The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells

Abstract

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage¹. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX7^2-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection^5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8⁺ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8⁺ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8⁺ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8⁺ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8⁺ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8⁺ T cell populations.

Ext. Data Fig. 1. P2RX7 is required for CD103^hi T_RM and T_CM generation upon acute viral infection, and for establishment of Ag-specific CD8⁺ T cells upon chronic viral infection

(a–e), 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. (data from 3–4 independent experiments, n=4 FRT and n=5–7 other organs from all experiments). (a), P2rx7^−/−/WT P14 ratios in different non-lymphoid organs over time. (b), Flow cytometric plots showing CD69 and CD103 co-expression by WT and P2rx7^−/− P14 cells from SI IEL and SI LP 4-weeks post-infection (representative of 3 experiments). (c), P2rx7^−/−/WT P14 ratios for CD103^hi, CD103^int and CD103^low subsets among SI LP over time. (d–e), We also evaluated the role of P2RX7 in the generation and maintenance of memory CD8⁺ T cell subsets based on CX3CR1 expression. (d) representative plot depicting the subpopulations studied (left) and P2RX7 median in these subsets (right). (e) Ratio of P2rx7^−/− to WT P14 CD8⁺ T cells in spleen, gated on indicated subsets. Data from 2–3 independent experiments, n=4–7 mice total. (f) WT and P2rx7^−/− OT-I CD8⁺ T cells were mixed 1:1 and co-adoptively transferred into B6 mice subsequently infected with VSV-OVA (two independent experiments, n=7 from all experiments). WT to P2rx7^−/− OT-I ratio in indicated tissues was determined 4-weeks post VSV-OVA infection. (g), The indicated radiation bone marrow chimeras were generated and infected with LCMV. Percentages of splenic D^b/gp33-tetramer binding (“gp33⁺”) CD8⁺ T_CM was determined 8-weeks post-infection with LCMV (data from two independent experiments, n=5 from all experiments). (h) WT or P2rx7^−/− mice were infected with LCMV-Arm or LCMV-Cl13, and the numbers (left) and percentages of CXCR5⁺PD1^low (right) gp33⁺ CD8⁺ T cells were evaluated 4 weeks after infection (n=6–13 from all experiments). (a,c–h) mean ± SEM is shown; (g–h), Two-tailed Student’s t-test, *P≤0.05, **P≤0.01, ***P≤0.001.

Ext. Data Fig. 2. Long-lived memory CD8⁺ T cells express higher levels of functional P2RX7

(a), Flow cytometry histograms for P2RX7 staining on naïve WT or P2rx7^−/− P14 cells (representative of 3 experiments). (b), Median average values for P2RX7 staining on naïve WT and P2rx7^−/−, and WT P14 cells 7d post-LCMV infection (data from three independent experiments, n=10 total). (c–h), Indicated subsets of adoptively transferred WT P14 CD8⁺ T cells from listed tissues were assayed for P2RX7 expression and functional response to the P2RX7 agonist BzATP following priming with LCMV for indicated time (data from three independent experiments, n=6–21 from all experiments). (c,d), Percentage of P2RX7^hi WT P14 cells in MPEC and SLEC (c) and T_CM and T_EM subsets (d) at 4-weeks post-infection. (e), P2RX7 median fold increase (relative to expression in naïve CD8⁺ T cells, showed as a dashed line) of P14 cells in blood, lymphoid and non-lymphoid organs (4-weeks post-infection). (f), P2RX7 median fold increase (relative to naïve CD8+ T cells) of CD103⁺ vs CD103⁻ P14 cells in SI IEL and SI LP. (g–h), adoptively transferred and LCMV primed WT and P2rx7^−/− P14 cells were incubated with DAPI then stimulated during flow cytometry with 300 µM Bz-ATP (which mediates P2RX7 pore opening and DAPI uptake). (g) shows flow cytometry plots of DAPI uptake by SI IEL and SI LP WT and P2rx7^−/− P14 CD8⁺ T cells over 30 minutes time (representative of 6 samples), while (h) shows compiled data for percentage DAPI⁺ P14 cells (defined as the percentage above P2rx7^−/− DAPI levels 5 minutes after Bz-ATP stimulation) in spleen, SI IEL and SI LP P14 cells (n=6 from all experiments). (b–f, h) mean ± SEM is shown; (b), One-way ANOVA with Tukey post-test; (c,d,f,h), Two-tailed Student’s t-test; *P≤0.05, **P≤0.01, ***P≤0.001.

