Acidic residues in the DR beta chain third hypervariable region are required for stimulation of a DR(alpha, beta...

We investigated the minimal requirements for stimulation of an antigen-specific HLA-DR(alpha, beta 1*0402)-restricted T-cell clone (Een217) by using transfectants expressing mutant DR beta chains as APCs. Antigen-specific stimulation of Een217 was induced with transfectants expressing DR(alpha, beta 1*0402) and DR(alpha, beta 1*0403) but not other DR4 subtypes that also bind the peptide recognized by this clone. Analysis of the effects of single amino acid substitutions in the beta chains of each of the DR4 subtypes revealed a requirement for acidic residues in the third HVR, particularly amino acid 71, in stimulation of clone Een217. Functional class II mutants were generated from nonstimulatory DR4 subtype beta chains by acidic residue substitutions within the third HVR. These data define the requirement for negatively charged residues in this region for peptide-induced stimulation of this T-cell clone. The required acidic residues can be located at either position 70, 71, or 74 in the DR beta chain. The negative charge in this segment of the DR beta chain alpha-helix may be required for direct interactions with the T-cell receptor of Een217 or may affect peptide conformation.