Selective regulation of IFN-γ and IL-4 co-producing unconventional T cells by purinergic signaling

Abstract

Unconventional T cells, including mucosal-associated invariant T (MAIT), natural killer T (NKT), and gamma-delta T (γδT) cells, comprise distinct T-bet+, IFN-γ+ and RORγt+, IL-17+ subsets which play differential roles in health and disease. NKT1 cells are susceptible to ARTC2-mediated P2X7 receptor (P2RX7) activation, but the effects on other unconventional T-cell types are unknown. Here, we show that MAIT, γδT, and NKT cells express P2RX7 and are sensitive to P2RX7-mediated cell death. Mouse peripheral T-bet+ MAIT1, γδT1, and NKT1 cells, especially in liver, co-express ARTC2 and P2RX7. These markers could be further upregulated upon exposure to retinoic acid. Blocking ARTC2 or inhibiting P2RX7 protected MAIT1, γδT1, and NKT1 cells from cell death, enhanced their survival in vivo, and increased the number of IFN-γ-secreting cells without affecting IL-17 production. Importantly, this revealed the existence of IFN-γ and IL-4 co-producing unconventional T-cell populations normally lost upon isolation due to ARTC2/P2RX7-induced death. Administering extracellular NAD in vivo activated this pathway, depleting P2RX7-sensitive unconventional T cells. Our study reveals ARTC2/P2RX7 as a common regulatory axis modulating the unconventional T-cell compartment, affecting the viability of IFN-γ- and IL-4-producing T cells, offering important insights to facilitate future studies into how these cells can be regulated in health and disease.

Figure S1.

P2RX7 expression analyses of T-cell subsets from human blood and liver samples. (A) Boxplots depict P2RX7 gene transcript levels within the indicated cell types (data source: Gutierrez-Arcelus et al. [2019], boxes show the first to third quartile with median, whiskers encompassing 1.5× the interquartile range, and data beyond that threshold indicated as outliers). TPM = transcripts per million. (B) Graph shows MFI of P2RX7 labeling by human blood MAIT cells (MR1-5-OP-RU tetramer⁺CD3⁺), Vδ2⁺ and Vδ2⁻ γδT cells (γδTCR⁺CD3⁺), NKT cells (CD1d-α-GalCer tetramer⁺CD3⁺), and conventional T cells (conv. T; defined as non-MAIT/NKT γδTCR⁻CD3⁺ T cells). A total of 13 donors were analyzed across four separate experiments. Each symbol represents an individual donor, where upward- and downward-pointing triangles represent freshly processed and cryopreserved samples, respectively. Graphs depict individual data points and mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001 using a Wilcoxon matched pairs signed rank test with a Bonferroni-Dunn correction for multiple comparisons. (C) Histograms depict the expression of P2RX7 by CD14⁺FSC^hiSSC^hi cells and CD3⁺ T cells from donors 03 and 04 processed and analyzed on the same day. Numbers within histograms represent MFI. FMO = fluorescence minus one. (D) Graphs depict the MFI of P2RX7 labeling by liver and blood T-cell subsets (blue square symbols) and their respective FMO controls (white circle symbols), as indicated by connecting lines between symbols. A total of four human liver donors and six blood donors were analyzed across two separate experiments. The FMO control for NKT cells within one liver donor was not analyzed due to low cell numbers. Half-shaded symbols represent matched blood and liver samples from one donor.

Figure 1.