Ext. Data Fig. 3. P2RX7 is required for optimal metabolism of MPECs and T_CM cells

WT (black) and P2rx7^−/− (red) 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. (a) Numbers of WT and P2rx7^−/− P14 MPECs and SLECs in spleens (Three independent experiments, n=5–6 from all experiments). (b) Gene expression profile from WT versus P2rx7^−/− P14 MPECs (sorted 2-weeks post-infection with LCMV). Gene expression in SLEC populations, sorted and analyzed at the same time, also showed minimal differences between WT and P2rx7^−/− groups (data not shown). (c–g), at day 8 (c,e–f) or 14d (d,g), splenic WT and P2rx7^−/− MPEC and SLEC subpopulations were stained for MTG (c), Cox-IV (d) TMRE (e) or for 2-NBDG uptake (f,g); two independent experiments, n=4–5 from all experiments. In (h), the ECAR levels in MPECs and SLECs (from the experiments described in Fig. 2d–f) are shown (three independent experiments, n=8–12). In i–l, 4-weeks following priming with LCMV, WT and P2rx7^−/− P14 CD8⁺ T_CM and T_EM subsets were assessed for expression of Cox-IV (i), Glut1 (j) or CPT1a (l), or uptake of the dye Bodipy (k). In (j–l), representative flow cytometric plots are shown for T_CM populations (relative to naïve WT host CD8⁺ T cells) on the left, and average median different in staining relative to naïve WT host CD8⁺ T cells. (i–l) Three independent experiments, n=5–10 from all experiments. (a,c–l) mean ± SEM is shown; (a,c,e–l) Two-tailed Student’s t-test; (d) One-way ANOVA with Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001.

Ext. Data Fig. 4. P2RX7 signaling is required for survival rather than homeostatic proliferation of long-lived memory CD8⁺ T cells

(a–g) WT (black) and P2rx7^−/− (red) 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 (data from three independent experiments, n=4–10 from all experiments). (a–d) the percentage of Ki-67⁺ cells (ex vivo staining; a), BrdU⁺ cells (b), Annexin V⁺ Live/Dead⁺ cells (c), and Bcl2 median fold increase (relative to median values of naïve CD8⁺ T cells) for bulk, T_CM and T_EM subsets (d) was determined for WT (black) and P2rx7^−/− (red) P14 CD8⁺ T cells 8-weeks post-infection (same color scheme used throughout figure). (e) shows median fold increase (relative to naïve CD8⁺ T cells) for expression of Tcf1 (left), Bcl2 (center) and Eomes (right) in MPECs and SLECs WT and P2rx7^−/− P14 CD8⁺ T cells at the indicated times. In (f), expression of Tcf1 (left) and Eomes (right) (shown as median expression relative to naïve CD8⁺ T cells) was determined in in splenic T_EM and T_CM subsets of WT and P2rx7^−/− P14 CD8⁺ T cells, 4-weeks post-infection). In (g), CD127 (left) and CD122 (right) median expression (relative to naïve CD8⁺ T cells) for splenic bulk, T_CM and T_EM subsets was determined 4-weeks post-infection. (h,i), WT and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1 and co-adoptively transferred into B6.SJL or IL-15^−/− mice, subsequently infected with LCMV (data from two independent experiments, n=4–5 from all experiments). (h) shows numbers of WT and P2rx7^−/− P14 CD8⁺ T cells in each host (4-weeks post-infection). (i) Ratio of total and T_CM P2rx7^−/− to WT P14 CD8⁺ T cells in spleens from WT and IL-15^−/− hosts at indicated times post-infection. (j,k), Congenically distinct WT and P2rx7^−/− P14 CD8⁺ T cells were stained with CFSE, mixed 1:1 and co-adoptively transferred into Rag2^−/− mice. Data from 2 independent experiments, n=6 total. (j) Ratio of P2rx7^−/− to WT P14 CD8⁺ T cells in the blood of Rag2^−/− hosts at indicated times post-transfer. (k), Percentages of P2rx7^−/− and WT P14 CD8⁺ T cells proliferating over 4 cycles in spleens of Rag2^−/− hosts 3 weeks post-transfer. (a–k) mean ± SEM is shown. (a–d,f–h,k), Two-tailed Student’s t-test; (e) Two-way ANOVA with Bonferroni post-test; *P≤0.05, **P≤0.01, ***P≤0.001.

