Extended Data Fig. 9: P2RX7 deficiency compromises memory CD8⁺ T cell function, and P2RX7 pharmacological blockade impairs generation of CD8⁺ T cell memory cells in vivo.

a, Wild-type or P2rx7^−/− mice were infected with LCMV-Cl13, and kidney PFU levels were quantified 4 weeks later. Data from 3 independent experiments, n = 7 total. b–f, 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 then challenged (or not) with Lm-gp33 6–8 weeks later (b). Data are from 2 independent experiments (n = 5–7 total). c, Ratio of P2rx7^−/− to wild-type splenic P14 CD8⁺ T cells before (0 d) and after challenge (5 d). d, Fold increase in numbers of wild-type or P2rx7^−/− P14 cells in indicated tissues, relative to mice that did not receive Lm-gp33 challenge. e, Percentage of cells in cell cycle, determined by Ki-67 staining. f, Frequency of dying cells indicated by the percentage of annexin V⁺Live–Dead⁺ cells in mice 5 d after Lm-gp33 challenge. g, h, Wild-type and P2rx7^−/− P14 CD8⁺ T cells were individually transferred into B6.SJL mice that were subsequently infected with LCMV. After 6–8 weeks, the mice were transcervically challenged with gp33 or PBS as in Fig. 4d. g, Flow cytometry plots for IFN-γ production by wild-type or P2rx7^−/− P14 cells in mice treated with PBS or gp33. h, Percentage of IFN-γ⁺ bystander (non-P14) CD8⁺ T cells (left) and percentage of CCR7⁺CD86⁺ dendritic cells (right) in the FRT 12 h later. g, h, Data are from three independent experiments, n = 4–11 total. i, Schematic of experimental scheme combining spared nerve injury (SNI), LCMV infection and A-438079 treatment. For surgery, two of the three branches of the sciatic nerve in one hind limb were exposed and cut (Fig. 4) or left uncut (sham). After 2 weeks mice were assayed for pain sensitivity, then infected with LCMV with or without A-438079 treatment for the first week after infection. Mice were assayed again for pain sensitivity (day 7) and subsequent development of central (CD62L⁺) and effector (CD62L⁻) memory cells specific for the LCMV epitope gp33 (after day 30). Data are compiled from two independent experiments, n = 6 from all experiments. j, Pain sensitivity of sham-surgery mice (pre- and post-treatment). k, Percentages of gp33-specific T_CM cells (left) and numbers of gp33-specific T_CM and T_EM cells (right) in sham surgery animals. l–p, In other studies, B6 or Balb/C mice were adoptively transferred or not with wild-type P14 cells, infected with LCMV, and treated with A-438079 in the time frames indicated, relative to infection. Data from 2–3 independent experiments, n = 5–10 total. m, Percentages of CD62L⁺CD44⁺ (T_CM) P14 cells per spleen (left) and spleen P14 T_CM and T_EM cell numbers (right) from the different treatment groups at 4 weeks post-infection. n, P14 recipient mice were treated with PBS or A-438079 for the first week following LCMV infection, then assayed at 3 weeks for numbers of MPECs and SLECs. o, Balb/C mice were infected with LCMV, treated with A-438079 throughout the first week post-infection, and the numbers of LCMV-specific (NPtet⁺) CD8⁺ T cells were quantified in the spleen, lymph nodes and SI-IEL at 4 weeks post-infection. p, B6 mice infected with LCMV were treated with A-438079 between days 40 and 50 post-infection and the numbers of gp33⁺ CD8⁺ T cells (T_CM and T_EM) per spleen were quantified at 8 weeks post-infection. Data shown as mean ± s.e.m.; a, c–f, k, n–p, two-tailed Student’s t-test; j, two-sided Mann–Whitney’s test; h, m, one-way ANOVA with Tukey’s post-test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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