P2Y11: The CD4 T cell mitochondrial shepherd

Graphical Abstract

Discussion on the article from Ledderose et al. (this issue), about how the ATP sensor P2Y11 shapes the intracellular mitochondrial re-orientation and T cell activation.

T cells are activated after engagement of their T cell receptors (TCRs) with cognate antigen presented by antigen-presenting cells (APCs), with the aid of co-stimulatory signals. These signals, which irradiate from a multi-protein structure formed by clusters of TCR, CD3, and co-stimulatory receptors such as CD28 and anchor molecules (e.g. LFA-1) called immunological synapse (IS), serve to amplify the intracellular cascades, leading to changes in protein phosphorylation and, ultimately, to module gene expression and protein synthesis [1]. Purinergic signaling, i.e. engagement of extracellular nucleotides by specific receptors, has been shown to promote CD4 T cell migration, proliferation and subset differentiation [2]. Quickly after activation, the extracellular ATP receptor P2X4 and Panx1 (a hemichannel that promotes ATP release to the extracellular milieu) concentrate in the IS and promote stable, continuous interaction APCs [3]. Among the events leading to T cell activation, the rearrangement of mitochondria in the cytoplasm is a fundamental step, where an accumulation of mitochondria around the IS provides a source of ATP needed for the chain reactions happening at that intracellular region. This includes ATP release via Panx1 and subsequent stimulation of P2X4 [3]. However, the intracellular mechanisms leading to the redistribution of mitochondria at the IS are poorly understood. In this issue of The Journal of Leukocyte Biology, Ledderose et al. addressed this question and described that the extracellular ATP receptor P2Y11 plays a fundamental role in promoting the migration of mitochondria toward the IS [4].

Using in vitro activation systems in human CD4 T cells, Ledderose et al. found that, similar to P2X4, pharmacological antagonism of P2Y11 significantly hampered cell proliferation. P2Y11 antagonism also decreased the expression of activation markers such as CD69, and IL-2 production in response to stimulation. Despite finding no effect of P2Y11 (or P2X4) on the early interactions between CD4 T cells and APCs, Ledderose et al. found instead that P2Y11 or P2X4 blockade diminished the levels of intracellular ATP quickly after T cell stimulation, suggesting both P2Y11 and P2X4 contribute to the initial burst of cell metabolism that follows the interaction between CD4 T cells and APCs, thus contributing to maintaining the metabolic program needed for the completion of T cell activation. In other experiments, the authors used siRNA silencing of P2Y11 receptors, which abrogated mitochondrial trafficking to the IS. Similar effects were found with blockade of P2X4 or by impairing microtubule polymerization, implying P2Y11, P2X4 and the microtubule network as players in the translocation of mitochondria toward the IS. P2Y11 activation alone, however, was not enough to promote organized mitochondrial migration toward the IS. Future experiments will have to be done to confirm the temporal role of distinct receptors, but from the results of Ledderose et al. it seems likely that TCR engagement with cognate antigen and co-stimulation are needed together with P2Y11 to promote the quick redistribution of mitochondria observed during early activation. It was also apparent that P2X4 or P2Y11 inhibition induced a partial (but not complete) abrogation of T cell proliferation. Targeting both molecules simultaneously will be crucial in the future to ensure these molecules are indeed operating in the same pathway to promote mitochondrial trafficking and subsequent T cell proliferation.

P2Y11 recognizes extracellular ATP (eATP), and this new report unveiled a potential role of this receptor as a component of a circuit of ATP export and autocrine/paracrine recognition. Previous work has shown that, after initial TCR stimulation, P2X4 receptors migrate toward the IS, and its engagement acts as a de facto co-stimulus for activation [3]. Simultaneously, the hemichannel Pannexin 1 (Panx1) also populates the IS and promotes ATP export into the pericellular environment [3]. This likely contributes to eATP recognition by P2X4, inducing Ca++ influx to the cell, which potentiates mitochondrial respiration. Reinforcing this cycle, ATP produced and released by IS-near mitochondria serves to increase the availability of ATP for Panx1-mediated export [5], simultaneously serving as fuel for the next steps of T cell activation. Unlike P2X4 (an ion channel), P2Y11 is a G-protein coupled receptor that promotes an intracellular increase in cAMP levels, which in turn activates the PKA and subsequently modulates T cell function. Indeed, the authors found that disruption of cAMP/PKA mimicked the results found with P2Y11 blockade, suggesting P2Y11 control of this pathway is needed for the promotion of CD4 T cell proliferation.

Importantly, P2Y11 does not promote P2X4 accumulation at the IS, indicating that, in the proposed purinergic signaling circuit, P2X4 is upstream of P2Y11. Surprisingly, P2Y11 is displaced from the membrane regions associated with the IS after T cell stimulation. Instead, they preferentially rearrange in the back of cells (i.e. opposite to the IS region). In summary, the authors propose a model where P2X4 migrates toward the IS, and P2Y11 migrates toward the opposite direction, after TCR engagement. Although the precise kinetics between P2X4 and P2Y11 migration is yet to be explored, it is clear that P2Y11 recognition of eATP is important to induce cAMP/PKA at distal regions of the cell, promoting a set of signals to favor mitochondrial migration toward the IS (Figure 1).

