Final Report Summary - NANODYNATCELLVATION (Nano -structural and -dynamic events in the T-cell activation)
Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by mayor histocompatibility complex (MHC) proteins. A productive interaction triggers the T cell signaling leading to the immunological synapse. Initially, the TCR is phosphorylated by the LCK, a membrane-anchored tyrosine kinase, producing membrane micro- and nano-clusters; another functional direct consequence of the triggering is the free cytosolic calcium release. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. There is poor quantitative information on the early activation process, mainly because there is no proper model that mimics the truly resting state. T cells are suspension cells, and under the microscope have always been studied in contact with a surface. For instance, cell basal membrane Super-resolution imaging techniques suggested protein pre-clustering into “nano-domains” in resting cells, contradicting the classical view of cluster formation upon activation. In the first stage of the project we focused in developing a model that mimics physiologically the true resting state, and that can be used to employ super-resolution structural and dynamical optical microscopy. We studied live T cells in suspension employing a hydrogel in a density gradient, and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of TCR and LCK in T-cells on surfaces and in suspension. We first used this protocol to understand the different dynamical and structural reorganisation states of the actin cytoskeleton during the early Tcell activation. This work has been recently published in Science Advances (Fritzsche et al, 2017). Next, we have focus in the localisation, and distribution of the TCR and LCK in suspension, and in the close contact regions, and we have followed the dynamics of these two crucial proteins individually and together, and we have observed the calcium release. For this purpose, we had to develop a method to suit our existing microscopes (scanning FCCS, and scanning STED FCCS), and we had to generate the software to analyse the data (FoCuS_scan). This new methodology has been recently published in the journal methods (Waithe et al, 2017) and the software is been made freely available to academic users via GitHub, and licenced under an IP with the University of Oxford. Employing this experimental approach we found that T cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of LCK and TCR. Nevertheless, they still displayed heterogeneity in the diffusion at the plasma membrane in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels. This work is currently in second round of revision in the journal Nature Immunology.