Fig. 4: Serine and glycine are limiting amino acids in the T cell compartment of tumors and are necessary for an effective T cell cytotoxic response.

a Schematic of experimental design. Female CD4-Cre:RiboTag mice (7-8 weeks-old) were treated with two doses of anti-PD1(2 µg/µl) or IgG (2 µg/µl). Two days after the last dose, tumors were flash frozen, lysed, and ribosomes from each cell type were immunoprecipitated using either anti-HA or anti-GFPs coated beads. b Diricore analysis of tumors treated as described in (a). Ribosome stalling in tumor-infiltrated T cells and E0771 tumor cells are shown in the upper and lower panels, respectively. Ribosome density at the A site is shown. *Out-of-frame analysis. p-values were determined using a two-tailed z-test. p < 0.01 for the TCA, TCC, TCG, TCT Ser codons and for the GGA, GGC, GGG, GGT Gly codons. Source data including exact p-values are provided as Source Data file. c Killing assay by CD8 + T cells in co-culture with E0771-OVA or TC1-OVA cells in serine/glycine (SG)-deprived medium. Data represent mean ± SD from biologically independent experiments (n = 3); p-values were calculated using a two-tailed unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. Source data including exact p-values are provided as Source Data file. d Quantification of IFNγ and GzmB levels produced by CD8 + T cells when co-culture with E0771-OVA cancer cells in full medium or SG-deprived medium. Data represent mean ± SD from biologically independent experiments (n = 3); p-values were calculated using a two-tailed unpaired t-test. **p < 0.01; ***p < 0.001. Source data including exact p-values are provided as Source Data file. e Schematic for ¹³C₆-Glucose tracing into the serine/glycine synthesis pathway. Blue circles represent ¹³C atoms while white circles represent ¹²C atoms. f Contribution of ¹³C₆-Glucose to serine M + 3 and glycine M + 2 in OT-1 CD8 + T cells and E0771-OVA breast cancer cells growing as mono- or co-cultures (CoC). Data represent mean ± SD from biologically independent experiments (n = 3); p-values were calculated using a two-tailed unpaired t-test. ***p < 0.001. Source data including exact p-values are provided as Source Data file. qRT-PCR quantification (g) and Western blotting (h) of the indicated genes in co-cultures (CoC) of OT-1 CD8 + T cells and OVA-expressing cancer cell lines. Cells were separated by size after 24 hours of co-culture (Supplementary Fig. 6c). Data represent mean ± SD from biologically independent experiments (n = 5 for T cells and n = 3 for cancer cells); NS not significant; p-values were calculated using a two-tailed unpaired t-test. **p < 0.01; ***p < 0.001. Source data including exact p-values are provided as Source Data file. i Protein synthesis rates based on OP-Puro incorporation in OT-1 CD8 + T cells and E0771-OVA breast cancer cells growing as mono- or co-cultures (CoC). Cells were grown in full medium (SG + ) or serine/glycine-deprived medium (SG−). Data represent mean ± SD from biologically independent experiments (n = 3); p-values were calculated using a two-tailed unpaired t-test. **p < 0.01; ***p < 0.001. Source data including exact p-values are provided as Source Data file. j Ser-tRNAs, Gly-tRNAs and control Leu-tRNAs aminoacylation analysis in OT-1 CD8 + T cells and E0771-OVA breast cancer cells growing as mono- or co-cultures (CoC).