Ext. Data Fig. 5. Defective metabolism of IL-15-polarized CD8⁺ T cells in the absence of P2RX7

(a), Viability of WT and P2rx7^−/− P14 cells maintained under culture conditions used for extracellular flux assays for 4h (data from two independent experiments, n=11 from all experiments). The same color scheme is used throughout the figure. (b–d), WT or P2rx7^−/− P14 cells were in vitro-activated and subsequently polarized in IL-2 or IL-15 for 72h and assayed for extracellular acidification rate (“ECAR”) to measure aerobic glycolysis (b); uptake of the dye TMRE to measure mitochondrial membrane potential (data normalized to MTG staining) (c); staining with MTG to determined total mitochondrial mass (d). (b–d) Data from three independent experiments, n=5–6 from all experiments). (e), WT and P2rx7^−/− P14 cells were activated in vitro for 72h, then stimulated with IL-15 (or not) for 30 min and immediately assayed for pSTAT5 expression. Data are representative of two independent experiments (n=6 total). (f), WT and P2rx7^−/− P14 cells were activated in vitro for 72h, then stimulated with IL-15 or IL-2 for 72h and assayed for expression of CD62L. Data are representative of three independent experiments (n=6 total). (g), Numbers of viable WT and P2rx7^−/− P14 cells following activation and subsequent culture in IL-15 or IL-2 for the indicated number of days. Values for WT and P2rx7^−/− cells were not significantly different (i.e. all p values were >0.05). Data from three independent experiments (n=3–6 total). (a–d,g) mean ± SEM is shown; (a,c–d,g); Two-tailed Student’s t-test; **P≤0.01.

Ext. Data Fig. 6. P2RX7 controls mitochondrial integrity in CD8⁺ T cells during immune responses