Unconventional T cells express P2RX7 to a greater extent than conventional T cells. (A) Graph shows MFI of P2RX7 labeling on MAIT cells (MR1-5-OP-RU tetramer⁺CD3⁺), Vδ2⁺, Vδ1⁺, and Vδ1⁻Vδ2⁻ γδT cells (γδTCR⁺CD3⁺), NKT cells (CD1d-α-GalCer tetramer⁺CD3⁺), and conventional T cells (conv. T; defined as non-MAIT/NKT γδTCR⁻CD3⁺ T cells) from human blood and liver. A total of 4 human livers and 18–19 blood donors were analyzed across six separate experiments. Half-shaded symbols represent matched blood and liver samples from one donor. Due to their paucity, NKT cells within one liver and one blood donor were not analyzed. (B) Graph shows percentages of ARTC2⁺P2RX7⁺ cells out of total MAIT, γδT, and NKT cells from C57BL/6 WT mouse organs. Each symbol represents an individual mouse. A total of eight mice were analyzed across three separate experiments. (C) Uniform Manifold Approximation and Projection (UMAP) representation of flow cytometric analysis of liver T cells. UMAP plots were generated by concatenation of all (n = 2) mice from one of two similar experiments. Red arrows within UMAP plots indicate various T-cell populations. (D and F) Graphs show percentages of ARTC2⁺P2RX7⁺ cells of T-bet⁺ MAIT1, γδT1, and NKT1 cells, RORγt⁺ MAIT17, γδT17, and NKT17 cells (D), and CD44^hi and CD44^neg non-MAIT/NKT αβT-cell CD4/CD8 subsets (F) from C57BL/6 WT mouse organs. (E and G) Flow cytometric analysis of ARTC2 and P2RX7 expression by indicated cell types. Red arrows in G indicate ARTC2^hi cells. (H) Flow cytometric analysis of PLZF and ARTC2, and T-bet and RORγt expression on indicated non-MAIT/NKT αβT-cell subsets. (A, B, D, and F) Graphs depict individual data points and mean ± SEM. ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 using a Wilcoxon matched-pairs signed-rank test with a Bonferroni-Dunn correction for multiple comparisons where required. (E, G, and H) Numbers in plots represent the percentage of gated cells. (D–F) The percentages of ARTC2⁺P2RX7⁺ thymic MAIT1 (D), iLN MAIT1 cells (E), and iLN CD44^neg CD4⁻CD8⁻ T cells (F) could not be reliably determined (n.d.) due to their paucity. (E) FACS plots generated by concatenation of all (n = 3) mice from one of three similar experiments. With the exception of H, all mice were injected with the anti-ARTC2 NB (clone: s + 16) prior to organ harvest.

Figure S2.

Expression of ARTC2 and P2RX7 on T-cell subsets across mouse organs. (A) UMAP representation of flow cytometric analysis of liver and spleen T cells. UMAP plots were generated by concatenation of data from n = 2 mice from one of two similar experiments. Red arrows within UMAP plots indicate various T-cell populations. (B) Representative gating of T-bet⁺RORγt⁻ MAIT1, γδT1, and NKT1 cells, and RORγt⁺T-bet⁻ MAIT17, γδT17, and NKT17 cells in C57BL/6 WT mouse liver, as indicated by red arrows. (C) Graph depicts the percentage of ARTC2⁺P2RX7⁺ cells of CD44^neg and CD44^hi γδT cells within C57BL/6 WT mouse organs. n = 3 separate experiments with a total of eight mice. ns P > 0.05, *P ≤ 0.05 using a Wilcoxon matched-pairs signed-rank test with a Bonferroni-Dunn correction for multiple comparisons where required. (D) Flow cytometric analysis of ARTC2 and P2RX7 expression on CD44^hi or CD44^neg non-MAIT/NKT CD4⁺, CD8⁺, and CD4⁻CD8⁻ αβT cells. Numbers in FACS plots represent the percentage of gated cells. CD44^neg CD4⁻CD8⁻ αβT cells in the iLNs were not determined (n.d.) due to their paucity. (E) Graphs depict the MFI of ARTC2 and P2RX7 expression by the indicated cell types. (F) Flow cytometric analysis of PLZF, CD44, and ARTC2 expression as indicated by non-MAIT/NKT αβT and γδT cells from the spleen and liver. (C and E) Graphs depict individual data points and mean ± SEM. With the exception of F, all mice were injected with the anti-ARTC2 NB s + 16 prior to organ harvest.

Figure S3.

