Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.