ATP release and autocrine signaling through P2X4 receptors regulate γδ T cell activation

Abstract

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional αβ T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the "unconventional" γδ T lymphocytes. We observed that γδ T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of γδ T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca(2+) signaling in response to TCR stimulation. qPCR revealed that γδ T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-α and IFN-γ in γδ T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human γδ T cells.

Figure 1.. γδ T cells release ATP upon in vitro stimulation.

Purified γδ T cells suspended in supplemented RPMI were stimulated with anti-CD3/CD28-coated beads (one bead/cell) or 25 μM IPP for the indicated time periods, and increase in extracellular ATP concentration poststimulation was determined with a luciferin/luciferase ATP bioluminescence assay kit. Data shown are representative of multiple experiments (n=4), and values indicate the increase in ATP concentrations in the culture media after cell stimulation. Basal ATP levels of unstimulated cells were 100 ± 6 nM; data shown are averages ± sd; *P < 0.01 as compared with unstimulated controls.

Figure 2.. γδ T cells release ATP through panx-1 and/or connexin hemichannels, as well as vesicular exocytosis.

Purified γδ T cells were pretreated for 20 min with ¹⁰panx-1 (400 μM), CBX (25 μM), Bf A (50 nM), or DIDS (200 μM) and then stimulated with anti-CD3/CD28-coated beads (one bead/cell; A) or IPP (25 μM; B) for 30 s. The increase in ATP concentration in the culture supernatant was measured with an ATP bioluminescence assay kit as described in Fig. 1. ATP release data are expressed as percentage of the ATP release by control cells stimulated in the absence of inhibitors. Basal ATP concentrations in culture supernatants of unstimulated cells were 87 ± 7 nM. Data shown are averages ± sd; n = 3; #P < 0.05; *P < 0.01 as compared with control.

Figure 3.. Blockade of panx-1 inhibits anti-CD3-induced Ca²⁺ signaling in γδ T cells.

Human PBMCs were loaded with Fluo-4 and labeled with anti-γδ TCR-APC antibodies, and Ca²⁺ signaling, in response to anti-CD3 (1 μg/ml) in the absence or presence of the indicated concentrations of ¹⁰panx-1, was recorded with flow cytometry. Baseline and maximum intracellular Ca²⁺ concentrations were determined using buffer control and ionomycin, respectively. Representative dot plots of Fluo-4 MFI readings over time (total duration: 512 s) are shown in A, and averaged data of a representative experiment are depicted in B. Arrows indicate the time-points at which buffer, anti-CD3, or ionomycin was added to the cells. Inhibition of Ca²⁺ signaling was calculated by combining data sets from three individual experiments (C). Data shown are averages ± sd; n = 3; #P < 0.05 as compared to stimulation with anti-CD3 alone.

Figure 4.. Peripheral human γδ T cells predominantly express the A2a, P2X1, P2X4, P2X7, and P2Y11 receptor subtypes.

Total RNA isolated from >98% pure γδ T cells was subjected to qPCR using primers specific for the 19 known human purinergic receptor subtypes. Analyses were performed in triplicate, and mRNA expression levels of individual receptors were normalized to β-actin. Data shown are averages ± sd; n = 3.

Figure 5.. P2X4 receptors play an important role in the activation of γδ T cells.

Purified γδ T cells were pretreated with NF023 (10 μM; P2X1 antagonist), TNP-ATP (30 μM; P2X4 antagonist), A438079 (10 μM; P2X7 antagonist), NF157 (10 μM; P2Y11 antagonist), or suramin (200 μM; nonselective P2 receptor antagonist) for 20 min. Then, the pretreated cells were stimulated with anti-CD3/CD28-coated beads (one bead/cell; left panels) or IPP (25 μM; right panels) for 4 h, and mRNA expression of CD69 (A and B), TNF-α (C and D), and IFN-γ (E and F) was assessed with qPCR. Individual expression levels were normalized to that of β-actin. Data shown are averages ± sd; n = 4; #P < 0.05; *P < 0.01; **P < 0.001.

Figure 6.. Proposed purinergic signaling in human γδ T cells.

In response to cell stimulation (1), purified γδ T cells release ATP through panx-1, connexin hemichannels, and vesicular exocytosis (2). P2X4 receptors bind to released ATP at the immunological synapse (3), and open to facilitate the influx of extracellular Ca²⁺ ions (4). The subsequent downstream signaling cascades culminate in up-regulated transcription and expression of cytokines such as IFN-γ and TNF-α (5). Blockade of ATP release or pharmacological inhibition of P2X4 receptors inhibits purinergic signaling and subsequent functional responses of stimulated γδ T cells.