Extended Data Fig. 5: RASA2 knockout promotes transcriptional reprogramming and RASA2 expression is differentially regulated by acute and chronic antigen stimulation.
From: RASA2 ablation in T cells boosts antigen sensitivity and long-term function
a, Fraction of Jurkat cells positive for three different mCherry reporter cell lines responsive to the transcription factor as indicated to the right of each panel, after TCR stimulation with titrated dilutions (1:1 = 25 µl/ml) of anti-CD3/CD28 complexes (columns) over 72 h (dots show 3 technical replicates, p-value from a two-sample Kolmogorov-Smirnov test). b, Volcano plot showing differentially regulated genes between RASA2 KO and control-edited stimulated T cells as determined by RNA-Seq. Genes highlighted have BH adjusted, two-tailed Wald test p-value < 0.0001 and absolute log2 fold change > 1, as determined by DESeq2 analysis (methods). c, d Gene set enrichment analysis (GSEA) of oxidative phosphorylation (c) and glycolysis (d) ranked genes, based on DESeq2, higher rank indicates enrichment in RASA2 KO over CTRL. p-value is shown as determined by a two-sided permutation-test. Top up-regulated genes in each enrichment are listed to the right of each panel e, GSEA of oxidative phosphorylation genes correlated with RASA2 expression in immune cells in the GEO expression database, as retrieved by correlationAnalyzeR (methods). Genes are ranked by the Pearson correlation coefficients between RASA2 and the query gene, p-value by two-sided permutation test, after FDR adjustment. f, Selected examples of expression of RASA2 and two mitochondrial fitness genes, MRPL27 (left panel) and MRPL14 (right panel) across GEO datasets from immune cells. Shown at the top is the Pearson’s correlation coefficient (R) and FDR adjusted p-value (padj) for each scatter plot. Values represent expression after variance stabilizing transformation (VST). g, Expression of RASA2 in stimulated T cells compared to unstimulated T cells, as measured by published single-cell RNA-Seq dataset, for two human donors13. h, i, Expression of Rasa2 compared to Pdcd1 in published RNA-Seq datasets from models of T cell exhaustion in murine T cells. Expression was scaled for a maximum of 1 and a minimum of 0 for each gene in each dataset (For LCMV samples in (h): n = 2 mice for Naive group, n = 3 mice for exhaustion group; for OVA samples, error bars are mean ± SEM (i): n = 3 mice for all groups, error bars are mean ± SEM). j, Log fold change (LFC) values for RASA2 sgRNAs in CRISPRa and CRISPRi screens for cytokine production42 (MAGeCK’s gene-level FDR listed for RASA2 in each screen). k, Western blot for level of RASA2 expression following RASA2 transgene or control (CTRL) transduction in two T cell donors. l, Normalized values for T cell expansion based on cell counts for T cells with RASA2 transgene versus GFP control (n = 3 human T cell donors, mean ± SEM, shape denotes donor). m, histogram for one example donor from cells described in (l), stained and FACS analyzed for CD69 activation marker. n, Summary data for CD69 levels in two T cell donors transduced with the RASA2 transgene versus control (n = 2 human T cell donors, shape denotes donor).