(a), Representative electron micrographs showing mitochondrial structures (left) and mitochondrial area measurements (right) of in vitro-activated WT and P2rx7^−/− P14 cells following culture for 72h in IL-2 or IL-15 as indicated. Representative of 3 independent experiments (n=8–9 in total). Black bars indicate 500nm. (b,c), WT and P2rx7^−/− P14 cells were activated in vitro for 72h and assayed at that time (b) or after a further 72h culture in IL-2 or IL-15 (as in Fig. 3b) (c) and oxygen consumption rate measured. Graphs show values for proton leak (i.e. the difference in OCR values after oligomycin and after Antimycin A/rotenone addition – see Fig. 3b for details of inhibitor addition). Data are from three independent experiments (n=11–18 total). (d) calculated proton leak derived from OCR measurements on in vivo activated WT and P2rx7^−/− P14 CD8⁺ MPEC described in Fig. 2d–f (data from three independent experiments, data pooled from 5 mice per experiment; n=4–6 wells in total). (e–h) WT and P2rx7^−/− P14 cells were activated in vitro and polarized with either IL-2 or IL-15 (as in Fig. 3b); total cell (e,f,g) and mitochondrial (g,h) protein extracts were collected for protein quantification experiments. Data from three independent experiments, samples pooled from n=6 mice total (2 mice per experiment). (e) Representative blot showing Opa1 expression in polarized WT or P2rx7^−/− P14 cells, in comparison with beta-actin (For gel source data, see Supplementary Figure 1). (f) Opa1 protein levels in polarized WT or P2rx7^−/− P14 cells, normalized to beta-actin. (g) mitochondrial concentration (normalized by total protein concentration) in polarized WT or P2rx7^−/− P14 cells. (h) Opa1 protein levels in polarized WT or P2rx7^−/− P14 cells, normalized to total mitochondrial concentration. (a–d,f–h), mean ± SEM is shown; (a–b,d) Two-tailed Student’s t-test; (c, f–h) Two-sided Mann-Whitney’s test; *P≤0.05, **P≤0.01, ***P≤0.001.

Ext. Data Fig. 7. Normal metabolic function of P2rx7^−/− naïve P14 CD8⁺ T cells, while pharmacological inhibition of P2RX7 compromises aerobic glycolysis of in vitro activated CD8⁺ T cells

(a–c), Naïve WT and P2rx7^−/− P14 cells were isolated and evaluated for different metabolism parameters (data from three independent experiments, n=6 total). (d,e), Human CD8⁺ T cells were in vitro activated in the presence of the P2RX7 inhibitor A-438079 (red) or vehicle alone (black) (from 20h of the beginning of the activation cultures), and assessed for extracellular acidification rate (“ECAR”) (d) or for viability of cells cultured in parallel under the same conditions used for extracellular flux assays (e). (d–e) Data are from three independent experiments, n=4–6 from all experiments. (a–e) mean ± SEM is shown; (b–c,e), Two-tailed Student’s t-test.

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.

Ext. 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), WT or P2rx7^−/− mice were infected with LCMV-Cl13, and the PFU levels in the kidneys were quantified 4 weeks post-infection. Data from three independent experiments, n=7 total. (b–f), WT and P2rx7^−/− P14 CD8⁺ T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV then challenged (or not) with Lm-gp33 6–8 weeks later (b). Data are from two independent experiments (n=5–7 total). (c) shows P2rx7^−/−/WT splenic P14 CD8⁺ T cell ratio before (0d) and post-challenge (5d), while (d) shows fold-increase in numbers of WT or P2rx7^−/− P14 cells in indicated tissues, relative to mice that did not receive Lm-gp33 challenge. The percentage of cells in cell cycle was determined by Ki-67 staining (e), and the frequency of dying cells indicated by the percentage of Annexin V⁺L/D⁺ cells in mice 5d following Lm-gp33 challenge (f). (g–h) WT and P2rx7^−/− P14 CD8⁺ T cells were individually transferred into B6.SJL mice that were subsequently infected with LCMV. At 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 WT or P2rx7^−/− P14 in PBS or gp33-treated mice (h) Percentage of IFN-γ⁺ “bystander” (i.e. non-P14) CD8⁺ T cells (left) and the percentage of CCR7⁺CD86⁺ DCs (right) in the FRT 12h later. (g–h) Data are from three independent experiments, n=4–11 total). (i–k), Schematic of experimental scheme combining spare nerve injury (SNI), LCMV infection and A-438079 treatment (i). For surgery, two of the three branches of the sciatic nerve in one hind limb were exposed and cut (SNI – see Fig 4)) or left uncut (sham). After 2-weeks recovery from surgery, mice were assays for pain sensitivity, then infected with LCMV with or without A-438079 treatment for the first week post-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 (>day 30). Data are compiled from two independent experiments, n=6 from all experiments. (j) shows pain sensitivity of sham-surgery mice (pre- and post-treatment) while (k) shows percentages of gp33-specific T_CM (left) and numbers of gp33-specific T_CM and T_EM (right) in sham surgery animals. (l–p), In other studies, B6 or Balb/C mice were adoptively transferred or not with WT 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. In (m), Percentages of CD62L⁺CD44⁺ (T_CM) P14 cells/spleen (left) and spleen P14 T_CM and T_EM numbers (right) from the different treatment groups at 4-weeks post-infection were assessed. In (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 MPEC and SLEC populations. In (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. In (p), B6 mice infected with LCMV were treated with A-438079 between days 40–50 post-infection and the numbers of gp33⁺ CD8⁺ T cells (T_CM and T_EM) per spleen were quantified at 8 weeks post-infection. (a,c–f,h,j–k,m–p) mean ± SEM is shown; (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.

