Extended Data Fig. 8: P2RX7-mediated eATP sensing is crucial for optimal CD8⁺ T cell immunometabolism by regulation of the AMPK–mTOR pathway.

a–g, Wild-type and P2rx7^−/− P14 cells were activated in vitro and polarized with IL-15 (as in Fig. 3b). Data from three independent experiments, samples pooled from n = 6 mice per experiment; n = 3–12 total samples. a–c, Numbers (left) and viability (right) of P14 cells in cultures supplemented with apyrase (a), oATP (b) or BzATP (c) during cell culture. d, e, g, IL-15-polarized wild-type or P2rx7^−/− P14 cells were assayed for OCR 1 h after addition of apyrase (d) or oATP (e), or 6 h after addition of A-438079 (g). f, IL-15-polarized cells or ex vivo wild-type P14 T_CM cells (isolated 4 weeks after LCMV infection) were incubated with DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of BzATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca²⁺ influx (right) over 30 min are shown. f, Data from two independent experiments, samples pooled from n = 5 mice total; n = 2–5 samples. h, In vitro-activated (72 h) wild-type and P2rx7^−/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n = 9 total. i, In vitro-activated, IL-15 polarized (24 h post-polarization) wild-type or P2rx7^−/− P14 cells were assayed for extracellular ATP concentration, following culture without or with the Panx1 inhibitor ¹⁰Panx. Data from two independent experiments, n = 3–4 total samples (pooled from six mice). j, Wild-type and P2rx7^−/− P14 cells were co-adoptively transferred and assayed 4 weeks after LCMV infection (as in Fig. 1a) and the ex vivo frequency of pS6-expressing cells was determined by flow cytometry. Data are from two independent experiments (n = 6 total). k, Expression of pACC in IL-15-polarized wild-type (black) and P2rx7^−/− (red) P14 cells (relative to Fig. 3l; representative of three independent experiments, n = 6 total). l, In vitro-activated and IL-15-polarized wild-type and P2rx7^−/− P14 cells were cultured for 6 h with the indicated concentrations of BzATP, then stained for pACC (left) and pS6 (centre), and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n = 6–8 total. m, In vitro-activated wild-type and P2rx7^−/− P14 cells were IL-15-polarized in the presence or absence of AICAR as in Fig. 3l. The percentage of viable cells at the indicated times following initiation of IL-15 culture with or without AICAR is indicated. Data are from three independent experiments (n = 3–6 total; samples pooled from n = 6 mice total). n–p, Wild-type and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1 and co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1. The mice were treated with metformin or PBS control during the first week of LCMV infection, and the cells were analysed at day 30. Data are compiled from three independent experiments (n = 11–12 total, n = 4 for FRT samples). n, Relates to Fig. 3m; ratio of P2rx7^−/− to wild-type P14 cells in the indicated non-lymphoid tissues (n = 9 except FRT, n = 4). o, p, Measurements of mitochondrial mass (measured using MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for the indicated splenocyte subsets (n = 3–6 total samples). q, Wild-type and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1 and co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells were identified as in Fig. 1. The mice were treated with rapamycin or PBS control between days 4 and 8 post-LCMV infection, and the cells were analysed at day 30. The numbers of wild-type and P2rx7^−/− P14 cells are shown (log-transformed). Data are compiled from three independent experiments (n = 15 total). a–j, l–q, Mean ± s.e.m.; a–c, h–j, m–p, two-tailed Student’s t-test; l, q, one-way ANOVA with Tukey’s post-test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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