Analysis of T-cell subsets in P2RX7-deficient mice. (A) Flow cytometric analysis of P2RX7 expression on B220⁻ lymphocytes from the thymus and spleen of C57BL/6 WT and P2rx7^−/− mice. Numbers in FACS plots represent the percentage of gated cells. (B) Graph depicts the absolute number of lymphocytes within C57BL/6 WT and P2rx7^−/− mouse organs. (C) Graphs depict the number and percentage of indicated T-cell populations of total T cells within WT and P2rx7^−/− mouse organs. CD4⁺, CD8⁺, and CD4⁻CD8⁻ T cells are non-MAIT/NKT αβT cells; ARTC2⁺ T cells are all T cells that express ARTC2. (D) Flow cytometric analysis of PLZF and ARTC2 expression on indicated T-cell populations from the livers of WT and P2rx7^−/− mice. FACS plots are representative of 8–12 mice per group analyzed across n = 2–3 separate experiments. (E and F) Graphs depict the percentage (E) and number (F) of indicated MAIT, γδT, and NKT-cell subsets from specified organs. Open symbols depict WT mice from a different animal house facility used to ensure sufficient numbers for comparison to P2rx7^−/− mice. (B, C, E, and F) Each symbol represents an individual mouse. Graphs depict individual data points and mean ± SEM. n = 2–3 separate experiments with a total of 8–12 mice/group. ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using a Mann–Whitney U test.

Figure 2.

RA induces expression of ARTC2 and P2RX7 on T cells. (A) Experimental schematic. Mouse thymocytes, splenocytes, or liver lymphocytes were cultured for 3 days in the presence of 20 nM all-trans retinoic acid (RA) or the vehicle control. Three to five thymuses were pooled prior to the complemented-mediated depletion of immature CD24⁺ thymocytes. Splenocytes and liver lymphocytes were obtained from mice injected with the anti-ARTC2 NB (clone: s + 16) prior to organ harvest. (B and C) Flow cytometric analysis of ARTC2 and P2RX7 expression by indicated thymic T-cell types 3 days after treatment with RA or vehicle control. Histograms and FACS plots show concatenated data from all replicate samples from one of three similar experiments. (B) Numbers in histograms represent percentages of P2RX7⁺ and ARTC2⁺ cells out of indicated T-cell types. (C) Numbers in FACS plots represent the percentage of gated cells out of T-bet⁺ MAIT1, γδT1, and NKT1 cells, or PLZF⁻ non-MAIT/NKT αβT cells. (D and E) Graphs show percentages of ARTC2⁺P2RX7⁺ cells of indicated T-cell types from thymus (D), spleen, and liver (E), and MFI of P2RX7 and ARTC2 expression (D) White- and purple-shaded circles represent data from the vehicle or RA-treated cells, respectively. Connecting lines represent paired data. Numbers adjacent to data points represent the average percentage for indicated data sets. For thymus data, each symbol represents data from three to five pooled thymuses where a total of nine pooled thymus samples were analyzed across three separate experiments. (E) For spleen and liver data, each symbol represents an individual mouse, where six to seven mice were analyzed across three separate experiments. MAIT17 and NKT17 cells were not analyzed within one liver sample due to their paucity. (D and E) ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01 using a Wilcoxon matched-pairs signed-rank test. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/b87o281.

Figure 3.

P2RX7 activation on unconventional T cells induces cell death and loss of surface CD27. (A) Experimental schematic. Liver and spleen cells from untreated or anti-ARTC2 NB (clone: s + 16)-treated mice were cultured at 4°C or 37°C for 30 or 90 min prior to analysis. Untreated mouse cells were cultured with or without the P2RX7i, A438079 (10 µM). (B) Flow cytometric analysis of liver MAIT, γδT, and NKT cells co-labeled with 7-AAD and Annexin V after a 90-min incubation. (C) Graphs depict the percentage of 7-AAD⁺ (dead) cells of total MAIT, γδT, and NKT cells from liver and spleen. n = 3 separate experiments with a total of four to eight mice/group. ns P > 0.05, *P ≤ 0.05 using a Mann–Whitney U test with correction for multiple comparisons for the untreated groups. (D) Flow cytometric analysis of surface CD27 expression on liver MAIT, γδT, and NKT cells. (E) Graphs depict percentages of CD27⁺ cells out of viable T-bet⁺ MAIT1, γδT1, and NKT1 cells, or RORγt⁺ MAIT17, γδT17, and NKT17 cells. n = 3–4 separate experiments with a total of 8–10 mice/group. ns P > 0.05, **P ≤ 0.01 using a Wilcoxon matched-pairs signed-rank test. (B and D) Numbers in FACS plots represent the percentage of gated cells. (C and E) Graphs depict individual data points and mean ± SEM. Each symbol represents an individual mouse. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/e39w615.