Ext. Data Fig. 10. Model of P2RX7 function in regulating long-lived CD8⁺ T cell memory

(a) Following CD8⁺ T cell activation, P2RX7 is stimulated by eATP (derived from damaged cells or exported from live activated cells). This induces calcium influx, increased mitochondrial metabolic activity, and activation of the ATP export channel PANX1 (bold arrows). Generation of eATP through PANX1 sustains P2RX7 activation, further promoting mitochondrial function and T cell homeostasis. (b) illustrates that the impact of P2RX7 function is magnified as the CD8⁺ T cell response progresses into memory phase, with P2RX7 deficiency having little effect on effector T cells, while severely compromising survival and metabolic function in memory T cells, impairing the generation of long-lived central memory (T_CM) and CD103^hi/CD69^hi resident memory (T_RM) populations. P2RX7 activation is proposed to stimulate AMPK activation, which may arise from both calcium influx and increased AMP/ATP ratios as a result of P2RX7/PANX1 activity. A requirement for eATP stimulation of P2RX7 persists throughout memory CD8+ T cell maintenance.

Fig. 1. P2RX7 is required for generation and maintenance of long-lived memory CD8⁺ T cells

(a–d) WT and P2rx7^−/− P14 were co-adoptively transferred and host mice infected with LCMV-Arm. (a) WT and P2rx7^−/− P14 numbers in spleens and lymph nodes (LN). (b) Frequency of splenic WT and P2rx7^−/− P14 T_CM and T_EM 8-weeks post-infection (representative of n=5). (c,d) P2rx7^−/−/WT P14 subset ratios in spleen (c) or small intestine intraepithelial lymphocytes (SI-IEL) (d). (e–f) After WT and P2rx7^−/− P14 cell co-adoptive transfer, mice were infected with LCMV-Cl13, and spleens analyzed 2–3 weeks later for donor cell numbers (e) and percentage CXCR5⁺PD1^low (f). (a–f) Mean ± SEM. (a–d) Three independent experiments, n=4–9 total. (e–f) Two independent experiments, n=11 total. (a,e) Two-tailed Student’s t-test; (f), Two-tailed Mann-Whitney test; **P≤0.01, ***P≤0.001.

Fig. 2. P2RX7 regulates mitochondrial homeostasis in memory CD8⁺ T cells

(a), P2rx7^−/−/WT P14 ratios among memory-precursor (MPEC) and short-lived (SLEC) effector cells. (b,c) Median fluorescence of Mitotracker Green (MTG) (b) and Tetramethylrhodamine (TMRE; normalized to MTG) (c) among MPEC and SLEC. (d–f), Sorted WT and P2rx7^−/− P14 MPEC and SLEC were analyzed for metabolic function, indicated by (d,e) oxygen consumption rate (OCR) parameters and (f) OCR/ECAR ratios. (g–i) subsets of splenic WT and P2rx7^−/− P14 were stained for MTG (g) or TMRE B at indicated times. In (i), median 2-NBDG uptake (relative to naïve CD8⁺ T cells) in memory P14 subsets is shown. (a–c,g–i) Three independent experiments, n=5–6 total/timepoint. (d–f) Three independent experiments, cells pooled from 5 mice/experiment; n=4–6 total. (a–i) Mean ± SEM; (b–c,f–h), Two-tailed Student’s t-test; (e,i), Two-tailed Mann-Whitney’s test; *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 3. P2RX7 ablation leads to aberrant metabolism and depressed AMPK activation in CD8⁺ T cells

