Ext. Data Fig. 8. P2RX7-mediated eATP sensing is crucial for optimal CD8⁺ T cell immunometabolism via AMPK/mTOR pathway regulation.

(a–g), WT and P2rx7^−/− P14 cells were in vitro activated 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) show the numbers of P14 cells (left) and viability (right) in cultures supplemented with (a) the eATP hydrolytic enzyme Apyrase, (b) the inhibitor oATP or (c) the eATP analog BzATP, during cell culture. In (d,e,g) IL-15-polarized WT or P2rx7^−/− P14 cells were assayed for OCR 1h after addition of apyrase (d) or oATP (e), or 6h after addition of A-438079 (g). In (f), IL-15-polarized cells or ex vivo WT P14 T_CM (isolated 4w after LCMV infection) were incubated with either DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of Bz-ATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca²⁺ influx (right) over 30m are shown. (f) Data from two independent experiments, samples pooled from n=5 mice total; n=2–5 samples. In (h), in vitro-activated (72h) WT and P2rx7^−/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n=9 total. In (i), In vitro-activated, IL-15 polarized (24h post-polarization) WT 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 6 mice). (j), WT and P2rx7^−/− P14 cells were co-adoptively transferred and assayed 4w post LCMV infection (as in Fig. 1a) and the ex vivo frequency of pS6-expressing cells determined by flow cytometry. Data are from two independent experiments (n=6 total). (k) Representative histograms showing expression of pACC in IL-15-polarized WT (black) and P2rx7^−/− (red) P14 cells (relative to Fig. 3l; representative from three independent experiments, n=6 total). (l) In vitro activated and IL-15-polarized WT and P2rx7^−/− P14 cells were cultured for 6h with the indicated concentrations of BzATP then stained for pACC (left) and pS6 (center) and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n=6–8 total. (m) In vitro-activated WT and P2rx7^−/− P14 cells were IL-15 polarized in presence or absence of the AMPK activator AICAR as in Fig. 3l. The percentage of viable cells at the indicated times following initiation of IL-15 +/− AICAR culture is indicated. Data are from three independent experiments (n=3–6 total; samples pooled from n=6 mice total). (n–p) WT and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1. The animals were treated with Metformin or PBS control during the first week of LCMV infection, and the cells analyzed at day 30. Data are compiled from three independent experiments (n=11–12 total, n=4 for FRT samples). Panel (n) relates to Fig. 3m and shows P2rx7^−/− /WT P14 ratio in indicated non-lymphoid tissues (n=9 except female reproductive tract – FRT – where n=4). (o,p) shows measurements of mitochondrial mass (measured by MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for indicated splenocyte subsets (n=3–6 total samples). (q) WT and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1. The animals were treated with Rapamycin or PBS control between days 4–8 post-LCMV infection, and the cells analyzed at day 30. The numbers of WT or P2rx7^−/− P14 cells are shown (log-transformed values). Data are compiled from three independent experiments (n=15 total). (a–j,l–q), mean ± SEM is shown; (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.