Figure S4.

Effects of P2RX7 activation on other T-cell subsets beyond MAIT1, γδT1, and NKT1 cells. (A) Graphs depict the percentage of 7-AAD⁺ MAIT, γδT, and NKT cells from thymus. n = 3 separate experiments with a total of four to eight mice/group. ns P > 0.05 using a Mann–Whitney U test, with a correction for multiple comparisons for the untreated groups. Mice were injected with the anti-ARTC2 NB (clone: s + 16) or were untreated prior to organ harvest. (B–F) Graphs depict the percentage of CD27⁺ cells out of each indicated cell type from C57BL/6 WT (B, D, E, and F), or C57BL/6 WT and P2rx7^−/− mice (C) after incubation for 30 min at 4°C or 37°C. Graphs depict individual data points and mean ± SEM. (C) n = 2 separate experiments with a total of six to eight mice/group. ns P > 0.05, *P ≤ 0.05 using a Wilcoxon matched-pairs signed-rank test. (B, D, E, and F) n = 3–4 separate experiments with a total of 8–10 mice/group. Each symbol represents an individual mouse. Graphs depict individual data points and mean ± SEM. ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01 using a Wilcoxon matched-pairs signed-rank test. (G) Representative overlay histograms of ARTC2 and P2RX7 expression by liver MAIT, γδT, and NKT cells. Dark and light-shaded histograms represent cells from NB-treated and untreated mice, respectively. Numbers within FACS plots represent the MFI. FACS plots are representative of n = 3–4 separate experiments with a total of 8–10 mice/group. (F) CD44^neg CD4⁻CD8⁻ αβT cells were not determined (n.d.) in iLNs due to paucity.

Figure 4.

Cognate antigen encounter partially rescues MAIT1 and NKT1 cells from the effects of NICD. (A) Experimental schematic. Mice were i.v. administered 5-OP-RU (200 pmol), α-GalCer (2 µg), or PBS and liver and spleens were harvested 45–60 min later. Liver and spleen cells were cultured at 4°C or 37°C for 40 min with or without the P2RX7i, A438079 (10 μM), prior to analysis. (B) Graphs depict the MFI of CD69 expression by liver and spleen T-bet⁺ MAIT1, γδT1, and NKT1 cells from indicated treatment groups. (C and D) Graphs depict percentages of viable CD27⁺ cells (C) or Zombie NIR⁺ (dead) cells (D) out of indicated cell types. (E) Representative flow cytometric analysis of ARTC2 and P2RX7 expression by the cell types specified. Numbers in FACS plots represent the percentage of gated cells. MAIT-cell FACS plots show concatenated data from all (n = 3-4) mice of the same treatment group from one of two similar experiments. (F) Graphs depict the MFI of ARTC2 and P2RX7 expression by indicated cell types. (B–D and F) A total of six mice per group were analyzed across two separate experiments. ns P > 0.05 (not shown on graphs), *P ≤ 0.05, **P ≤ 0.01 using a Mann–Whitney U test with a Bonferroni-Dunn correction for multiple comparisons. Graphs depict individual data points and mean ± SEM. Each symbol represents an individual mouse. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/j46m750.

Figure 5.

