Fig 2: Repetitive tumor stimulation assay shows that RASA2 ablation rescues T cells from a dysfunctional state.a, Metrics of NY-ESO-1-specific 1G4 TCR-T cells after each repetitive co-culture with A375 tumor cells, including percent positive for CD8, NY-ESO-1 1G4 TCR, and activation marker CD25 (assessed by flow cytometry, n = 4 T cell donors, lines are mean ± SEM). b, Gene expression levels for selected genes, including RASA2, with repeated tumor stimulation measured by RNAseq (n = 3 donors TCR-T cells and n = 3 donors CAR-T cells, mean ± SEM). c, T cell viability, measured by flow cytometry with Live/Dead stain, compared between RASA2 KO and control (CTRL) T cells (n = 4 donors, mean ± SEM, *p < 0.05 and **p < 0.01 for two-sided Wilcoxon test). d, RASA2 KO T cells following multiple stimulations show higher levels of phosphorylated ERK and CD69 compared to control cells (n = 2 donors). e, Fraction of T cells positive by flow cytometry for p-ERK and CD69 after 6 repeated co-cultures with A375 tumor cells (n = 2 donors, mean ± SEM, *p < 0.05 for two-sided Wilcoxon test). f, Western blot analysis for p-ERK and p-MEK levels in T cells after each repeated CD3/CD28 stimulation. g, Flow cytometry data for multiple effector cytokines (labeled on bottom) in NY-ESO-1-specific TCR-T cells (top row) and CD19-specific CAR-T cells (bottom row) after 6 repeated co-cultures with target tumor cells. h, Histograms showing CD62L levels in NY-ESO-1-specific T cells in 2 donors after 6 repeated co-cultures with A375 tumor cells. i, Percent of cells expression exhaustion-associated markers as measured by flow cytometry of T cells after multiple stimulations show similar levels between RASA2 KO and control-edited (CTRL) T cells (n = 4 donors, mean ± SEM, *p < 0.05 and ns is p > 0.05 for two-sided Wilcoxon test, shape denotes donor). Source data

Fig 3: RASA2 ablation in TCR-T and CAR-T cells improves tumor control in vivo.a, Individual subcutaneous tumor growth by caliper measurements over time in NSG mice engrafted with 1x106 A375 melanoma cells and intravenously injected with 1x106 NY-ESO-1-specific 1G4 TCR-T cells (n = 6 mice per group). b, Survival of mice shown in (a), exact p-value by log-rank test. c, Individual tumor growth by bioluminescence (BLI as total flux) measurements over time in NSG mice engrafted with 0.3x106 Nalm6 leukemia cells (engineered to express NY-ESO-1), and injected with 0.5x106 NY-ESO-1-specific TCR-T cells (n = 5 mice for RASA2 group, n = 4 for CTRL group). d, Survival of mice from two cohorts shown in (c), exact p-value by log-rank test. e, Flow cytometry data showing levels of CD19-CAR positivity in T cells immediately prior to injection into Nalm6 bearing mice. Percentages were used to adjust with the goal of equal numbers of CAR+ T cells per mouse. f, Individual tumor growth over time in NSG mice engrafted with 0.5x106 Nalm6 leukemia cells and injected with 0.2x106 CD19-specific CAR-T cells (n = 7 mice per group). g, Tumor progression in (f) was monitored using bioluminescent imaging (BLI). h, Individual tumor growth over time in a cohort of NSG mice bearing Nalm6 leukemia cells injected with CD19-specific CAR-T from an independent human blood donor (n = 8 mice per group for control-edited CAR-T cells and n = 7 mice per group for RASA2 edited CAR-T cells). i, Survival of mice shown in (h), exact p-value by log-rank test. j, CD4+ and CD8+ percentage composition of CAR-T cells isolated from the bone marrow of Nalm6-bearing mice day 7 and 16 after CAR-T cell infusion (Day 7: n = 5 for CTRL, n = 6 for RASA2, Day 16: n = 6 for CTRL, n = 6 for RASA2, error bars mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 for two-sided Wilcoxon test). k, Differentiation status based on CD45RA and CD62L staining for CAR-T cells from cohort in (j) (n = 6 per group, mean ± SEM, *p < 0.05, **p < 0.01 for two-sided Wilcoxon test, TSCM = CD62L+CD45RA+, TCM = CD62L+CD45RA-, TEM = CD62L-CD45RA-, TEMRA/EFF = CD62L-CD45RA+). l, Gating for input mixed cell populations for in vivo competition assay. T cells are first gated on CAR+ cells, then EGFR+/- populations. Input mix 1 and mix 2 each had ~50/50% AAVS1 and RASA2 KO CAR-T cells, with the EGFRt on opposite populations in each mix for identification. m, Percentage of the two different mixed CAR-T cell populations after isolation from bone marrow at days 7 and 16 post infusion into Nalm6-bearing mice and stained for CAR and EGFRt markers to determine CTRL/RASA2 KO CAR-T cells percentages in vivo (Day 7: n = 6 for CTRL, n = 6 for RASA2, Day 16: n = 6 for CTRL, n = 6 for RASA2, error bars are mean ± SEM, *p < 0.05 and **p < 0.01 for two-sided Wilcoxon test). Source data

