Despite all the structural and functional data that have been accumulated regarding major histocompatibility complex (MHC) class II molecules during recent years, the relative contribution of putative T cell receptor (TcR)-contacting residues and peptide-binding MHC polymorphisms to MHC-restricted and allospecific T cell responses remains a point of contention. Some authors emphasize the importance of direct interaction between the allospecific TcR and polymorphic MHC residues whereas other emphasize the role of naturally processed MHC-bound peptides. We have previously described a new HLA-DRB1 allele: DR BON (DRB1*0103). This gene differs from DRB1*0101 by six base pairs clustered in the third variable region of the second exon leading to three amino acid changes at positions 67, 70 and 71 of the beta chain of the HLA-DR molecule. To define the respective role of these residues in allorecognition, we have performed site-directed mutagenesis on the DRB1*0103 allele to create six mutants which are intermediary between the DR BON and the DR1 alleles. These mutant cDNA were expressed in mouse fibroblasts and the transfectants with the highest expression of class II molecules were used as stimulators for a panel of ten anti-DR BON and five anti-DR1 alloreactive T cell clones. We demonstrate that the residue at the peptide-binding position 71 is of paramount importance in the alloresponse of these clones. In addition some clones were sensitive to amino acid substitution at the TcR-contacting position 70, while substitution at position 67 affects very few clones. The dominance of residue 71 was also observed with an influenza hemagglutinin-specific HLA-DR BON-restricted T cell line.
The binding ability of 23 overlapping peptides, all derived from the CB11 fragment of CII, was tested on several HLA-DR molecules associated or not with disease susceptibility. These experiments were performed on a variety of cells expressing different HLA-DR molecules, using both indirect and direct binding assays. The CII (256-271) fragment was shown to bind to a restricted population among which the HLA-DR molecules associated with susceptibility to rheumatoid arthritis. The results also clearly indicate that the binding specificity of CII (256-271), among the DR4 molecules, is controlled by the nature of the HLA-DR molecule beta-chain residues 71 and 74, residues previously shown by X-ray crystallography to be involved in the HLA-DR/peptide interaction. The human CII (256-271) peptide is thus likely to play a role in the disease process.
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