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. 2024 Jun 17;27(7):110290.
doi: 10.1016/j.isci.2024.110290. eCollection 2024 Jul 19.

The ATP-exporting channel Pannexin 1 promotes CD8+ T cell effector and memory responses

Trupti Vardam-Kaur  1 , Alma Banuelos  1 , Maria Gabaldon-Parish  1 , Bruna Gois Macedo  1 , Caio Loureiro Salgado  1 , Kelsey Marie Wanhainen  2 , Maggie Hanqi Zhou  1 , Sarah van Dijk  1 , Igor Santiago-Carvalho  1 , Angad S Beniwal  1   3 , Chloe L Leff  1 , Changwei Peng  2 , Nhan L Tran  3 , Stephen C Jameson  2 , Henrique Borges da Silva  1   3
Affiliations

The ATP-exporting channel Pannexin 1 promotes CD8+ T cell effector and memory responses

Trupti Vardam-Kaur et al. iScience. .

Abstract

Sensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo, however, has not been previously addressed. Here, we report that T-cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation, which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast, Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement, leading to improved mitochondrial metabolism. In summary, Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways.

Keywords: Biochemistry; Biological sciences; Immune response; Immune system; Immunology; Natural sciences.

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Conflict of interest statement

H.BdS. and N.L.T. are advisors for International Genomics Consortium. The remaining authors have no financial disclosures. The authors declare no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
CD8+ T-cell-specific Panx1 is required for T cell activation and effector differentiation (A) Panx1 mRNA expression from RNA-seq samples in the indicated subsets of LCMV-gp33 transgenic P14 CD8+ T cells. (B) Single-cell RNA-seq (scRNAseq) analysis of effector P14 cells showing UMAP cluster distribution (left) and expression of Panx1 mRNA in each cluster (right). (C) Western blot analysis of Panx1 expression in WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1fl/fl) CD8+ T cells at 0 h or 72 h after activation with anti-CD3/CD28 + IL-2. The full blot versions can be found in Figure S2. (D) WT (CD4-Cre) or CD4-Cre Panx1fl/fl mice were infected with LCMV-Arm. Numbers of total (left) and TE and MP (right) spleen gp33+ CD8+ T cells at day 7 post-infection are shown. (E–G) A 1:1 mix of bone marrow cells from CD4-Cre (WT; CD45.1+) and CD4-Cre Panx1fl/fl (Panx1-KO; CD45.2+) mice was transferred into lethally irradiated C57BL/6 mice (CD45.1/2+). After two months, BM chimeric mice were infected with LCMV-Arm. The red dotted lines show the Panx1-KO:WT ratios after two months of reconstitution. (E) Graphical scheme. (F) Representative flow cytometry plots showing WT and Panx1-KO total, TE, and MP spleen gp33+ CD8+ T cells at day 7 post-infection. (G) Panx1-KO/WT ratios of total (left) and of naive, TE and MP spleen gp33+ CD8+ T cells (right) at day 7 post-infection. (H) Heatmaps showing the expression of TCR signaling pathway genes from RNA-seq data of activated WT and Panx1-KO CD8+ T cells. (I) CD4-Cre or CD4-Cre Panx1fl/fl mice were infected with LCMV-Arm and evaluated at 3 days after infection. Representative histograms (left) and average percentages (right) of Ki-67+ gp33+ CD8+ T cells are shown. (J and K) CD4-Cre (WT; CD45.1/2+) and CD4-Cre Panx1fl/fl (Panx1-KO; CD45.2+) P14 cells were labeled with CellTrace Violet and cotransferred into LCMV-infected recipient C57BL/6 (CD45.1+) mice. (J) Histograms showing CellTrace Violet dilution (left) and graphs showing average gMFI values (right) are shown. (K) Representative flow cytometry plots showing expression of CD44 and CD69 are shown in the left. In the right, average percentages of CD44+CD69+ cells (top) and of CD62L+ cells (bottom) are shown. (L–Q) WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1fl/fl) CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2 and in some cases PMA/Ionomycin) for up to 72 h. In some experiments (O), WT Nur77-GFP CD8+ T cells were activated in the presence of vehicle (PBS; Control) or trovafloxacin (Panx1 inhibitor; Panx1i). (L) Representative flow cytometry plots showing expression of CD69 and dilution of CellTrace Violet at 24 h, 48 h, and 72 h after activation. (M) Percentages of CellTrace Violet (CTV) divided cells (top left), average gMFI values for CD44 (top right), and percentages of CD69+ (bottom left) and CD25+ (bottom