Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.

Identification of the amino acid residues contributing to monoclonal antibody-defined DQw1 epitopes.

The reactivity of monoclonal antibodies (mAbs) R1, S1, and S5, shown previously to recognize polymorphic epitopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQB1 alleles. mAb S5 reacts with cells expressing DQB1*0503, 0601, 0602, 0603, or 0604 alleles while R1 and S1 react with all DQB1 alleles except *0201 and 0301. In the case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitopes. This prediction has been confirmed by showing that a G----E mutation in position 45 of the DQB1*0302 gene eliminates binding of both mAbs.

Epitope analysis with monoclonal antibodies directed against class II molecules subdivides HLA-DR2, DR3 and DR4: correlations with cellularly defined Dw specificities.

Monoclonal antibodies (Mabs) defining 14 distinct polymorphic epitopes have been produced against the class II antigens of HLA-DR3Dw3DQw2 cells. Population analysis indicates that Mab C1 is directed against the DQw2 specificity and Mab M6 against the DRw52 specificity. The remaining Mabs define epitopes shared by the class II molecules of DR3 and various other specificities. Seven DR3Dw3DQw2 haplotypes were examined and could be divided into two types based on the presence of the epitope defined by Mab M3. Analysis of DR2 and DR4 homozygous cells with these Mabs revealed several distinct patterns of epitope expression. These subdivisions were found to correlate with the cellularly defined Dw specificities.