in need thereof.

18. The method of claim 17, wherein the patient is a cancer patient.

19. The method of claim 17 or 18, wherein the cells are autologous.

20. The method of claim 17 or 18, wherein the cells are allogeneic.

21. A method comprising: (a) expanding T cells that comprise a recombinant immune receptor ex vivo to produce expanded T cells, wherein the T cells comprise: (i) a nucleic acid encoding an A20 protein that lacks a functional ZnF7 domain; and / or a genome in which the endogenous A20 gene has been knocked out; and (ii) a nucleic acid encoding a recombinant immune receptor, and (b) harvesting the expanded T cells to produce a T cell population.

22. The method of claim 21, wherein the recombinant immune receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).

23. The method of claim 21 or 22, wherein the expanded T cells are harvested within 3-8 days of the initiation of step (a).

24. The method of claim 21 or 22, wherein the expanded T cells are harvested within 8-16 days of the initiation of step (a).

25. The method of any of claims 21-24, wherein the T cell population of step (b) comprises 100,000-1 Bn of the expanded T cells.

26. The method of any of claims 21-25, wherein the method further comprises: (c) administering a therapeutic amount of the T cell population harvested in step (b) to a cancer patient in need thereof.

27. The method of claim 26, wherein the T cells are autologous.

28. The method of claim 26, wherein the T cells are allogeneic.

29. A cell population manufactured according to the method of any of claims 21-25.

30. A method of treatment comprising: administering an A20 gene-targeted CRISPR / Cas9-based therapeutic to a patient in need thereof, where the therapeutic either knocks out the endogenous A20 gene in the patient or introduces a point mutation, insertion, deletion, or substitution in region of the A20 gene that encodes the A20 ZnF7 domain.

31. A method for modifying a T cell comprising: knocking out the endogenous A20 gene in the T cell or introducing a point mutation, insertion, deletion, or substitution in region of the A20 gene that encodes the A20 ZnF7 domain of the T cell.

32. The method of claim 31, wherein the T cell is ex vivo.

33. The method of claim 31 or 32, wherein the knocking out or introducing is done by an A20 gene-targeted CRISPR / Cas9 system.

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