right) cells at 24 h, 48 h, and 72 h after activation. (N) Representative histograms showing expression of IFNγ (top) and average percentages of IFNγ+ CD8+ T cells (bottom) at 72 h after activation. (O) Representative histograms showing Nur77-GFP expression in control of Panx1i-treated cells (top), and average percentages of Nur77-GFP+ CD8+ T cells (bottom) at 3 h after activation are shown. (P) Average percentages of CD44+ (left) and CD69+ (right) cells at 3 h after activation with either anti-CD3/CD28 or PMA/Ionomycin are shown. (Q) CD4-Cre and CD4-Cre Panx1fl/fl mice were infected with LCMV-Arm; at day 7 after infection, the percentages of FMK/FLICA+ and PI+ cells were assessed in gp33+ CD8+ T cells. In the left, representative flow cytometry plots are shown; in the right, average percentages of FMK/FLICA+ cells are depicted. (C) Data representative of two independent experiments; n = 4–5 per group. (D–J, Q) Data from two to three independent experiments; n = 5–12 per experimental group. (K) Each replicate is a pool of activated CD8+ T cells from three mice; n = 3 replicates per experimental group. (L–P) Data from four independent experiments; n = 7–11 per experimental group per time point. ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; unpaired t test (D, J, K, N, and O), one-way ANOVA with Tukey’s post-test (D, G, I, K, P, and Q), two-way ANOVA with Bonferroni’s post-test (G, M). See also Figures S1–S4.
Figure 2
Figure 2
Panx1 controls the intracellular metabolism of effector CD8+ T cells (A and B) Naive CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were analyzed for mitochondrial function. (A) Measurements of oxygen consumption rate (OCR) levels after sequential addition of oligomycin, FCCP, and rotenone/antimycin A (top right); average OCR values at spare respiratory capacity (bottom right, SRC; see STAR Methods) are shown. (B) MitoTracker Green (Mito Green) and TMRE; representative histograms are shown above, whereas average gMFI values are shown below. (C and D) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for up to 72 h. (C) Mito Green and TMRE average gMFI levels at 3 h (top) and 72 h (bottom) after activation are shown. (D) Measurements of extracellular acidification rate (ECAR) levels after addition of oligomycin (top left); measurements of oxygen consumption rate (OCR) levels after sequential addition of oligomycin, FCCP, and rotenone/antimycin A (top right); average ECAR values at baseline (bottom left), average OCR values at baseline (bottom middle), and spare respiratory capacity (bottom right, SRC; see STAR Methods) are shown. (E) Heatmap showing expression of genes from the glycolysis pathway in 72-h-activated CD8+ T cells; expression in memory-like CD8+ T cells (with additional IL-15 treatment; more on Figure 6) are shown in comparison. (F and H) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for 72 h, then further polarized into effector-like cells with IL-2 for additional 72 h. (F) Experimental design. (G) Measurements of extracellular acidification rate (ECAR) levels from effector-like CD8+ T cells after sequential addition of glucose, oligomycin, and 2-DG (top left); in the top right, kinetics of OCR values. On the bottom, average baseline ECAR and OCR values and the ratios between ECAR and OCR values from effector-like CD8+ T cells are shown, respectively. (H) Average Mito Green and TMRE gMFI values for effector-like CD8+ T cells. (I) CD4-Cre and CD4-Cre Panx1fl/fl P14 cells were transferred into C57BL/6 mice infected with LCMV-Arm. At day 7 post-infection, TE P14 cells were sorted. ECAR and OCR kinetics (top), ECAR values (bottom left), OCR values (bottom center), and ECAR/OCR ratios (bottom right) from sorted TE gp33+ CD8+ T cells are shown. (A–D, F–I) Data from two to three independent experiments; n = 4–21 per experimental group. (E) Data from 2 to 3 biological replicates per experimental group (from n = 5–8 mice per group). ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; unpaired t test (A–D, F–I). See also Figure S5.
Figure 3
Figure 3
