Protein Dimensions and Interactions at Immune-Cell Contacts

Sammanfattning: Immune cells, such as B and T cells, fight pathogens infecting the human body. To fulfil this function the immune cells need to form a contact during which different membrane proteins play an important role. A key event in this contact is the interaction between the T-cell receptor (TCR) and an antigenic peptide-presenting major histocompatibility complex (MHC) protein. If this interaction is sufficiently strong then the T cell gets activated leading to an immune response. The TCR-MHC together with other partaking proteins are highly organised within the cell- cell contact in respect to their size and function. However, information about their lateral interactions, height- dependent orientation and the consequences of this for binding are largely missing, but important in order to understand how an immune response is initiated on a molecular scale. The aim of this thesis was to shed light on this. For investigating protein size and lateral interactions an important negative regulator of TCR signalling, the phosphatase CD45, was studied. It was postulated by the kinetic segregation model that the tall glycoprotein CD45 is excluded from the contact area. The segregation from the TCR in the contact results in a net positive signalling event and thus in T-cell activation. The size of CD45 is a key factor but complete structural information at the cell surface is lacking. By using a technique called hydrodynamic trapping I determined the dimensions of CD45 and quantified its lateral interactions with other CD45 molecules in a cell membrane model. This was done by trapping CD45 anchored to a supported lipid bilayer (SLB) causing high local protein densities below a micropipette through which a negative pressure was applied. Using this method, it was found that CD45 has a lower apparent height than estimated previously which is accounted for by a high flexibility of the protein on the model membrane.When investigating protein binding in this thesis the focus was on the binding strength (affinity) of TCR-MHC interactions. Each TCR has a specific affinity for their cognate peptide MHC, which varies from very weak to strong depending on the presented peptide. Agonist (pathogenic) peptides typically generate strong binding and an activation of the immune response. Measuring affinities in a cellular system is however complicated due to various protein interactions taking place at the same time. By using a model system in which cells bind to an SLB, the two- dimensional affinity of a TCR binding to an agonist MHC on the opposing cell was measured herein. It was found that high ligand densities affected the cells’ cytoskeletal reorganisation, observed as strong lamellipodia formation, which caused a significant increase in the amount of bound ligands. Additionally, it was observed that the binding strength of the TCR-MHC interaction was influenced by the presence of the auxiliary protein CD2 and decreased with increasing amounts of CD2 in the contact. Using the methods described in this thesis allows to measure and interpret weak interactions such as to ‘self’ (body’s own) peptide MHCs. In addition, my hydrodynamic trapping studies of proteins on functionalised SLBs could give new information about membrane protein behaviour, for example how protein flexibility can decrease the effective protein height and thus influence molecular exclusion at cell-cell contacts, and how glycosylation can increase the repulsion, which might prevent protein aggregation.