Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CD8 cell repertoire induced by a minor transplantation antigen

T cell immunity is often focused on one peptide segment of a complex protei n Ag, with other epitopes inducing weaker, low frequency responses or no re sponses at all. Such determinant hierarchy has been well characterized for MHC class II-restricted CD4 cell immunity, but is less well understood for class I-restricted CD8 cell responses. We studied class I determinant recog nition in a skin transplant model with beta-galactosidase (beta-gal) as a m inor transplantation Ag, CD8 T cells from C57BL/6 mice that rejected congen ic C57BL/6 beta-gal transgenic skin were tested in enzyme-linked immunospot assays for recall responses to single-step, overlapping, 9-mer peptides th at spanned a 94-aa region of the beta-gal sequence. This approach provided every possible class I-restricted peptide for CD8 cell recognition, allowin g us to define the in vivo frequency of CD8 cells specific for each of the 86 individual peptides. While four peptides were predicted to bind to the K -b or D-b molecules, only one (beta-gal(96-103)) actually induced an immune response. No peptides outside of the motifs were recognized. Tolerization to beta-gal(96-103) significantly prolonged beta-gal transgenic skin graft survival, confirming its immune dominance. Therefore, single-determinant do minance characterized this CD8 cell response. The data demonstrate the feas ibility of large-scale, comprehensive, class I determinant mapping, an appr oach that should be indispensable in measuring CD8 cell immunity in humans.