T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP—such as in response to inflammation, sepsis, and cancer—disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.

T cells form an immune synapse (IS) with antigen-presenting cells (APCs) to detect antigens that match their TCR. Mitochondria, pannexin-1 (panx1) channels, and P2X4 receptors congregate at the IS where mitochondria produce the ATP that panx1 channels release in order to stimulate P2X4 receptors. P2X4 receptor stimulation causes cellular Ca 2+ influx that up-regulates mitochondrial metabolism and localized ATP production at the IS. Here we show that P2Y11 receptors are essential players that sustain these T cell activation mechanisms. We found that P2Y11 receptors retract from the IS toward the back of cells where their stimulation by extracellular ATP induces cAMP/PKA signaling that redirects mitochondrial trafficking to the IS. P2Y11 receptors thus reinforce IS signaling by promoting the aggregation of mitochondria with panx1 ATP release channels and P2X4 receptors at the IS. This dual purinergic signaling mechanism involving P2X4 and P2Y11 receptors focuses mitochondrial metabolism to the IS where localized ATP production sustains synaptic activity in order to allow successful completion of T cell activation responses. Our findings have practical implications because rodents lack P2Y11 receptors, raising concerns as to the validity of rodent models to study treatment of infections and inflammatory conditions.

Cell-cell junctions of blood endothelial cells are critical barriers in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin, and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2) phosphorylation by localising GEF-H1 to cell junctions. Using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function in diseases.