Use of mutants to analyze regions on the H-2Kb molecule for interaction with immune receptors

MHC variants isolated both in vivo (by tissue graft rejection) and in vitro (by antibody selection) were utilized to study sites on the H-2Kb molecule involved in interaction with antibodies and with the TCR. Kb mutants selected by antibodies were found to have single point mutations, which when analyzed in the context of the three-dimensional structure of a Kb molecule modeled from HLA-A2 coordinates showed that the altered residues were localized mostly to the alpha 1 and alpha 2 helices. The side chains of the variant amino acid residues pointed upward and away from the antigenic site. Analysis of the altered amino acids in the previously described tissue-graft-selected Kb mutants showed that the side chains of the variant residues occur either in the alpha-helical regions or in the beta-pleated-sheet floor of the antigen groove, but every mutant contained at least one and sometimes several acid side chains projecting into the antigen-binding groove. Monoclonal antibody studies showed that the available monoclonal antibodies mapped to discrete domain-specific sites. Analysis of CTL recognition sites using cloned mutant anti-parent or allogeneic combinations showed that all CTL clones examined interacted with amino acid side-chain residues on both the alpha 1 and alpha 2 helices. Thus, we concluded that the CTLs must simultaneously interact with the amino acid residues in both the alpha-helical stretches of the alpha 1 and alpha 2 domains. Our analyses with the point mutants imply that the TCR must interact with the MHC molecule over a relatively large surface area in such an orientation that it interfaces with the two alpha helices, as well as with the foreign or self-peptide in the antigen-binding site between the helices. These findings, together with the observation that several of the in vivo Kb mutants induce strong alloreactions yet have changes only in the bottom of the antigen-binding groove and no alterations in the alpha-helical residues, are consistent with the hypothesis that in some cases alloreaction can be the result of T-cell recognition of an altered pattern on the MHC molecule due to a changed peptide in the antigen groove.