Analysis of the role of CD8{490} in co-receptor function.

The T cell receptor is found on the surface of the T cell in association with a complex of invariant proteins that are essential for functional signal transduction. One such molecule, CD8 , is a co-receptor that binds to invariant parts of the MHC Class I molecule. CD8 comprises a disulphide linked a and p chain. Although the a chain has been shown to interact with the MHC as a CD8 a a homodimer, the p chain is implicated in this interaction. The majority of CD8 molecules exist as ap heterodimers rather than a a homodimers. Previous studies have identified biochemical alterations in CD8 p upon T cell activation, predicted to occur in the hinge region between the Ig like domain and the transmembrane domain. In order to study the nature of this region specifically, a transgenic mouse was created expressing an altered CD8 p chain. The mutated CD8 p chain comprises the Ig like, transmembrane and cytoplasmic domain of CD8 p, but the hinge region of CD8 a. By comparing functional and biochemical data from these mice with wild type data, the project aims to define how TcR-CD8 / MHC interactions regulate T cell differentiation and activation. In this thesis it is shown that the mutated CD8 p chain cannot restore selection in the thymus when expressed as a transgene on a CD8 p knockout background, similarly to mice lacking CD8 p altogether. Polyclonal mice were backcrossed onto the Rag'' F5 TcR monoclonal background, where many characteristic maturation markers are comparable with wild type in the mutated mice. However certain phenotypic markers suggest there is a defect in the cells at the double positive stage, where they fail to advance through positive selection. There is a corresponding drop in the percentage and overall numbers of CD8 ̂T cells in the periphery in mice expressing the mutated transgene, similar to that of mice lacking CD8 p. Antibodies specific for the Ig-like domain of CD8 were able to bind the chimeric molecule, the stoichiometry of the molecule was comparable to wild type, and that it was able to associate with the signaling molecule LCK. Like wild type, the mutated molecule became a faster migrating species on activation, probably due to de-sialylation, and data collected using lectins showed that in post translational modification, glycans are attached to the mutated molecule via the same specific linkages as wild type. Nevertheless there were striking differences between cells expressing the mutated molecule and those expressing wild type CD8 ap. Double positive thymocytes were significantly less able to bind cognate MHC, while the calcium flux in response to TcR/ co-receptor crosslinking was also severely reduced in lymphocytes expressing the mutated molecule compared to wild type. When challenged with antigen peptide the response of lymphocytes expressing the mutated molecule is slightly different to that of wild type cells. This difference in response is also demonstrated when blocking antibodies are included in the assay. Cells expressing the mutated molecule go through more cell cycles and have a higher percentage of CD25^ cells than wild type controls, and blocking antibodies have little effect on this response. These data suggest that the glycosylation of CD8 p has a very specific influence on development and selection in the thymus, and that CD8|3 could be acting in a regulatory fashion in the periphery.