Calcium signaling dynamics in T cells - role of intra- & extracellular adenine nucleotides

Abstract

For cells to survive, proliferate, and differentiate, it is essential that both extracellular and intracellular signaling are tightly regulated. In T cells, signaling pathways that depend on Ca2+ control their effector functions, resulting in either pro- or anti-inflammatory responses based on how long [Ca2+]i remains elevated. Ca2+ microdomains that are adhesion-dependent and independent of T cell receptors play a role in the initial sensitizing process, which is marked by local signals with reduced Ca2+ amplitudes, such as ATP-dependent Ca2+ microdomains through P2X4. The engagement of the TCR/CD3 complex leads to the rapid generation of NAADP, a second messenger that mobilises Ca2+ and induces Ca2+ microdomains through the action of type 1 ryanodine receptors. These microdomains eventually converge into a global Ca2+ response, which is augmented by store-operated Ca2+ entry and further intensified by IP3 and subsequent cADPR signaling. Additionally, the purinergic Ca2+ channels P2X4 and P2X7, as well as ATP release by pannexin 1 channels, further influence the initial Ca2+ microdomain formation in CD4+ T cells, fine-tuning the T cell response. The ability of NAADP to induce Ca2+ release from intracellular stores such as the ER and lysosomes has been a topic of debate over the past decade. The recent discovery of NAADP-binding proteins HN1L/JPT2 and Lsm12, along with their binding receptors on the ER (RyR1) and lysosomes (TPCs), underscores the need for a tool to directly investigate NAADP-dependent signaling across various cell types. One main aim of this thesis was to characterise the recently developed compound MASTER-NAADP, a membrane-permeable and bioreversible NAADP derivative, and its respective control, in their capacity to evoke local Ca2+ microdomains and global Ca2+ signals in Jurkat T cells by high-resolution live-cell imaging (Publication 2.3). Additionally, using different cell types, e.g. Neuro2A, KHYG-1 and primary murine CD4+ T cells, working concentrations of MASTER-NAADP in those cell types were established, as well as using knockout models of Hn1l/Jpt2 to validate the involvement of the NAADP binding protein. Furthermore, comparison of MASTER-NAADP to the commercially available NAADP-AM in live-cell-imaging, as well as HPLC studies, contributes to the characterisation of MASTER-NAADP. Finally, the liberated derivative was validated in a permeabilized cell assay and in lipid planar bilayer measurements containing HN1L/JPT2 and RyR1. Additionally, the involvement of purinergic signaling pathways in initial Ca2+ microdomain formation as well as downstream effects was identified in CD8+ T cells (Publication 2.2). P2X4 and P2X7 signaling were essential for Ca2+ microdomain formation, as both knockouts and extracellular ATP removal by apyrase resulted in diminished Ca2+ microdomain formation, whereas the global Ca2+ response was only mildly affected. Further, NFAT-1 translocation was impaired in P2rx4-/- and P2rx7-/- CD8+ T cells compared to the WT, along with reduced Nur77, INF-γ and granzyme B expression. Finally, proliferation was also negatively impacted by P2rx4-/- and P2rx7-/- in these cells. As the analysis and quantification of Ca2+ microdomains proved to be time-consuming, a modular open-source Python pipeline for (time) efficient analysis was developed (Publication 2.1). DARTS (Deconvolution, Analysis, Registration, Tracking and Shape normalisation) integrates state-of-the-art image postprocessing options, with variable deconvolution algorithms to choose from depending on image quality, deep-learning based cell detection and tracking and bleaching correction of FuraRed. Further, the integrated cell shape normalisation module allows for aggregation of the spatio-temporal information of Ca2+ microdomains onto a circular template (dartboard projection) allowing for accessible comparison of Ca2+ microdomains across groups. In summary, the updated method for high-resolution live-cell Ca2+ imaging, along with the post-processing and analysis pipeline described here, enables Ca2+ microdomain detection across cell types. In CD8+ T cells, both P2X4 and P2X7 influence not only Ca2+ microdomain formation, but Ca2+ microdomains via these channels are necessary for a proper immune response. Further, MASTER-NAADP is established as a bona fide NAADP-mimic to unravel NAADP-mediated Ca2+ signaling.