Research ArticleT CELL MEMORY

TRM maintenance is regulated by tissue damage via P2RX7

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Science Immunology  14 Dec 2018:
Vol. 3, Issue 30, eaau1022
DOI: 10.1126/sciimmunol.aau1022
  • Fig. 1 P2RX7 is selectively expressed by CD8

    + TRM. (A) RNA-seq of naïve and LCMV-specific memory CD8+ T cell subsets. Heatmap of PRR mRNA that is differentially expressed between the memory T cell subsets (TN: CD44 CD62L+; TCM: CD44+ CD62L+ CD69; TEM: CD44+ CD62L CD69; TRM: CD44+ CD62L CD69+). (B) Mice were infected with LCMV or (C) LM-OVA and day 30+ postinfection pathogen-specific CD8+ T cells were analyzed [GP33 (KAVYNFATC) tetramer+ for LCMV or OVA (SIINFEKL) tetramer+ for LM-OVA]. Thirty minutes before sacrifice, mice were injected with ARTC2.2-blocking nanobody s+16a to prevent P2RX7 ADP ribosylation (60), and P2RX7 expression on pathogen-specific TCM, TEM, and TRM in the different organs was analyzed (representative of more than three experiments). (D) WT-P2RX7 KO mixed BM chimeras were infected with LM-OVA. More than 30 days after infection, mice were injected with anti-ARTC2.2 nanobody, and the expression of P2RX7 and ARTC2.2 was analyzed by flow cytometry. LM-OVA–specific (SIINFEKL-tetramer) WT CD8+ T cells are displayed. P2RX7 KO CD8+ T cells from the same mouse were used for gating (representative of n = 5). (E) At day 30+ after LCMV infection, LCMV-specific (GP33-tetramer) CD8+ T cells were analyzed (n = 4/5).

  • Fig. 2 P2RX7 deficiency does not impair memory differentiation of CD8

    + T cells. (A and B) WT or P2RX7 KO mice were infected with LCMV. More than 30 days after infection (A), the frequency of LCMV-specific CD8+ T cells and (B) the phenotype of the LCMV-specific CD8+ T cells were analyzed [n = 4 to 10, one (BM) or two experiments]. (C) WT:P2RX7 KO mixed BM chimeras were infected with LCMV. More than 30 days after infection, the phenotype of LCMV-specific (GP33 tetramer) WT or P2RX7 KO donor CD8+ T cells was analyzed (n = 5, one experiment). Sal. gl., salivary glands.

  • Fig. 3 P2RX7 activation by NAD

    + induces cell death of TRM but not of TCIRC. (A to E) IEL or splenocytes were cultured in medium or in the presence of different concentrations of NAD+. After 20 hours, the number of viable CD8αβ+ TRM (IEL) or TCIRC (spleen) was assessed by flow cytometry and normalized to the medium control. (A and B) TRM or TCIRC of WT mice (n = 10/11, four experiments). (C) TRM of WT or P2RX7 KO mice. (D) TRM or TCIRC of WT and P2RX7 KO mice cultured in medium with or without 10 μM NAD+ (n = 7 to 11 per group, five experiments). ns, not significant. (E) TRM or TCIRC of WT mice were treated with P2RX7 inhibitors (A438079, KN-62) or anti-ARTC2.2 nanobody s+16a or dimethyl sulfoxide (DMSO) as control for 30 min before addition of 10 μM NAD+ (n = 4 to 5, three experiments). Unpaired t test (B and D) and paired t test (E). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 4 P2RX7 expression is modulated by inflammatory cytokines and TCR activation.

    (A) SI IEL were cultured for 3 days with homeostatic cytokines (recombinant murine IL2, IL7, and IL15) alone or additional IFN-α or IL-12 (10 ng/ml each), and the expression of P2RX7 on CD8αβ+ TRM was assessed by flow cytometry (n = 6, two experiments). (B) IEL or splenic CD8+ T cells were stimulated with plate-bound anti-CD3 or left untreated and cultured for 3 days with homeostatic cytokines. P2RX7 expression on CD8ab+ TRM (IEL) or TCIRC (spleen) was assessed (n = 5, three experiments). (C and D) CD8+ TRM (IEL) or TCIRC (spleen) of OT1 mice were stimulated with plate-bound anti-CD3 or SIINFEKL peptide or left untreated and cultured for 1 day with soluble anti-CD28 and homeostatic cytokines. (C) P2RX7 expression on CD8αβ+ OT1 cells was assessed, or (D) cells were treated with 10 μM NAD+ for 20 hours and the number of viable CD8αβ+ T cells relative to medium control was quantified (n = 4, two experiments). Unpaired t test (A to D). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 5 P2RX7 activation depletes T

