Extended Data Fig. 5. Endogenous antioxidant production is limiting for T cell proliferation.

(a) Motif analysis of sites with increased accessibility in tumor-infiltrating CD8+ T cells (L7) as compared to T cells from Listeria-infected mice (E7) showing NFATc1 as among the motifs whose accessibility was most preferentially increased in L7 cells¹⁵. (b) Intracellular calcium flux as measured by ratio of bound to unbound Indo-1-AM in acutely and chronically stimulated T cells, at baseline, in response to monomeric anti-CD3, and in response to receptor clustering (streptavidin). (c) Gene set enrichment plot showing that chronically stimulated OT-I T cells are enriched for NFAT target genes. (d) Expression of NFAT target genes (“nfat score”) in independent CD8+ T cell clusters. (e) Correlation of expression of NFAT target genes (“nfat score”) with expression of oxidative stress-related metabolic genes (“ROS score”) in tumor-infiltrating CD8+ T cells from melanoma patients treated with immune checkpoint inhibitors. (f) Fluorescence intensity of acutely and chronically stimulated T cells cultured with or without βME supplementation after loading with CM-H₂DCFDA to measure ROS. Light-grey-shaded peak represents negative control. (g) Proliferation of T cells acutely stimulated in the presence or absence of BSO or diamide as measured by dilution of Cell Trace Violet fluorescence. (h) Expression of TCF-1 and TOX in chronically stimulated T cells cultured in the presence or absence of BSO. P values were calculated by one-sided Student’s t-test relative to base mean (d-e). ****P<0.0001