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Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

Summary Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

(B) Surface expression of CD3 in unstimulated T-cells, or T-cells stimulated in complete medium (+Arg) or in the absence of arginine (-Arg).
(G) Growth of THP1 cells incubated in complete medium (untreated) or in equivalent medium treated with recombinant human Arginase 2 with (+rhARG2 +Citr) or without (+rhARG2) the addition of citrulline. Cells were counted every 24h for 96h. Data are represented as mean ±SD, n=4.
(H) CFSE labeling analysis of THP1 cell growth in arginine-free medium, supplemented with the indicated concentrations of citrulline.
(I) Number of upregulated, downregulated and not significantly changed (NS) genes in stimulated T-cells and THP1 cells following 72h incubation in arginine-free medium. See Table  S1.
(J) Change in expression (log2(fold-change)) of genes associated with the T-cell receptor signaling KEGG pathway term in stimulated T-cells under arginine starvation. Midline shows median logFC, with upper and lower hinges showing 25 th and 75 th percentile, respectively. Upper and lower whiskers extend to the largest and smallest datapoints within 1.5 times the interquartile range of either hinge. Gene-specific logFC data are provided in Table S2. (A) qRT-PCR analysis of transporter gene expression in THP1 and T-cells stimulated in complete medium (+Arg) or in the absence of arginine (-Arg) for 72h. Data are normalized to YWHAZ, relative to +Arg T-cells, represented as mean ± SEM, n=4 (THP1), n=6 (T-cell). **** p<0.0001, ** p<0.01, * p<0.05, ns: no significant difference (Dunnett's multiple comparison test).
(D) LC-MS chromatograms showing intracellular arginine and citrulline levels in THP1 cells incubated in complete medium (+Arg) or in arginine-free medium with (-Arg +Citr) or without (-Arg) the addition of citrulline.
(F) Representative western blot of ASS1 expression in Tem, Tcm and naïve CD4+ T-cells following 72h incubation in complete medium (+), medium containing 20 µM arginine (low) or lacking arginine (-). Non-specific band is indicated by *. Lower: quantification of western blot replicates. Protein levels are normalized to GAPDH, relative to +Arg Tem cells. Data are represented as mean ± SD, n=2.
(G) Significantly enriched transcription factor motifs at the promoters of Class III genes (upregulated in THP1 but not T-cells; see Fig 1D).

RNA expression
Open circles indicate an unmethylated CpG, closed circles indicated a methylated CpG. Six rows are shown for each condition to indicate the proportion of methylation at each CpG.
(G) Levels of TCA cycle intermediates detected by LC-MS, taken from T-cells stimulated in complete medium (+Arg), medium containing 20 µM arginine (low Arg) or lacking arginine (-Arg), or CD4+ T-cells analyzed without stimulation. Midline shows median, n=5, with upper and lower hinges showing 25 th and 75 th percentile, respectively. Upper and lower whiskers extend to the largest and smallest datapoints. ** p<0.01, * p<0.05, ns: no significant differences; comparisons made with +Arg (Tukey's multiple comparison test). ND, not determined.
(H) Seahorse data from Fig 7H are replotted to show the relative differences in mitochondrial and glycolytic rate under baseline and stressed (after addition of FCCP and oligomycin) conditions.
(I) Change in expression (log2(fold-change)) of genes associated with the indicated metabolic pathways in stimulated T-cells under arginine starvation. Midline shows median, with upper and lower hinges showing 25 th and 75 th percentile, respectively. Upper and lower whiskers extend to the largest and smallest datapoints within 1.5 times the interquartile range of either hinge. Gene-specific logFC data are provided in Table S2.