Lipid interactions of the T cell receptor complex

Every cell of our body is surrounded by a plasma membrane that separates the inside of a cell from the outside. This is not just a static barrier but the stage of an intricate interplay of invisibly small structures, proteins and even smaller lipids, that move and assemble themselves in complex ways to mediate membrane function. A special type of cells, T-cells, are major players in our immune system. One protein in their plasma membrane, the T-cell receptor (TCR), recognizes a part of a pathogen (the antigen), which is presented by another cell, that has previously found this intruder in our body. Upon this recognition process, the T-cell becomes activated and a series of events is initiated ultimately leading to an immune response. We have already a good understanding of the protein players involved in this process, but the role of lipids is largely unclear. This is because lipids are extremely hard to study, particularly in living cells. Lipids and proteins are much too small to be directly imaged using optical microscopic methods, and they are constantly moving around. However, by labelling only a tiny subset of these molecules with fluorescent dyes they can be detected as individual diffraction limited spots and we can follow their movement over time and watch, how they interact with each other. Still, this is not good enough for lipids, because their interactions with proteins are often too short. Further, they cannot only influence proteins by direct, one-on-one interaction, but they also make up the 2-dimensional matrix of the plasma membrane itself. There are thousands of different lipid species with very different properties and their organization and dynamics shape the properties of the membrane. Here, a novel experimental approach will be employed and will allow, for the first time, to interrogate the interaction of lipids with the TCR. T-cells will be interfaced with microstructured substrates which mimic antigen presenting cells; this allows to immobilize and enrich the TCR specifically within certain areas of the T-cell plasma membrane in live cells. Thus, it becomes possible to extract quantitative parameters on the interaction of the immobile interaction partner (the TCR) with a mobile interaction partner (the lipid). Several powerful single molecule fluorescence techniques will be used to for the first time observe the behavior of the lipids with respect to the TCR to elucidate their role in the process of T-cell activation.

The plasma membrane is a hub for cellular signaling, where the intricate interplay of proteins and lipids creates a multitude of interconnected functional units. A prominent and well-studied example of this is T-cell antigen recognition, which takes place in the area of contact between a T-cell and an antigen presenting cell (APC). Upon recognition of specific antigen-carrying MHC molecules (pMHC) on the APC surface, T-cell receptors (TCRs) rapidly reorganize to form microclusters in which signaling relevant proteins become concentrated, eventually leading to T-cell activation. In this project, we aimed to engineer novel micro- and nanostructured biointerfaces to study T-cell plasma membrane organization and the associated cellular signaling processes directly in living cells. We used a protein micropatterning assay recently developed by us, which is based on confining a protein of interest to defined areas in the cellular plasma membrane and monitoring its interaction with lipids or proteins via single molecule fluorescence microscopy. In this fashion, we could show that the interplay of membrane-anchored proteins and lipids was governed by their physical size rather than the formation of nanoscopic ordered membrane domains. Next, we modified the micropatterning assay to study the interaction kinetics between the TCR and an intracellular signaling protein. Our new experimental approach allowed the isolation of specific protein binding from background events thus eliminating sources of error and considerably simplifying analysis. Performing single molecule fluorescence microscopy experiments with the TCR, we realized that its nanoscopic organization was a relevant parameter in T-cell signaling. To study this aspect at a quantitative and mechanistic level, we developed an APC-mimicking biointerface, which allowed the experimenter to adjust protein distances with nanoscale precision as a means to disturb signaling. Specifically, we used DNA nanotechnology to generate ligand-functionalized DNA origami platforms and anchored these to uid planar supported lipid bilayers. We found that ligand spatial cues did not generally dictate T-cell activation, but that this effect was strongly dependent on ligand-TCR interaction kinetics. In case of high-affinity interactions, close proximity of ligands within a distance of less than 20 nanometers was required for efficient T-cell activation. However, for the physiological ligand, transiently binding pMHC, the spatial organization was not a relevant parameter for T-cell activation as single, well-isolated pMHC molecules efficiently stimulated T-cells. This finding has wide-ranging implications for our mechanistic understanding regarding T-cell antigen recognition and, as a consequence, for the design of T-cell-based immunotherapies.