Fig. 3: DR antagonizes terminal CD8+ T cell exhaustion in the TME.
a, WnnUMAP of 7,005 activated (CD44+) CD8⁺ TILs from B16 melanoma tumours (combined AL and DR) (n = 4). Legend indicates unique T cell clusters called based on RNA expression and ADTs. T cell clusters with high and low Tox expression are highlighted by circles. b, Overlay of wnnUMAP for activated CD8+ TIL from a and MSigDB gene expression signatures for TexEff, cell cycle and Gene Ontology (GO) Immune Response pathways for TILs isolated from B16 tumours (combined AL and DR). Joint density plot indicates the highest combined expression/cell of Ifng, Tnfa and Gzmb among activated CD8⁺ T cells from B16 tumours. c, wnnUMAP of activated CD8⁺ TILs divided by dietary conditions (AL, 3,348 cells; DR, 3,657 cells). Prominent CD8⁺ T cell clusters are indicated. d, Stacked bar graphs showing the percentage of CD8⁺ T cell populations divided by Tox expression (Toxlo versus Toxhi). e, RNA velocity plots inferring cellular differentiation trajectory for CD8⁺ T cells infiltrating B16 tumours from AL-fed or DR-fed mice. Trajectories were derived from the dynamical prediction model scVelo, with representative directionality for T cells under each diet shown in the inset. f, PD1 and TOX expression in B16 tumour-infiltrating CD8⁺ T cells isolated from AL or DR mice 14 days PTI. Left, representative flow cytometry plots for PD1 versus TOX expression; right, bar graph showing the percentage of PD1⁺TOX⁺ CD8⁺ T cells isolated from tumours. Data represent the means; error bars, s.e.m. (n = 8 AL and 7 DR mice). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. g, Expression of stemness and exhaustion markers in TOX⁺ CD8⁺ T cells isolated from B16 tumours from AL-fed or DR-fed mice. Top, representative histograms of LY108, PD1 and TOX expression. Geometric mean fluorescence intensity averaged across all biological replicates is indicated. Bottom, bar graphs quantifying the percentage of CD8+ cells expressing each of the indicated proteins. Data represent the means; error bars, s.e.m. (n = 8 AL and 7 DR mice). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. h, Expression of LY108 versus TIM3 among PD1⁺ CD8⁺ TILs from B16 tumours. Left, representative flow cytometry plots showing LY108 and TIM3 expression; right, bar graph showing percentages of progenitor (TexProg, LY108+TIM3−) and terminal (TexTerm, LY108−TIM3+) Tex cell subsets under AL and DR conditions. Data represent the means; error bars, s.e.m. (n = 8 mice per group). Statistical significance was calculated with a two-way ANOVA multiple comparisons test. i, PD1 and TOX expression in EO771 (breast cancer) tumour-infiltrating CD8⁺ T cells isolated from AL or DR mice 14 days PTI. Left, representative flow cytometry plots for PD1 versus TOX expression; right, bar graph showing the percentage of TOX⁺ CD8⁺ T cells isolated from tumours. Data represent the means; error bars, s.e.m. (n = 4 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. j, CD44 and IFNγ expression in EO771 (breast cancer) tumour-infiltrating CD8⁺ T cells isolated from AL or DR mice 14 days PTI. Left, representative flow cytometry plots for CD44 versus IFNγ expression; right, bar graph showing the percentage of IFNγ-producing cells from PD1+TOX⁺ CD8⁺ T cells isolated from EO771 tumours. Data represent the means; error bars, s.e.m. (n = 4 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are as follows: f, P = 0.0002; g, left, P < 0.0001, middle, P = 0.0469, right, P = 0.0004; h, left, P = 0.0003, right, P < 0.0001; i, P = 0.032; j, P = 0.0275.
