Extended Data Fig. 10: Metabolic dependence of PD-1 expression on intratumoral T_reg cells, and model of metabolic adaptation.

a, Left, flow-cytometry analysis, and right, quantification of PD-1 MFI on intratumoral T_reg cells from Foxp3^CreFasn^+/+ or Foxp3^CreFasn^+/fl control (n = 9) and Foxp3^CreFasn^fl/fl (n = 10) mice bearing B16 melanoma on day 17. b, Resting T_reg cells were sorted from Foxp3^CreHmgcr^+/fl and Foxp3^CreHmgcr^fl/fl mice under steady state (n = 4 samples per genotype), labelled with CTV, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Shown is relative PD-1 MFI on CTV^lo cells. c, d, Foxp3–YFP⁺CD4⁺ cells were sorted from Foxp3^CreScap^+/fl and Foxp3^CreScap^fl/fl mice, labelled with CTV, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days in the presence of cholesterol or vehicle (T_reg cells were pretreated with cholesterol or vehicle for 1 h before activation; n = 6 samples per group). Shown are flow-cytometry analysis (left) and relative MFI (right) of CTLA4 (c) and PD-1 (d) in CTV^lo cells. e, Foxp3–YFP⁺CD4⁺ cells were sorted from Foxp3^Cre mice, treated with vehicle or simvastatin (2 μM), and then stimulated with anti-CD3/28 antibodies plus IL-2 for 18 h in the presence of mevalonate (500 μM), FPP (50 μM), GGPP (5 μM) or DMSO for real-time PCR analysis of Pdcd1 mRNA expression (n = 4 per group). f, T_reg cells from Foxp3^Cre mice were labelled with CTV and pretreated with vehicle, FTI-277 (FTI) (10 μM) or GGTI-2147 (GGTI) (5 μM) for 1 h and then stimulated with anti-CD3/28 antibodies plus IL-2 for 72 h. Shown is relative MFI of PD-1 on CTV^lo cells (n = 8 per group). g, Resting T_reg cells were sorted from young (1–3 weeks) Foxp3^CrePggt1b^+/fl and Foxp3^CrePggt1b^fl/fl mice (n = 4 samples per genotype) under steady state; young mice were used to limit the influence of systemic inflammation. T_reg cells were labelled with CTV and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, relative MFI of PD-1 on CTV^lo cells. h, Resting T_reg cells were sorted from young (1–3 weeks) Foxp3^CreFntb^+/+ (n = 2) and Foxp3^CreFntb^fl/fl (n = 3) mice under steady state. T_reg cells were labelled with CTV and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, relative MFI of PD-1 on CTV^lo cells. i, Resting T_reg cells were sorted from Foxp3^Cre control and Foxp3^CreRac1^fl/flRac2^–/– mice (n = 4 mice per genotype) under steady state, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, and relative MFI of PD-1. j, Foxp3–YFP⁺CD4⁺ cells were sorted from Foxp3^Cre mice (n = 5) and stimulated with anti-CD3/28 antibodies plus IL-2 for 3 days, in the presence of vehicle control, simvastatin (2 μM), GGTI (5 μM) or two different AP-1 inhibitors (Sr11302 (30 μM) or T-5224 (120 μM)). Top, flow-cytometry analysis, and bottom, relative MFI of PD-1. k, SREBP-driven metabolic and functional adaptation of T_reg cells in the TME. SREBPs are activated in intratumoral T_reg cells as a key driver of the functional specialization of T_reg cells in the TME, which leads to suppression of effective antitumour immune responses and the reduced efficacy of immune-checkpoint therapy. Mechanistically, SREBP-dependent de novo fatty-acid synthesis and PD-1 signalling in T_reg cells enforce the suppressive function of T_reg cells in tumours. T_reg cells show enhanced Pdcd1 expression in tumours, and this upregulation is dependent on SREBP-dependent mevalonate metabolism that further signals to Pggt1b-driven protein geranylgeranylation. PD-1 induction is required to repress overt production of IFNγ by T_reg cells in response to TCR activation in the TME, suggesting that SREBP signalling supports the functional fitness of intratumoral T_reg cells, in part by preventing the fragility of T_reg cells. Therefore, SREBP-dependent metabolic reprogramming enforces the functional specialization of T_reg cells in tumours by coordinating lipid synthesis and inhibitory receptor signalling pathways. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (a, b, g–i) or one-way ANOVA (c–f, j). Data are means ± s.e.m. in a–j. Data are compiled from three (b–d, f, g) or two (a, e, h–j) independent experiments. Values in control samples were set to 1 (a–d, f–j).

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