    RM but not TCIRC in vivo. (A to C) Mice were injected with PBS or NAD+, and organs were analyzed 24 hours later. (A) Frequencies of CD8αβ+ T cell subsets in the liver and (B) in the BM as well as (C) absolute numbers were analyzed by flow cytometry (n = 4 to 6 per group, three experiments). (D and E) More than 30 days after LCMV infection, mice were injected with PBS or NAD+. Frequencies of LCMV GP33-specific CD8+ T cells with TEM or TRM phenotype were quantified in the (D) IEL and (E) liver 24 hours after NAD+ treatment (n = 4 per group, one experiment). (F) WT:P2RX7 KO mixed BM chimeras were infected with LCMV. More than 30 days after infection, mice were treated with NAD+ or PBS as control. Twenty-four hours after NAD+ treatment, GP33-specific donor CD8+ T cells were quantified (n = 7 per group, two experiments). IEL, small intestine intraepithelial lymphocytes. Unpaired t test (C to F). *P ≤ 0.05, ***P ≤ 0.001.

  • Fig. 6 Tissue damage depletes TRM but not TCIRC.

    (A) Liver CD8+ T cells from untreated mice or mice that were injected with ARTC2.2-blocking nanobody s+16a were isolated and CD8+ T cell subsets were sorted. Subsets were then cultured for 6 hours, and viability was assessed by flow cytometry (n = 9/12 per group, four experiments). (B) WT or P2RX7 KO mice were infected with LCMV, and 30+ days after infection, mice were treated with acetaminophen to induce liver injury or PBS as control. Twenty-four hours after acetaminophen injection, GP33-specific CD8+ T cells in the liver were analyzed, and the frequency of TRM was quantified (n = 3/4 per group). (C) WT-P2RX7 KO mixed BM chimeras were infected with LCMV. More than 30 days after infection, mice were treated with acetaminophen or PBS as control. More than 30 days after acetaminophen treatment, LCMV-specific donor CD8+ T cells (GP33 tetramer) in the liver were analyzed, and the frequency of TRM within the WT and P2RX7 KO compartment was quantified (n = 4 per group, one experiment). (D and E) Mice were injected with a 1:1 mixture of naïve WT and P2RX7 KO OT1 CD8+ T cells and infected with rLCMV-OVA. More than 30 days after infection, mice were treated with acetaminophen, and 30+ days later, the frequencies of TRM in transferred WT and P2RX7 KO OT1 CD8+ T cells in the (D) liver and (E) kidney were quantified (n = 8 per group, one experiment). Mean ± SEM. Unpaired t test. *P ≤ 0.05, ***P ≤ 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/30/eaau1022/DC1

    Fig. S1. Gating strategies.

    Fig. S2. CD69+/CD62L CD8+ T cells have features of TRM cells.

    Fig. S3. P2RX7 and ART2.2 are highly expressed by TRM cells.

    Fig. S4. Expression of P2RX7, ARTC2.2, CD38, and CD39 on lymphocyte subsets.

    Fig. S5. P2RX7 deficiency does not impair effector differentiation of CD8+ T cells.

    Fig. S6. P2RX7 deficiency does not impair memory differentiation of CD8+ T cells.

    Fig. S7. P2RX7 deficiency does not impair ARTC2.2 and CD38 expression of memory CD8+ T cells.

    Fig. S8. Modulation of P2RX7 expression cytokines and TCR activation.

    Fig. S9. P2RX7 activation depletes TRM but not TCIRC in vivo.

    Fig. S10. Effect of tissue damage on TRM in IEL and spleen.

    Table S1. Selected PRR genes expressed in RNA-seq data set.

    Table S2. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Gating strategies.
    • Fig. S2. CD69+/CD62L CD8+ T cells have features of TRM cells.
    • Fig. S3. P2RX7 and ART2.2 are highly expressed by TRM cells.
    • Fig. S4. Expression of P2RX7, ARTC2.2, CD38, and CD39 on lymphocyte subsets.
    • Fig. S5. P2RX7 deficiency does not impair effector differentiation of CD8+ T cells.
    • Fig. S6. P2RX7 deficiency does not impair memory differentiation of CD8+ T cells.
    • Fig. S7. P2RX7 deficiency does not impair ARTC2.2 and CD38 expression of memory CD8+ T cells.
    • Fig. S8. Modulation of P2RX7 expression cytokines and TCR activation.
    • Fig. S9. P2RX7 activation depletes TRM but not TCIRC in vivo.
    • Fig. S10. Effect of tissue damage on TRM in IEL and spleen.
    • Table S1. Selected PRR genes expressed in RNA-seq data set.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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