Imaging T Cell Antigen Recognition

T-cell antigen recognition is fundamental to establishing adaptive immunity and essential for neutralizing pathogens. It is also inherent to allergy and autoimmunity. T-cells have thus been intensely studied for the last 30 years, yet we still lack a fundamental understanding to explain their exquisite sensitivity towards antigen. One difficulty in addressing this subject is rooted in the fact that T-cell antigen recognition takes place in the interface between a T-cell and its conjugated Antigen Presenting Cell (APC), termed the immunological synapse. It is here, where T-cell activation is driven through molecular interactions of T-cell receptors (TCRs) with their nominal antigens, i.e. antigenic peptides presented by MHC molecules (pMHC), but where conventional biochemistry, which requires a membrane-free environment, loses much of its resolving power. We have recently overcome this limitation with a powerful live cell imaging approach, which allows us to deduce the molecular dynamics of T-cell antigen recognition in situ on a truly quantitative level. T-cells are confronted with planar glass-supported lipid bilayers as surrogate APC and TCR-pMHC interactions are measured through single molecule Förster resonance energy transfer (FRET) microscopy. Furthermore, by combining the bilayer- approach with super-resolution microscopy we were able to determine the distribution of the TCR and signaling molecules in living T-cells below the diffraction limit. We now wish to continue these successful lines of experiments to identify and characterize T-cell intrinsic factors controlling synaptic TCR-pMHC binding and ensuing TCR-proximal signaling events. We expect to determine how these factors affect T-cell antigen sensitivity and signaling properties and how they are regulated in T-cell differentiation. We will primarily focus on the influence of cell adhesion (aim 1), co-stimulation (aim 2) and the activity of galectins (aim 3), a class of lectins, on TCR-pMHC binding, proximal signaling events and cell surface distribution of the TCR and accessory molecules on a nanoscopic level. Of particular interest are also homotypic interactions between TCRs and between pMHCs and to quantify the formation of higher order TCR structures in living T-cells, as these might affect TCR-pMHC binding and T-cell signaling (aim 4). To assess the extent to which the T-cell intrinsic factors are subject to regulation in T-cell development, we will compare their importance for TCR-engagement and TCR-proximal signaling in synapses of T-cells of different developmental stages including thymocytes, nave T-cells, effector and memory T-cells.

No additional funding sources recorded.