Investigation of Antigen Processing Using Partially Assembled

The processing of antigens for presentation to CD8+ T-cells in association with MHC class I molecules involves many chaperones and accessory proteins in the ER. Once processed into a trimeric complex of beta-2-microglobulin (p2m), heavy chain and antigenic peptide the MHC class I molecule is complete and can traffic to the cell surface where it interacts with the T cell receptor of CD8+ T-cells. The main focus of this work is the use of MHC class I fusion proteins, which by means of a covalent linker form partially assembled class I molecules. Using a fusion protein in which an antigenic HLA-A2 binding peptide is linked to p2m (PB), it has been possible to show by immunoprecipitation that class I molecules can continue to interact with the peptide-loading complex after high affinity peptide has bound. This data is further supported by peptide release assays in which class I molecules are not released from the peptide-loading complex upon provision of high affinity peptide. The expression of fusion proteins in which p2in is linked to the heavy chain alleles HLA-B44 (P2m-B44) or HLA-A2 (P2in-A2), has highlighted an allelic difference in the requirement of MHC class I molecules for chaperones in the antigen processing pathway. Specifically it has been possible to show that while P2m-A2 is able to express in murine fibroblast K41 cells, p2m-B44 is not. This inability to express is not as a result of the absence o f human tapasin, as previously reported, but because of some other unknown factor that is present in human cells but not in murine K41 cells. Interestingly the expression o f p2m-B44 in K41 cells also disrupts native MHC class I expression implying that the construct may be occupying chaperone molecules in the ER and delaying processing of mouse class I molecules. Furthermore, in the same system it has been possible to show that the major function of calreticulin is not the recruitment of P2in to heavy chain, since the p2m-A2 fusion protein, like normal class I expressed 8-10 fold less well in calreticulin deficient cells than in ‘wild type’ cells.