The influence of adhesion molecules on binding and protein organization in cell contacts

Sammanfattning: Interactions between immune cells such as T cells and antigen-presenting cells (APCs) are integral for mounting an adaptive immune response. The interaction between the T cell receptor (TCR) and the antigen-presenting major histocompatibility complex (pMHC) on a contacting T cell and APC, is widely accepted to be the key interaction. If the interaction is favourable, then T cell activation occurs. A large pool of research has been aimed at characterizing this interaction by measuring the binding kinetics and relating it to the T cell response. A simplified model membrane system called a supported lipid bilayer (SLB) is often used to mimic the membrane of the APC. In many T cell activation studies, the SLB contains the nickel-chelating lipid DGS-NTA(Ni) to functionalize the SLB with histidine-tagged proteins. In the first part of this thesis I show that interactions between DGS-NTA(Ni) and the T cells can lead to, unwanted, T cell signaling. It was found that increasing the concentration of DGS-NTA(Ni) both increased cell adhesion and the fraction of signaling cells. Adding bovine serum albumin (BSA) functioned as a blocking agent, preventing unspecific cell adhesion and decreased the fraction of signaling cells down to a basal level. A low level of signaling was also obtained when functionalizing the blocked SLBs with adhesion molecules binding to receptors on the T cell. In contrast, without blocking these functionalized SLBs again signaled at a similar level to the unblocked, not functionalized DGS-NTA(Ni) SLBs. The DGS-NTA(Ni) signaling was argued to be due to TCR-DGS-NTA(Ni) interactions and stressed the importance of adequately blocking these interactions in T cell activation studies.In the second part of the thesis, a new method to measure the two-dimensional dissociation constant (2D Kd) of ligand-receptor interactions on single cells is presented. This is measured on individual cell-SLB contacts, providing an accurate new means of measuring binding affinity and to study differences in the 2D Kd in the cell population. In the final part of the thesis, the interaction of TCR-pMHC in the presence of adhesion molecules of different length and density is studied. Adhesion molecule pairs of similar height as TCR-pMHC have been argued to facilitate the TCR-pMHC interaction by physically keeping the opposing membranes at an optimal distance for binding. However, adhesion pairs of different height than that of TCR-pMHC are also important for cell-cell contact formation and have been shown to result in an impaired T cell response if removed. To better understand how, and if, adhesion molecules of different lengths influences TCR-pMHC binding the 2D Kd of TCR-pMHC in the presence of differently-sized adhesion molecules was studied. For this purpose, a SLB functionalized with TCR and an adhesion ligand, was allowed to bind cell with pMHC and the corresponding adhesion receptor. It was found that the 2D Kd of the TCR-pMHC interaction could be up to an order of magnitude higher (weaker) than the corresponding value for TCR-pMHC alone when having height-mismatched molecules. In addition, the TCR-pMHC distributed non-homogeneously in the cell-SLB contacts when having height-mismatched adhesion molecules, but homogeneously when having height-matched adhesion molecules. Furthermore, even for height-matched adhesion molecules the 2D Kd of the TCR-pMHC interaction was found to be dependent on the relative density fraction of TCR to adhesion molecules, with low fractions of TCR molecules giving 2-3 times weaker binding. This indicates that TCR-pMHC binding in cell contacts depends significantly on the local environment and not only on the protein-protein interaction per se.