Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse

Abstract

Engagement of T cells with antigen-presenting cells requires T-cell receptor (TCR) stimulation at the immune synapse. We previously reported that TCR stimulation induces the release of cellular adenosine-5'-triphosphate (ATP) that regulates T-cell activation. Here we tested the roles of pannexin-1 hemichannels, which have been implicated in ATP release, and of various P2X receptors, which serve as ATP-gated Ca(2+) channels, in events that control T-cell activation. TCR stimulation results in the translocation of P2X1 and P2X4 receptors and pannexin-1 hemichannels to the immune synapse, while P2X7 receptors remain uniformly distributed on the cell surface. Removal of extracellular ATP or inhibition, mutation, or silencing of P2X1 and P2X4 receptors inhibits Ca(2+) entry, nuclear factors of activated T cells (NFAT) activation, and induction of interleukin-2 synthesis. Inhibition of pannexin-1 hemichannels suppresses TCR-induced ATP release, Ca(2+) entry, and T-cell activation. We conclude that pannexin-1 hemichannels and P2X1 and P2X4 receptors facilitate ATP release and autocrine feedback mechanisms that control Ca(2+) entry and T-cell activation at the immune synapse.

Figure 1

Expression of P2X receptors by human T cells. (A) P2X receptor mRNA expression in Jurkat cells and human peripheral CD4⁺ T cells determined by real-time reverse transcriptase PCR analysis. (B) Immunocytochemical assessment of P2X1 and P2X4 receptor expression of Jurkat cells and human primary CD4⁺ T cells evaluated by fluorescence microscopy. (C) P2X1 and P2X4 receptor mRNA expression levels of Jurkat cells in response to stimulation by PHA (50 ng/mL) and PMA (5 ng/mL). (D) Immunoblotting of P2X1 and P2X4 receptors before and after stimulation of Jurkat cells with PHA (50 ng/mL) and PMA (5 ng/mL). Data represent means ± SEM from triplicate experiments.

Figure 2

Translocation of P2X1 and P2X4 receptors to the immune synapse of activated T cells. Time-lapse confocal live-cell microscopy images showing the expression of EGFP-tagged P2X1 (A) and P2X4 receptors (B) in Jurkat cells stimulated with anti-CD3/CD28 antibody-loaded beads (asterisks). Localization of receptors persists at least 1 hour after stimulation (Right panels: fluorescent microscopy images 60 minutes after stimulation).

Figure 3

P2X1 and P2X4, but not P2X7, receptors translocate to the immune synapse of T cells. Laser scanning microscopy images of primary human T cells activated with anti-CD3/CD28 antibody–loaded beads (asterisks). Cells were fixed after the indicated stimulation time and stained with anti-P2X1 (A), P2X4 (B), or P2X7 (C) receptor antibodies. While P2X4 receptors rapidly translocate to the immune synapse, P2X1 receptors first aggregate in clusters, and then translocate to the synapse after 15-30 minutes. P2X7 receptors do not redistribute upon TCR/CD28 stimulation and maintain their uniform distribution on the cell surface.

Figure 4

P2X1 and P2X4 receptors are expressed with Orai1 and STIM1 at the immune synapse. Confocal live-cell images of Jurkat cells coexpressing EGFP-tagged P2X1 or P2X4 receptors and either Orai1-EYFP (A,C) or STIM1-ECFP (B,D). Unstimulated cells show uniform Orai1, STIM1, and P2X1 and P2X4 receptor distributions (top panels) that localize at the immune synapse within 30 minutes of stimulation with anti-CD3/28 antibody-loaded beads (marked with asterisks in bottom panels). Scale bars represent 10 μm.

Figure 5

P2X1 and P2X4 receptors regulate Ca²⁺ signaling in response to TCR stimulation. Effects of silencing P2X receptors in Jurkat cells (A) or pharmacologic P2X receptor inhibition of human primary CD4⁺ T cells (PBMCs; B) on Ca²⁺ signaling. Cells were stimulated by TCR activation (0.5 μg/mL anti-CD3 antibody). The response was assessed by flow cytometry using Fluo-3 as a Ca²⁺ indicator. Triple silencing was carried out using one-third of siRNA per gene compared with silencing of individual genes. Graphs depict representative results from at least 3 experiments performed with cells from different donors.

Figure 6

P2X1 and P2X4 receptors contribute to NFAT activation and IL-2 expression of T cells. NFAT activation of Jurkat cells overexpressing wild-type (A) or mutated P2X1 or P2X4 receptors (B). NFAT activation (C) and IL-2 mRNA expression (D) of Jurkat cells after silencing of P2X1 or P2X4 receptors. Cell responses were assessed after stimulation with anti-CD3/CD28 antibody-coated beads for 8 hours. (E) IL-2 mRNA expression in Jurkat cells after combined silencing of P2X1, P2X4, and P2X7 receptors. (F) IL-2 mRNA expression in human PBMCs and mouse splenocytes stimulated for 4 hours in the presence or absence of P2X1 receptor–selective (NF023, 10μM), P2X1 and P2X4 receptor–selective (TNP-ATP, 30μM), P2X7 selective (A438079, 10μM), or nonselective (suramin, 100μM) P2 receptor antagonists. #P ≤ .05 compared with resting cells, *P ≤ .05, **P ≤ .01 compared with CD3/CD28- stimulated controls.

Figure 7

Pannexin-1 hemichannels facilitate ATP release at the immune synapse. Stimulation of the TCR leads to redistribution of pannexin-1 to the immune synapse (A). Treatment with the gap channel inhibitor carbenoxolone reduces ATP release and IL-2 gene transcription upon CD3/CD28 stimulation. (B). The pannexin-1 specific inhibitor ¹⁰panx-1 reduced Ca²⁺ entry compared with the control peptide (^scpanx-1), or to CD3 stimulation without any peptide (control) in human PBMCs (C) and IL-2 transcription in human PBMCs and mouse splenocytes (D). *P ≤ .05, **P ≤ .01 compared with CD3/CD28-stimulated controls.