(a–d) In vitro activated WT and P2rx7^−/− P14 were activated then polarized with IL-2 or IL-15 (cells pooled from 2 mice/experiment). (a) Cell viability during IL-2 or IL-15 cultures (p≥0.2 for all IL-2 polarized cells and IL-15 polarized at 24h). (b,c) OCR (b) and calculated SRC (c) for IL-2 or IL-15-polarized cells. (d) electron microscopy of mitochondria (arrows) in IL-15-polarized WT or P2rx7^−/− P14 (black bars =500nm). (e–k) mouse (e,i–k) or human (f–h) CD8⁺ T cells were stimulated in vitro in the presence of A-438079 (e–h), BzATP (i), Probenecid (j,k), or vehicle controls. Mouse cells activated as in (a), human cells assayed 72h post-stimulation. OCR (e,f,i,j) and SRC (k) were measured and human cells assayed for proliferation (Ki67) (g) and Granzyme B/IFN-γ (h). (l) pACC in IL-15-polarized WT and P2rx7^−/− P14 (representative flow cytometric histograms and median average values (right). In (m), cells were incubated (6h) with/without AICAR prior to OCR measurement. (n–o) Mice receiving co-transferred WT and P2rx7^−/− P14 were LCMV-infected primed and treated with metformin (1–7 dpi; n) or rapamycin (4–8 dpi; o). P2rx7^−/−/WT ratios for splenic memory subsets (n) and P14 T_CM numbers were determined (o). (a–o) Three independent experiments, n=3–6 (a), 8–9 (b–c), 6 (d,g–h,l–m), 4–5 (e,i), 4 (f), 5–6 (j–k), 10–11 (n), 12–17 (o) total. (a–c,e–o) Mean ± SEM; (a,c,g–h,l,n), Two-tailed Student’s t-test; (k,o) One-way ANOVA + Tukey post-test, *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 4. P2RX7 promotes memory CD8⁺ T cell function and P2RX7 blockade compromises CD8⁺ T cell memory maintenance

(a–c) Memory WT or P2rx7^−/− P14 were independently transferred into naïve recipient mice, which were then VacV-gp33-infected. (b) Splenic WT or P2rx7^−/− P14 numbers (left) and fold increase (right) 7 days post-challenge. (c), VacV-gp33 ovary viral titers 7 days post-challenge (log-transformed). (d), WT and P2rx7^−/− P14 cells were independently transferred and recipient mice primed with LCMV. After >30d, a transcervical challenge with gp33 or PBS was conducted and female reproductive tract (FRT) P14 12h later for IFN-γ production and Gzm-B expression. (e–f) B6 mice subjected to spare nerve injury were subsequently LCMV infected with/without A-438079 treatment. (e) mice were tested for pain sensitivity and (f) subsequent development of LCMV-specific (“gp33⁺”) splenic memory subsets. (g) mice were co-transferred and primed as in Fig. 1 and A-438079-treated 40–50 dpi. P14 numbers/spleen (left) and T_CM percentages (right) are shown. (a–e) Three independent experiments, n=9–10 (a–b), 7–10 (c), 4–11 (d) total. (e–g) Two independent experiments, n=12 (e–f), 8–10 (h) total. (b–c,d–g) mean ± SEM; (b,e), Two-tailed Student’s t-test; (e–f), Two-tailed Mann-Whitney t-test; (c,g) One-way ANOVA + Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001.