Time-resolved role of P2X4 and P2X7 during CD8⁺ T cell activation

CD8⁺ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca²⁺ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca²⁺ microdomains in CD4⁺ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8⁺ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca²⁺ microdomains, global Ca²⁺ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca²⁺ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8⁺ T cells lacking P2X4 and P2X7 channels, global Ca²⁺ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8⁺ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8⁺ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca²⁺ events during CD8⁺ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.