Cell surface clusters of MHC class I molecules

Applicant FWF: Dr. Peter Lanzerstorfer, Faculty of Engineering, University of Applied Sciences Upper Austria, Austria. Applicant DFG: Univ. Prof. Dr. Sebastian Springer, Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany. MHC-class I-proteins (MHC-proteins) can be found on the surface of almost all cell types and are mainly responsible for antigen presentation for cytotoxic T cells (T-killer cells). Infected and degenerated cells that produce foreign proteins can thus be specifically identified by the T-killer cells and subsequently eliminated. MHC-proteins are therefore essential for cellular immunity against viruses, intracellular bacteria, and tumours. The cell surface form of the MHC-protein, which binds the T cell receptor, is a non-covalent protein complex consisting of three subunits (trimer): the heavy chain (HC) anchored to the cell membrane, the smaller soluble subunit beta-2-microglobulin (ß2 m) and an antigenic peptide. Under physiological conditions, the peptide presented on the cell surface dissociates from the trimer after a few hours to days (depending on affinity) and an `empty dimer` of HC and ß2 m remains. From this dimer, ß2 m dissociates within a few minutes, and the "free" HC (FHC) remains. We have recently shown that FHCs assemble into clusters on the cell surface. These clusters consist of several FHCs that interact in cis (on the same membrane). Cluster formation has been demonstrated for the first time using a `two-hybrid` antibody-micropattern assay. Our preliminary data suggest that the a3 -domain of the FHC is sufficient for interaction. Furthermore, it appears that FHC clusters are of transient nature, mainly consisting of protein dimers and are responsible for efficient endocytosis of MHC-proteins. Furthermore, possible functions in the transmission of stress signals to other cells of the immune system have been discussed but not yet proven. In the present project, the following points should be clarified: i) Which MHC-allotypes are subject to FHC cluster formation?, ii) How are FHC clusters formed spatially and what are their dynamics?, and iii) What is the biological and physiological function of those clusters? Different methodological approaches will be applied to answer these questions. In order to detect FHC clusters and to get first insights into the interaction dynamics, an adapted antibody-micropattern assay is used. The spatial elucidation of the potential clusters is achieved by means of in-silico models and X-ray crystallography. Single-molecule spectroscopy will provide detailed information about the size, dynamics and affinity of the clusters. Thus, the project lays the basis for a systematic investigation of the physiological role of peptide-free MHC-class I-molecules on the cell surface.

Immune proteins cluster together to stay at the cell surface and avoid being discarded. This was found by the group of Peter Lanzerstorfer (FHOÖ) in collaboration with the group of Sebastian Springer (Constructor University Bremen). MHC proteins work like the proverbial red flag on virus-infected or cancerous cells to alert the immune system. But it is not understood what regulates the lifetime of MHC proteins. How long do they stay at the cell surface? How are they disposed of? Or do they get recycled and re-used? Here we have made an important step on the way of finding out. We investigated one particular shape of the MHC proteins called the free heavy chain (FHC), because we had found earlier that FHCs group together to form clusters at the cell surface. Now, in this project, the question was what these clusters are good for. We hypothesised that the clusters had something to do with the endocytosis MHC proteins, that is, their uptake back into the cell. To test this hypothesis, we made use of the fact that MHC proteins vary greatly between different people. We decided to test whether these variants showed different tendencies to form clusters, and/or different lifetimes at the cell surface, and whether these two properties were related in some way. To do so, a new way of looking at the clusters in the microscope has been invented. We coated a glass plate with antibody micropatterns that hold onto the MHC protein molecules, and we observed other MHC molecules assemble around them. This approach allowed to measure the tendency to form clusters, for each MHC protein variant separately. We found that one MHC protein variant called H 2Kb is especially good in forming clusters, whereas another one, called H 2Ld, forms few to no clusters. At the same time, the group of Sebastian Springer in Bremen, measured the lifetime of the same MHC protein variants at the cell surface with a method called flow cytometry. To do this, they stopped the transport of MHC proteins from the interior of the cell to the surface and then watched the MHC proteins that already were at the cell surface slowly disappear into the cell interior. Importantly, they found that H 2Ld disappears fast, whereas H 2Kb remains at the surface for a long time. This suggests that the cluster formation keeps the MHC proteins at the cell surface. To test this hypothesis, they connected two H 2Ld molecules with an artificial link, and indeed, they stayed at the cell surface for much longer. Next, we will next investigate how these differences between the MHC protein variants contribute to their immunological function such as the activation of immune cells against cancer.