Extended Data Fig. 1: Multiple genome-wide CRISPR screens for T cell resistance.

a, Dropout of essential genes¹⁹ across screen conditions. X-axis is scaled and binned log2 fold change of CFSE low over CFSE high cells in each screen, Y-axis is the number of essential genes in each bin. As expected, essential genes tend to have a negative LFC in these CRISPR KO screens of primary human T cells (n = 4 human donors for Stim and Tregs screens, n = 2 for Adenosine, Cyclosporine and Tacrolimus screens, and n = 1 for the TGFβ screen. Normalized Enrichment Score (NES) and adjusted p-value by GSEA and two-sided permutation test). b, Screen hits are expressed in human T cells. X-axis is scaled and binned log2 fold change in each screen, Y-axis shows the expression in activated human T cells²⁰. Both negative and positive hits tended to be highly expressed in human T cells, suggesting these pooled KO screens point to relevant T cell biology (n = 4 human donors for Stim and Tregs screens, n = 2 for Adenosine, Cyclosporine and Tacrolimus screens, and n = 1 for the TGFβ screen, dots are mean +/− SEM). c, Shared hits (y-axis) (z-score > 1.5, methods) across the screen conditions (x-axis) including hits unique to each individual screen. Shared hits for each subset are detailed in Supplementary Table 1. d, Heatmap of the pairwise Pearson’s correlation coefficient for gene-level z-scores for all screen conditions. e, Volcano plots showing p-value (MAGeCK RRA one-sided test and methods) on the y-axis and gene-level z-scores on the x-axis, comparing highly dividing cells in each suppressive condition to highly dividing in the vehicle condition. Highlighted are genes found to be specific to adenosine and TGFβ screens, selected for further validation. f, Gene targets from screens were selected as either general (PAN) or more specific to certain suppressive contexts and were knocked out individually in T cells. CFSE stained, Cas9 RNP-electroporated edited T cells were stimulated and cultured in the different suppressive conditions. Percent of cells proliferating for each gene KO compared to control cells are displayed for each suppressive condition (n = 2 donors, 2 sgRNAs per gene target in triplicates. We highlighted gene KOs found to confer significant resistance in predicted conditions (adenosine, TGFB, and calcium/calcineurin inhibitors – Tacrolimus, and Cyclosporine), using a cut-off of FDR adjusted p-value < 0.05. For clarity, displayed are the significant p-values for gene targets according to their suppressive screen condition of origin, but results for all genes across conditions are detailed in Supplementary Table 3). As expected, ADORA2A, TGFBR1 and TGFBR2, FKBP1A, and PPIA KOs conferred resistance in the adenosine, TGFB, Tacrolimus, and Cyclosporine conditions, respectively. PDE4C and NKX2-6 KOs were found to confer relatively selective resistance in the adenosine condition, and NFKB2 KO was found to increase resistance in the calcineurin inhibitor (tacrolimus and cyclosporine) conditions. TMEM222, while scoring very highly in the screens, did not increase proliferative advantage in this arrayed validation (dots are individuals replicates, black vertical lines are the mean, *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001 for two-sided unpaired Student’s t-test). g, Log fold change (LFC) of guides targeting RasGAP genes or the RasGEF RASGRP1, across the different suppressive screen conditions shown here. h, Expression levels (scaled to minimum of 0 and a maximum of 1) of the RasGAP family members available in the BioGPS dataset²⁷, including RASA2, across healthy human tissues revealed RASA2 as selectively expressed in CD8⁺/4⁺ human T cells. Data also shown for RASGRP1, a RasGEF with defined roles in TCR signaling and an expression pattern strikingly similar to that of RASA2.

Source data