ARTC2 blockade improves recovery of adoptively transferred unconventional T cells. (A) Experimental schematic. Liver and spleen cells from anti-ARTC2 NB (clone: s + 16)-treated or untreated mice were labeled with CTV (NB-treated) or CFSE (untreated) prior to co-transfer at a 1:1 ratio into recipient mice. Donor liver and spleen cells were recovered from recipient mouse livers and spleens 8 days later. Donor and recipient spleen cells were subjected to magnetic bead depletion of B220⁺ and CD62L⁺ cells, or B220⁺ cells, respectively. (B and D) Flow cytometric analysis of donor CD45.1⁺ MAIT, γδT, NKT, and non-T/B cells sourced from liver (B) or spleen (D) and recovered from liver and spleens of recipient mice 8 days after adoptive transfer. Numbers in FACS plots represent the percentage of gated cells. MAIT and γδT-cell FACS plots were generated by concatenation of data from all (n = 3) mice of the same group from one of two similar experiments. (C and E) Stacked bar charts depict the percentages of donor CTV⁺ (purple) and CFSE⁺ (green) cells upon recovery from recipient mice on day 8. Graphs depict individual data points and mean ± SEM. n = 2 separate experiments with a total of six recipient mice per group. ns P > 0.05, *P ≤ 0.05 using a Wilcoxon matched-pairs signed-rank test for comparison between NB-treated versus untreated. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/z81c196.

Figure S5.

Recovery of adoptively transferred unconventional T cells after ARTC2 blockade from pLNs and the subset distribution of adoptively transferred unconventional T cells from spleen and liver. (A) Graphs show the ratio between percentages of CTV⁺ cells relative to CFSE⁺ cells pre- and post-adoptive transfer. (B) Flow cytometric analysis of donor CD45.1⁺ MAIT, γδT, and non-T/B cells sourced from liver or spleen and recovered from the pooled pLNs of recipient mice 8 days after adoptive transfer. MAIT and γδT-cell FACS plots were generated by concatenation of data from all (n = 3) mice of the same group from one of two similar experiments. NKT cells were not analyzed due to low LN cell numbers. Stacked bar charts depict the percentage of recovered donor CTV⁺ and CFSE⁺ cells sourced from liver and spleen after adoptive transfer. ns P > 0.05, *P ≤ 0.05 using a Wilcoxon matched-pairs signed-rank test for NB-treated versus untreated. Graph shows the ratio between the percentage of CTV⁺ cells relative to CFSE⁺ cells pre- and post-adoptive transfer. (A and B) Graphs depict individual data points and mean ± SEM. Post: each symbol represents an individual mouse, where a total of six mice/group were analyzed across n = 2 separate experiments. Pre: each symbol represents a separate experiment where cells from eight mice were pooled. (C) Graphs depict the absolute numbers of recovered CTV⁺ and CFSE⁺ MAIT, NKT, and γδT-cell subsets after adoptive transfer. MAIT1, γδT1, and NKT1 cells defined as CD44⁺CD319⁺. MAIT17 and NKT17 cells defined as ICOS⁺CD319⁻. γδT17 cells defined as CD44^hiCD319⁻. Remaining CD44⁻CD319⁻ γδT cells defined as “other.” Connecting lines represent paired data (cells recovered from the same organs). ns P > 0.05, *P ≤ 0.05 using a Wilcoxon matched-pairs signed-rank test. n.d. = not determined.

Figure 6.

