Fig. 7: DR synergizes with anti-PD1 immunotherapy to enhance anti-tumour immunity.

a, Kaplan–Meier plot comparing B16 melanoma tumour onset in AL-fed or DR-fed mice that received anti-PD1 or IgG control antibodies by intraperitoneal injection (200 μg per dose). Antibody treatment was administered every 3 days for a total of five injections, beginning 7 days PTI (n = 11–20 mice per group). Statistical significance was assessed by a log-rank test with Bonferroni correction. b, PD1 and TOX expression in B16 tumour-infiltrating CD8⁺ T cells from DR-fed mice treated with anti-PD1 immunotherapy. DR-fed mice harbouring B16 tumours were administered anti-IgG or anti-PD1 antibodies (200 μg intraperitoneally) every 3 days for five doses, beginning on day 7 PTI. CD8⁺ T cells were isolated from B16 melanoma tumours at 21 days PTI. Representative flow cytometry plots for TOX versus PD1 expression. c, Bar plot representing the per cent of T_eff cells (PD1⁻TOX⁻CD8⁺ TILs). Data represent the means; error bars, s.e.m. (n = 8 mice per group). Bottom, bar plot representing the per cent of T_ex^Term cells (PD1⁺TOX⁺CD8⁺ TILs). Data represent the means; error bars, s.e.m. (n = 8 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. d, Representative histograms of GZMB expression of T_eff from DR-fed mice harbouring B16 tumours that were administered anti-IgG or anti-PD1 antibodies. Inset, gMFI values for GZMB. Data represent the means; error bars, s.e.m. (n = 8). Right, bar graph showing the percentage of PD1⁻TOX⁻ CD8⁺ T cells (T_eff) expressing GZMB following IgG or anti-PD1 treatment. Data represent the means; error bars, s.e.m. (n = 8 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. e, Representative flow cytometry plots of TNF versus IFNγ expression of PD1⁺TOX⁺ CD8⁺ TILs (T_ex^Term) from DR-fed mice harbouring B16 tumours that were administered anti-IgG or anti-PD1 antibodies. Right, bar graphs quantifying the percentage of PD1⁺TOX⁺ CD8⁺ T cells expressing IFNγ and TNF (IFNG⁺TNF⁺). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. Data represent the means; error bars, s.e.m. (n = 7 for AL and n = 8 for DR). f, Representative histograms of GZMB expression of T_ex^Term T cells from DR-fed mice harbouring B16 tumours that were administered anti-IgG or anti-PD1 antibodies from a. Inset, gMFI values for GZMB. Data represent the means; error bars, s.e.m. (n = 8). Right, bar graph showing the percentage of GZMB production from T_ex^Term CD8⁺ TILs that were previously treated with anti-IgG or anti-PD1 from a. Data represent the means; error bars, s.e.m. (n = 8 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. g, Kaplan–Meier analysis of tumour-free survival in dietary-restricted WT and ketolysis-deficient DKO mice (Cd4-Cre driven) treated with anti-PD1 or isotype control IgG. DR-conditioned WT (blue) and DKO (red) mice bearing subcutaneous B16 melanoma allografts received either control IgG (solid lines) or anti-PD1 antibody (dashed lines) once tumours reached 7 days PTI. Survival is plotted as days PTI. n = 9–13 mice per group. Statistical significance was assessed by a log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are as follows: a, AL (IgG) to DR (IgG), P < 0.0001; DR (IgG) to DR (anti-PD1), P = 0.005; c, top, P = 0.0175; bottom, P < 0.0001; d, P = 0.0157; e, P = 0.0376; f, P = 0.0279; g, WT (IgG) to WT (anti-PD1), P = 0.0083; WT (anti-PD1) to DKO (anti-PD1), P = 0.0391.

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