Fig 4: RASA2 validates as a T cell target to engineer resistance to suppressive conditions.a, Western blots showing level of RASA2 ablation in T cells from 4 human blood donors. b, RASA2 KO T cells have a stimulation dependent proliferative advantage over control T cells, for two independent human donors (D1 and D2). c, CFSE staining traces showing how suppressive conditions inhibited T cell proliferation compared to vehicle conditions, for one representative human T cell donor. We note that although both RASA2-KO and control-edited (CTRL) T cells were inhibited by the suppressive signals to varying degrees, RASA2 KO T cells retain a consistent proliferation advantage in each condition. d, Summary of gated CFSE low (dividing) cells on y-axis across a range of suppressive conditions on x-axis (mean +/- SEM, n = 2 donors in 3 replicates). e, Cancer killing assay by NY-ESO-1-specific 1G4 TCR-T cells in the presence of suppressive molecules. Lines show the count of A375-RFP+ cancer cells as detected by live-cell microscopy, normalized by their count at t = 0. Grey area represents a 95% confidence interval of one representative donor with 3 technical replicates. f, Treg suppression assay. Stimulated CD8 T cells were stained by CFSE to track their proliferation in co-culture with suppressive Tregs. T cells from four distinct human donors are shown across the columns, with Tregs:CD8 ratio shown in the rows. g, Schematic of cancer killing assay by effector T cells in the presence of Tregs. h, Cancer cell killing (calculated by 1 - scaled AUC for the cancer cell growth curve) shown on the y-axis for a range of Tregs:CD8 ratios. Horizontal lines are the mean, dots are individual wells (CTRL = non-targeting guide, n = 4 human donors, each in 2 replicates, **p < 0.01 and ***p < 0.001 for two-sided Wilcoxon test). Source data

Fig 5: Multiple genome-wide CRISPR screens in primary human T cells identify RASA2 as a modulator of resistance to immunosuppressive conditions.a, Schematic of genome-wide screens for resistance gene targets in human T cells. b, Top shared gene hits (z-score >1.5) between 5 (blue) and all 6 (pink) of the screens are labelled. Bar height is the number of shared genes among the screens, connected by dots in the lower panel (n = 4 human donors for stimulated (stim) and Treg cell screens, n = 2 for adenosine, cyclosporine and tacrolimus, and n = 1 for the TGFß screen). c,d, log2 fold change (FC) for individual guide RNAs (vertical lines); background shows the overall guide distribution in greyscale. c, Guides targeting RASA2 (pink) across all suppressive conditions. d, Guides targeting RasGAP family members other than RASA2 were not enriched consistently in either direction, whereas guides targeting the RasGEF RASGRP1 were depleted from dividing cells as expected. e, Distribution of CFSE staining in RASA2-KO versus control (Ctrl; non-targeting guide RNA) T cells across all suppressive conditions. f, Cancer cell growth during in vitro cancer cell-killing assay under suppressive conditions. AUC, area under the growth curve. n = 2 donors in triplicate, shape denotes donor. g, Suppression assay confirms that RASA2 ablation rendered T cells resistant to Treg cell suppression of proliferation in vitro. Bars show the CD8+ cell count 4 days after stimulation (n = 4 donors per group; mean ± s.e.m.; **P < 0.01 and ***P < 0.001, two-sided paired Student’s t-test). h, RASA2 ablation rendered T cells resistant to Treg cell suppression compared with control T cells in an in vitro cancer cell-killing assay for one representative donor out of four (summary statistics shown in Extended Data Fig. 2g). Line is the mean and shaded area is 95% confidence interval for 3 technical replicates.Source data