Inhibition of ARTC2 or P2RX7 preserves IFN-γ–producing unconventional T cells. (A) Experimental schematic. Liver and spleen cells from untreated or anti-ARTC2 NB (clone: s + 16)-treated mice were stimulated with PMA and ionomycin and analyzed 4 h later. Untreated mouse cells were stimulated with or without the P2RX7i, A438079 (10 µM). (B) Flow cytometric analysis of IL-17A and IFN-γ expression by CD44⁺ MAIT, γδT, and NKT cells. Numbers in FACS plots represent the percentage of gated cells. (C and D) The percentage (%) and absolute number (#) of IL-17A⁻IFN-γ⁺ and IL-17A⁺IFN-γ⁻MAIT, γδT, and NKT cells out of total MAIT, γδT, and NKT cells from WT (C) and WT and P2rx7^−/− mice (D), as indicated, were graphed. (D) n = 2 separate experiments with a total of six to seven mice/group. n.s. P > 0.05 (not shown on graph), *P ≤ 0.05, and **P ≤ 0.01 using a Mann–Whitney U test with a Bonferroni-Dunn correction for multiple comparisons. (E) Graphs depict the percentage and absolute number of IL-17A⁻IFN-γ⁺ and IL-17A⁺IFN-γ⁻ cells among the specified CD44⁺ non-MAIT/NKT αβT-cell CD4/CD8 subsets. (C and E) Each symbol represents an individual mouse. n.s. P > 0.05 (not shown on graph) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 using a Wilcoxon matched-pairs signed rank test for +P2RX7i versus −P2RX7i or using a Mann–Whitney U test with a Bonferroni-Dunn correction for multiple comparisons for all other comparisons between conditions. n = 3–5 separate experiments with a total of 8–12 mice/group. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/v48p152.

Figure 7.

Blockade of ARTC2-mediated P2RX7 activation reveals unconventional T cells that co-produce IFN-γ and IL-4. (A) Flow cytometric analysis of IL-4 and IFN-γ expression by CD44⁺ MAIT, γδT, and NKT cells, and by CD44⁺ non-MAIT/NKT αβT-cell CD4/CD8 subsets. Numbers in FACS plots represent the percentage of gated cells. (B–D) The percentage (%) (B) and absolute number (#) (C and D) of IL-4/IFN-γ subsets among the specified T-cell subsets were graphed. Each symbol represents an individual mouse. n = 3 separate experiments with a total of seven to eight mice/group. n.s. P > 0.05 (not shown on graph), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using a Wilcoxon matched-pairs signed-rank test for +P2RX7i versus −P2RX7i or using a Mann–Whitney U test with a Bonferroni-Dunn correction for multiple comparisons for all other comparisons between conditions.

Figure 8.

NAD selectively depletes liver T-bet⁺ unconventional T cells in vivo. (A) Experimental schematic. Anti-ARTC2 NB (clone: s + 16)-treated or untreated mice were i.v. administered with NAD (10 mg) or PBS 30 min prior to organ harvest. (B and C) Flow cytometric analysis of PLZF and T-bet expression by liver T cells (B), and RORγt and T-bet expression by liver MAIT, CD44⁺ γδT, and NKT cells (C). (D and E) Graphs show the percentages (D) and absolute numbers (E) of T-bet⁺ MAIT1, γδT1, and NKT1 cells, and RORγt⁺ MAIT17, γδT17, and NKT17 cells out of total MAIT, γδT, and NKT cells, and of PLZF⁺ARTC2^hi CD4⁺ and CD4⁻CD8⁻ cells out of total CD4⁺ and CD4⁻CD8⁻ CD44^hi non-MAIT/NKT αβT cells. (F) Flow cytometric analysis of PLZF and ARTC2 expression. (G) Graph depicts the absolute number of the indicated liver PLZF/ARTC2 T-cell subsets. (H) Graphs depict the fold change in the MFI of T-bet expression in the specified liver T-cell types relative to PBS control mouse samples, or MFI of RORγt expression, as indicated. Dotted line represents a fold change of 1. (I) Flow cytometric analysis of CD69 against T-bet, PLZF, and ARTC2 expression. (J) Graphs depict the numbers of CD69⁺ and CD69⁻ MAIT, γδT, and NKT cells, and non-MAIT/NKT αβT-cell subsets from liver. (D, E, G, H, and J) n = 2 separate experiments with a total of six to seven mice/group. Graphs depict individual data points and mean ± SEM. Each symbol represents an individual mouse. n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01 using a Mann–Whitney U test with a Bonferroni-Dunn correction for multiple comparisons. (B, C, F, and I) Numbers in FACS plots represent the percentage of gated cells. All experimental schematics were in part created in BioRender. Xu, C. (2024) https://BioRender.com/i91e494.