Grueneberg, Ulrike; (1998) The interaction of HLA-DM with conventional MHC class II molecules. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The subject of this thesis is the interaction of the MHC class II encoded protein HLA-DM with conventional MHC class II proteins such as HLA-DR and HLA-DQ. MHC class II molecules in DM-negative cells cannot present antigenic peptides but remain loaded with the class II-associated invariant chain derived peptide (CLIP). In order to study the involvement of HLA-DM in the generation of this phenotype an insect cell baculovirus expression system was set up with the goal of producing soluble DM protein. The recombinant HLA-DM was purified and used in peptide exchange assays using CLIP-loaded DR3 molecules from the DM-negative cell line T2.DR3. Recombinant HLA-DM facilitated the exchange of CLIP for antigenic peptides in a catalytic, pH-dependent manner. In addition to CLIP, all those peptides that did not have an allele-specific binding motif were released by DM. Thus, HLA-DM acts a peptide editor, facilitating selection of peptides that stably bind to class II molecules for eventual presentation to the immune system from the pool of available peptides. Certain alleles of another MHC class II isotype, HLA-DQ, are highly associated with autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). I set out to explore whether an abnormal interaction of certain HLA-DQ alleles with HLA-DM might lead to an altered peptide repertoire. To this end an antiserum recognizing the conserved C-terminus of the DQα chain was generated and the association of different HLA-DQ alleles with DM was assessed using co-immunoprecipitations. No gross differences between alleles were observed. In order to establish a more sensitive system to study the interaction of HLA-DM with DQ, I tried to transfect MHC class II-negative T2 cells with different DQ alleles and generated soluble DQ molecules using the system that was developed for the production of HLA-DM. Soluble DQβ appeared to be unstable indicating putative structural deficiencies of certain DQ alleles.

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