Antigen presentation to specific T cells by Ia molecules selectively altered by site-directed mutagenesis

A functional analysis of mutant class II molecules was conducted to identify regions important for antigen-specific T cell activation. Site-directed mutagenesis was used to construct a panel of mutant A beta k genes containing either single or multiple d allele substitutions in the beta 1 domain. The product of each of these genes was expressed with either the A alpha d or A alpha k polypeptide in the Ia-negative B cell lymphoma M12.C3. These mutant class II molecule-bearing cells were tested for their ability to present antigen to a panel of Ak-restricted T cell clones specific for various epitopes of myoglobin. Results from this analysis demonstrate that T helper clones recognized complex determinants interacting with multiple residues on the beta 1 domain and also requiring the haplotype-matched alpha 1 domain. This is in contrast to monoclonal antibodies that recognize a domain-specific, immunodominant region involving residues 40, 63, and 65-67. Every T helper clone was found to interact with a distinct pattern of residues, even among clones recognizing the same combination of peptide and major histocompatibility complex (MHC) molecule. The 3 for 1 residue substitution between k and d alleles at residues 65-67 was one of the most important, because it resulted in loss of ability to present antigen to 7 of 7 I-Ak-restricted T cell clones. These residues have been shown previously to comprise the immunodominant allo-specific serological determinants and to stimulate some alloreactive T cell clones. Substitution at residues 12 and 13 also abrogated antigen presentation to all the T cell clones, but this may be a consequence of a conformational change due to altered alpha beta chain pairing. Substitution at position 9, which is predicted to be located in the floor of the peptide-binding groove where it should not interact directly with the T cell receptor, enhanced presentation of the antigenic site 102-118 to some T cells and diminished it to others. This finding suggests a most interesting conclusion that the same antigenic site may bind in different conformations or orientations to the same MHC molecule, although an indirect effect on the conformation of the MHC molecule itself cannot be excluded. Substitutions at residues 85, 86 and 88 also abrogated the response of one T cell clone but not others specific for the same peptide with the same Ia molecule.(ABSTRACT TRUNCATED AT 400 WORDS)