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.

Antigen recognition by T-cells is integral to our immune system in the fight against pathogens. However, when misguided, T- cells cause severe, at times fatal autoimmune diseases. We are currently witnessing a remarkable progress in the development of cancer immunotherapies based on reprogramming T-cells, highlighting even more their enormous clinical potential. Nonetheless T-cell antigen recognition is not well understood at the molecular and subcellular level. It is tied to the formation of an immunological synapse (IS), the site of contact between a T-cell and its conjugated antigen presenting cell (APC), and driven by the binding of the T-cell antigen receptor (TCR) to fragments of protein antigens, i.e. antigenic peptides, processed and presented by APCs in the context of MHC-molecules. Remarkably, T-cells are exquisitely sensitive as they can detect the presence of just a single antigenic peptide-MHC complexes (pMHCs) on the surface of APCs. This is despite the fact that TCR- pMHC interactions are very weak, and that antigenic pMHCs are vastly outnumbered by non-stimulatory yet structurally similar pMHC molecules featuring non-antigenic endogenous peptides. So how can we then explain the efficacy of T-cell antigen recognition? Importantly, TCR-pMHC binding takes place within the unique binding environment of the IS with molecular binding dynamics considerably altered due to the way TCRs and their ligands are facing each other and also the simultaneous binding of other accessory proteins providing adhesion. To account for these factors, we have developed highly resolving molecular imaging modalities, which allow us to monitor TCR-pMHC binding within the IS. We have identified the adhesion molecule ICAM-1 to be instrumental for proper TCR-pMHC binding. We are currently investigating the extent to which TCR-pMHC -association and - dissociation as well as TCR-proximal signaling are affected by LFA-1-ICAM-1 binding. Quite unexpectedly we also discovered that T-cells modified with anti-tumor antigen-reactive chimeric antigen receptors (CARs) to target cancer cells require ICAM-1 for the recognition of low levels of tumor antigens, even though CARs feature unlike TCRs high affinities towards their antigen. Hence, both adhesion and signaling via LFA-1 contribute to T- cell antigen recognition when antigen is low. Challenging previous reports, we observed TCRs to act as individual receptors and not as physically associated clusters. We also found that pMHC molecules move laterally as monomeric entities with a high diffusion rate on the surface of APCs. Combined our data imply that TCR-pMHC interactions do not involve higher order structures or induced formation thereof. We are currently quantifying the extent to which so-termed co-agonists, i.e. pMHC molecules considered to promote the detection of rare antigenic pMHCs via barely measurable TCR-affinities, affect TCR-binding of antigenic pMHCs within the immunological synapse. Combined our data contribute to a better understanding of T-cell antigen recognition on a cellular and molecular level, which is instrumental for optimizing already existing T-cell based immunotherapies as well as for devising novel clinical approaches involving T-cells.