Figure 1. Working model on the role of P2Y11 to favor the initial activation of CD4 T cells.

At steady-state (left), distribution of the purinergic receptors P2Y11, P2X4, and the hemichannel Panx1 is relatively uniform, and no relevant eATP signaling occurs. After initial engagement of antigen and co-stimulus (co-stim) via TCR complexes (right), both P2X4 and Panx1 concentrate around the immunological synapse (IS), leading to concentrated eATP export via Panx1. The eATP exported is enough to activate both P2X4 in the IS, and P2Y11 which migrate towards the back of the cell. P2Y11 receptors, upon eATP recognition, induce the cAMP/PKA pathway. This pathway ultimately promotes mitochondrial migration towards the IS through cytoskeleton modifications; mitochondrial accumulation around the IS provides increased ATP, which is exported by Panx1 and signal through P2X4 and P2Y11, and simultaneously harness the signaling events needed for full engagement of the effector program in CD4 T cells. Thus, the re-arrangement of P2X4, P2Y11 and Panx1 on the cellular membrane of activated CD4 T cells constitutes a closed amplification circuit that perpetuates autocrine eATP sensing, aiding in the activation of CD4 T cells.

The mechanism by which P2Y11 promotes mitochondrial translocation to the IS is still unknown. A probable candidate is cAMP/PKA mediated phosphorylation of anchor proteins located in the outer mitochondrial membrane, which in turn induce mitochondrial transport via kinesin and dynein proteins. In addition to promoting mitochondrial motility, cAMP/PKA has been shown in other models to favor cristae integrity and mitochondrial function [6]. Considering this, it will also be important to know whether signals through P2Y11 can directly promote mitochondrial respiration, in addition to other signals from the IS.

The affinity to ATP is equal between P2X4 and P2Y11, in the low micromolar range. Hence, P2X4 being activated upstream (due to its positioning in the IS) can reinforce Panx1 release of ATP which, in turn, would favor P2Y11 to be activated. This way, Panx1 release of eATP would influence P2Y11, gradually. Experiments using Panx1 blockade will be important to gauge to what extent P2Y11 relies on Panx1-derived ATP. Another possibility could be the release of ATP via other channels such as Connexins or exosomal release. It is unknown, however, to what extent those mechanisms of ATP release occur on a T cell, and if true, where in the T cell membrane this would happen (i.e. concentrated in the IS or not). Additionally, the mechanisms behind the redistribution of P2Y11 outside of the IS are unknown and will need further research. P2X7 is another purinergic receptor expressed by T cells, and due to its lower affinity for ATP, P2X7 has been considered a receptor for paracrine ATP source [7]. A role for autocrine ATP in activating P2X7 cannot be fully discarded, however, especially at later time points. Experiments combining knockouts/inhibition of P2X7 and Panx1 will be needed in the future to fully elucidate this question. Also, in several immune/non-immune models it has been implied that P2X7 can regulate ATP release through Panx1. Assessment of whether this is also true during T cell activation will be important to understand whether this participates in the circuit proposed by Ledderose et al. in this article.

Unlike human CD4 T cells, P2Y11 is not expressed in murine T cells. Unveiling the exact components of purinergic signaling that play the same role in mice will be crucial to develop models to better understand how mitochondria are redirected to the IS during initial activation, and what are the consequences of that for long-term T cell responses. Ledderose et al. suggested that adenosine receptors, which also induce cAMP/PKA signaling and are expressed in mouse T cells, can play this role. However, extracellular adenosine signaling is preferentially engaged in steady-state conditions [8]. Thus, unless new evidence suggests adenosine signaling does indeed occur in the early stages of activation it is unlikely this could play a role. Instead, non-purinergic receptors (or an unknown purinergic sensor) could take the place of P2Y11 in mouse models. However, the data showed by Ledderose et al. suggest that induction of mitochondrial movement toward the IS controlled by the P2X4-Panx1-P2Y11 circuit in a non-redundant way, suggesting that alternative mechanisms (i.e. not mediated by purinergic signaling) for induction of mitochondrial trafficking are unlikely.

Another interesting scenario not explored in the current article is how this purinergic system circuit would act in response to an environment high on eATP, for example in the presence of extensive local tissue damage. Ledderose et al. proposed that the excess of eATP would overwhelm the P2X4 and P2Y11 signaling pathways, rendering T cells unable to mount proper immune responses. At the same time, T cells often express ectonucleotidases which may curb these effects [9]. Nevertheless, disruption of P2Y11/P2X4 function may be a fundamental reason why T cells are dysfunctional in chronic infections or cancer, where extracellular ATP is high [10]. Future studies to address this situation will help test this hypothesis, with a potential clinical impact. In summary, this elegant study has added to the notion that sensing of extracellular nucleotides plays a substantial role to ignite and sustain the initial activation of T cells. This study will have a significant impact in the purinergic signaling field, as well as in the understanding of how T cells respond to the surrounding environment when encountering a pathogen or foreign agent.