imaging-initial-ca2-microdomains-in-primary-t-cells

Imaging Initial Ca<sup>2+</sup> Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

University Medical Centre Hamburg-Eppendorf

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca microdomains. Purinergic signaling is known to be involved in Ca influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca signals but also promote initial Ca microdomains tens of milliseconds after T cell stimulation. These Ca microdomains were significantly decreased in T cells from  and  mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1-dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca microdomains already in the first second of T cell activation.

P2X4 and P2X7 are essential players in basal T cell activity and Ca signaling milliseconds after 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 Ca microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca 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 Ca microdomains, global Ca responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca microdomain formation was significantly reduced in the first milliseconds to seconds in CD8 T cells lacking P2X4 and P2X7 channels, global Ca 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 Ca concentration ([Ca]). 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 Ca 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.

Article	Year
P2X4 and P2X7 are essential players in basal T cell activity and Ca signaling milliseconds after T cell activation. Science advances\| PubMed ID: 35119925	2022
Time-resolved role of P2X4 and P2X7 during CD8 T cell activation. Frontiers in immunology\| PubMed ID: 38426095	2024

Valerie J. Brock

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