Adhesion mediated pre-activation of T cells

Abstract

T cells are an essential part of the adaptive immune system and play a central role in cell-mediated immune response. Their activation leads to an increase of the free cytosolic Ca2+ concentration ([Ca2+]i), which can be described by two main pathways: (i) the release of Ca2+ from intracellular Ca2+ stores into the cytosol and (ii) Ca2+ influx from the extracellular space through Ca2+ channels located at the plasma membrane (PM). Once activated, T cells proliferate and migrate into the inflamed tissue, thereby binding to other cells or extracellular matrix (ECM) proteins via integrins. Already in 2018, our lab observed formation of local Ca2+ microdomains in the absence of TCR stimulation. However, up to now their detailed molecular mechanism could not be fully elucidated. In this thesis, the involvement of various components (such as FAK, IP3R, and SOCE proteins) in adhesion-induced pre-activation of T cells was investigated. Therefore, we utilized three different advanced optical methods: fluorescence resonance energy transfer (FRET), Super Resolution via Optical Re-assignment (SoRa) and Stimulated Emission Depletion (STED) microscopy. Overall, STED microscopy provided the most accurate information for protein localization due to the achievable spatial resolution of up to 40 nm. Furthermore, the formation of clusters could be determined and it was even possible to dissect them into loose and tight clusters. Here, a tight cluster is defined as an interaction between proteins that is visible as a single spot due to its tight localization. Loose clusters, on the other hand, display an accumulation of these spots in a defined region. Based on the existent crystal structures of individual proteins, we were able to examine individual clusters in more detail. Therefore, it was possible to estimate the number of proteins that accumulate at a site. Next, for the first time, adhesion-dependent co-localization with ORAI1, as well as clustering of FAK, the three IP3R subtypes and SOCE proteins (ORAI1 and STIM1/2) could be determined. The increased co-localization and cluster formation of ORAI1, STIM1, FAK and IP3R1, due to adhesion, suggests an essential role of these proteins in pre-activation of T cells. In addition, the data supports the hypothesis that SOCE activation by STIM1 is critical after adhesion, whereas STIM2 serves to regulate Ca2+ levels. In conclusion, these results demonstrate a pre-activation state of T cells evoked by adhesion to ECM proteins, involving FAK, IP3R1, and activation of SOCE. Therefore, our findings can be summarized in a three-state model of T cells: (i) quiescent state, (ii) adhesion pre-activated state and (iii) fully activated state upon TCR stimulation.