Panx1 controls the activation of effector CD8+ T cells through export of eATP and late extracellular lactate accumulation (A) WT Nur77-GFP CD8+ T cells were activated in the presence of PBS or Panx1i, with the addition of vehicle or eATP. Nur77-GFP representative histograms (left) and average Nur77-GFP+ percentages (right) are shown. (B and C) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for up to 48 h, with the addition of vehicle or eATP at either the beginning of cultures (B) or at 20 h after activation (C). (B) Average percentages of CD69+ cells at 3 h after activation. (C) Average percentages of CD44+ and CD69+ cells at 24 h after activation (left); average percentages of CellTracer Violet− cells (CTV % divided) at 48 h after activation (right). (D) CD4-Cre or CD4-Cre Panx1fl/fl effector-like CD8+ T cells (with prolonged exposure to IL-2) were incubated in the presence or absence of eATP; average percentages of CD69+ cells and of ECAR values are shown. (E) WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1fl/fl) effector-like and memory-like CD8+ T cell cultures were harvested, and intracellular lysates and supernatants were submitted for untargeted metabolomics (GC-MS) analysis. Enrichment analysis showing pathways preferentially represented in the metabolites from the supernatants of WT effector-like CD8+ T cells (WT > Panx1-KO). Levels (arbitrary units [AU]) of l-lactate in the supernatants (left) and intracellular lysates (right) of effector-like WT and Panx1-KO CD8+ T cells are shown below. (F–G) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for 72 h, and l-lactate measurements (mM) were done. (F) Intracellular lactate levels. (G) Extracellular lactate levels, in the presence or absence of inhibitors for MCT1 (SR13800) and MCT4 (VB124). (H) Average percentages of IFNγ+ CD8+ T cells at 72 h after activation, with addition of sodium lactate +/− MCT1i. (I) Representative histograms (left) and average percentages of CTV % divided and CD69+ cells at 48 h after activation, with addition of vehicle, MCT1i, MCT4i, or MCT1/MCT4i. (G–K) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for up to 72 h, in the presence of the indicated metabolites or inhibitors. (J) Average percentages of CD44+ and CD69+ cells after 24 h of activation (left) and of CTV % divided cells at 48 h after activation (right), with the addition of vehicle or sodium lactate. (K) ECAR and OCR kinetics (left) and average baseline values (right) at 72 h after activation, with the addition of vehicle or sodium lactate. (L) Average percentages of CD69+ cells at 48 h after activation, with addition or sodium lactate +/− oligomycin. (M) CD4-Cre or CD4-Cre Panx1fl/fl effector-like CD8+ T cells were incubated in the presence or absence of sodium lactate (right); average percentages of CD69+ cells and of ECAR values are shown. (N) WT Nur77-GFP CD8+ T cells were activated in the presence of PBS or Panx1i, with the addition of vehicle or sodium lactate; average percentages of Nur77-GFP+ cells (left), CD69+ cells (center), or CD44+ cells (right) are shown. (O) WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1fl/fl) P14 cells (CD45.2+) were transferred into LCMV-infected WT CD45.1+ mice. Some mice were treated with sodium lactate between days 1 and 3 post-infection, and spleen P14 cells were analyzed at day 7 post-infection. Flow cytometry plots showing expression of CD127 and KLRG1 (left) and the average numbers of TE, MP, and DN P14 cells per spleen (right) are shown. (A–D, F–O) Data from two to three independent experiments; n = 3–14 per experimental group. (E) Data from three biological replicates per experimental group (from n = 3 mice per group). ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post-test (A–D, F–O) unpaired t test (E), two-way ANOVA with Bonferroni’s post-test (M). See also Figures S5 and S6.
Figure 4
Figure 4
Panx1 promotes carbon source uptake and leads to maintenance of lipid rafts in activated CD8+ T cells (A) Levels (arbitrary units– [AU]) of pyruvate, malate, aspartate, and citrate in the intracellular lysates of effector-like WT (CD4-Cre) or Panx1-KO (CD4-Cre Panx1fl/fl) CD8+ T cells from untargeted metabolomics are shown. (B and C) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for 72 h; in the last 12 h, either 13C-Glucose (B) or 13C-Lactate (C) were substituted into the cultures for metabolite carbon tracing. (B) Percentages of 13C-Glucose incorporation into citrate, isocitrate, a-ketoglutarate, and alanine. (C) Percentages of 13C-Lactate incorporation into citrate and isocitrate. (D) Top enriched intracellular pathways in WT effector-like CD8+ T cells (compared to Panx1-KO), depicted from the untargeted metabolomics from (A). (E) Levels (AU) of total PIs in CD4-Cre versus CD4-Cre Panx1fl/fl effector-like CD8+ T cells; polyunsaturated (two or more double bonds) or saturated (one or less double bonds) PIs are depicted in white and gray, respectively. In the right, levels (AU) of PIP(34:1) in CD4-Cre versus CD4-Cre Panx1fl/fl effector-like CD8+ T cells. (F) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of sodium lactate. Representative histograms (left) and average gMFI values (right) for cholera toxin B are shown. (G) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of BSA-conjugated oleic acid. Average percentages of IFNγ+ CD8+ T cells are shown. (H) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of sodium lactate and/or the CDIPT inhibitor inostamycin (CDIPTi). Average percentages of IFNγ+ CD8+ T cells are shown. (I) RNA expression levels (AU) of the indicated PLCγ-MAPK signaling pathway genes in CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells after 72 h of activation. (J) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of sodium lactate. Representative histograms (left) and average gMFI values (right) for phosphorylated ERK1/2 (pERK1/2) are shown. (K) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of sodium lactate. Average gMFI values for Glut1 are shown. (L and M) CD4-Cre and CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) in the presence or not of additional Glucose. (L) Average percentages of IFNγ+ CD8+ T cells are shown. (M) ECAR kinetics, OCR kinetics, baseline ECAR, and SRC OCR values are respectively shown. (A–E, I) Data from two to three biological replicates per experimental group (from n = 3 mice per group). (F–H, J–M) Data from two to three independent experiments; n = 4–7 per experimental group. ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; unpaired t test (A, E, I), one-way ANOVA with Tukey’s post-test (F–H, J–M). See also Figure S5, Data S1.
Figure 5
Figure 5
Panx1 promotes the long-term establishment of memory CD8+ T cells (A and B) CD4-Cre or CD4-Cre Panx1fl/fl mice were infected with LCMV-Arm, and gp33+ CD8+ T cells were tracked over time. Numbers of total, TCM and LLEC spleen gp33+ CD8+ T cells at day 42 post-infection are shown in (A), while the numbers of SI IEL gp33+ CD8+ T cells at day 42 post-infection are shown in (B). (C and D) A 1:1 mix of bone marrow cells from CD4-Cre (WT; CD45.1+) and CD4-Cre Panx1fl/fl (Panx1-KO; CD45.2+) mice was transferred into lethally irradiated C57BL/6 mice (CD45.1/2+). After two months, BM chimeric mice were infected with LCMV-Arm. The red dotted lines show the Panx1-KO:WT ratios after two months of reconstitution. Panx1-KO/WT ratios of TCM and LLEC gp33+ spleen CD8+ T cells over time post-infection are shown in (C). In (D), the Panx1-KO/WT ratios of SI IEL, Salivary gland, and kidney TRM gp33+ spleen CD8+ T cells over time post-infection are shown. (E–I) Recipient C57BL/6 mice (CD45.1+) were adoptively transferred with a 1:1 mix of P14 ERT2-Cre LSL-YFP Panx1+/+ (WT; CD45.1/2+) and P14 ERT2-Cre LSL-YFP Panx1fl/fl (CD45.2+) cells, then infected with LCMV-Arm. Infected mice were treated with vehicle or tamoxifen (Tx) at the indicated time intervals. (E) Experimental design. (F) Panx1fl/fl/Panx1+/+ P14 cell ratios in the blood over time post-infection, in mice treated with vehicle or Tx between days 28–35 (d28-35) post-infection. Treatment periods is indicated by the blue shaded box. (G) Representative flow cytometry plots showing the distribution of spleen TCM, TEM and LLEC Panx1+/+ and Panx1fl/fl P14 cells from mice treated with Tx d28-35 at 63days post-infection. (H) Panx1fl/fl/Panx1+/+ ratios of spleen TCM, TEM and LLEC P14 cells from mice treated with vehicle or Tx d28-35, at 63days post-infection. (I) Panx1fl/fl/Panx1+/+ P14 cell ratios in SG and SI IEL at day 63 post-infection, in mice treated with Tx between days 28–35. (A–D, F–I) Data from three independent experiments; n = 6–12 per time point per experimental group. ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post-test (A, H, I), Unpaired t test (B), two-way ANOVA with Bonferroni’s post-test (C, D, F). See also Figure S7.
Figure 6
Figure 6
Panx1 promotes memory CD8+ T cell maintenance through eATP export and activation of the AMPK pathway (A and B) WT (CD4-Cre) or P2RX7-KO (CD4-Cre P2rx7fl/fl) P14 cells (CD45.2+) were in vitro activated for 24 h, then electroporated with the Cas9 protein combined with sgRNAs for Cd4 (sgCd4; control) or Panx1 (sgPanx1; Panx1-KO). After 48 h of resting with IL-2, P14 cells were transferred into recipient mice (CD45.1+) infected with LCMV-Arm. (A) Experimental design. (B) Numbers of total spleen P14 cells (left), spleen TCM P14 cells (middle), and spleen LLEC P14 cells (right) at day 42 post-infection. (C) CD4-Cre and CD4-Cre Panx1fl/fl P14 cells were transferred into C57BL/6 mice infected with LCMV-Arm. At day 7 post-infection, MP P14 cells were sorted. OCR kinetics (left), average baseline OCR values (middle), and average SRC OCR values (right) are shown. (D–H) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2) for 72 h, then further polarized into memory-like cells with IL-15 for additional 72 h. (D) Experimental design. (E) In the left, OCR kinetics for both groups are shown. In the right, average baseline OCR values, average SRC OCR values, and average ECAR/OCR ratios are shown. (F) OCR kinetics (left) and average baseline OCR values (right) are shown for memory-like cells with additional treatment with eATP +/− A-438079. (G) Average TMRE gMFI values for memory-like cells with additional treatment with eATP +/− A-438079. (H) OCR kinetics (left) and average TMRE gMFI values for memory-like cells with additional treatment with sodium lactate. (I) CD4-Cre or CD4-Cre Panx1fl/fl mice were infected with LCMV-Arm and analyzed at 60 days post-infection. Histograms showing pACC staining (top) and average pACC gMFI values (bottom) are shown for memory gp33+ CD8+ T cells. (J) Heatmap showing expression of genes from the AMPK and FOXO1 pathways in memory-like CD8+ T cells; expression in CD8+ T cells (with 72 h post-activation) are shown in comparison. (K and L) CD4-Cre (WT; CD45.1/2+) or CD4-Cre Panx1fl/fl (Panx1-KO; CD45.2+) P14 cells were transferred into WT (CD45.1+) mice infected with LCMV-Arm; between days 1 and 7 post-infection, mice were treated with AICAR. (K) pACC expression in P14 cells. (L) Panx1-KO/WT P14 cell ratios for spleen total (top) and TCM (bottom) cells are shown. (M and N) CD4-Cre or CD4-Cre Panx1fl/fl CD8+ T cells were activated in vitro (anti-CD3/CD28 + IL-2), then polarized into memory-like cells, with additional treatment with vehicle or AICAR. (M) pACC expression in CD8+ T cells. (N) Average TMRE gMFI values (left), OCR kinetics (middle), and average baseline OCR values (right) are shown. (B and C, E–I, K–N) Data from two to three independent experiments; n = 4–13. (J) Data from two to three biological replicates per experimental group (from n = 5–8 mice per group). ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post-test (B, F–I, M and N), Unpaired t test (C, E, K, and L).
Figure 7
Figure 7
Cell-specific Panx1 promotes antitumor CD8+ T cell responses and graft-versus-host disease in vivo (A–E) WT (CD4-Cre) and Panx1-KO (CD4-Cre Panx1fl/fl) P14 cells were activated in vitro (α-CD3, α-CD28, IL-2, and IL-12) for 72 h. After this period, P14 cells were transferred individually (A–C) or cotransferred (D and E) into B16.gp33 tumor-bearing mice. The tumor sizes and survival of individually transferred mice were tracked over time post-tumor inoculation; the numbers and phenotype of transferred P14 cells were assessed at 20 days post-tumor inoculation in cotransferred mice. (A) Individual tumor growth values over time. (B) Average tumor areas at day 14 post-tumor inoculation are shown. (C) Survival curves of individually transferred mice over time. (D) Average Panx1-KO/WT P14 cell number ratios in the indicated organs. (E) Average values for Mito Green (MTG) gMFI (left), TMRE gMFI (middle), and percent of P14 cells with depolarized mitochondria (MTGhiTMRE−; right). (F and G) In other experiments, BALB/c mice were lethally irradiated and reconstituted with BALB/c BM cells (no transfer), BM + spleen CD8+ T cells from CD4-Cre mice (+ WT CD8), and BM + spleen CD8+ T cells from CD4-Cre Panx1fl/fl mice (+Panx1-KO CD8). (F) Experimental design. (G) Average survival values over time after cell transfer. (A–G) Data from two to three independent experiments; n = 5–20 per experimental group. ns: not significant (p > 0.05); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Kaplan-Meier Survival curve analysis (E), unpaired t test (F and G), one-way ANOVA with Tukey’s post-test (B), two-way ANOVA with Bonferroni’s post-test (G).
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