Comprehensive Assessment of Determinant Specificity, Frequency, and Cytokine Signature of the Primed CD8 Cell Repertoire Induced by a Minor Transplantation Antigen

Abstract

T cell immunity is often focused on one peptide segment of a complex protein Ag, with other epitopes inducing weaker, low frequency responses or no responses 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 recognition in a skin transplant model with β-galactosidase (β-gal) as a minor transplantation Ag. CD8 T cells from C57BL/6 mice that rejected congenic C57BL/6 β-gal transgenic skin were tested in enzyme-linked immunospot assays for recall responses to single-step, overlapping, 9-mer peptides that spanned a 94-aa region of the β-gal sequence. This approach provided every possible class I-restricted peptide for CD8 cell recognition, allowing 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 Kb or Db molecules, only one (β-gal96–103) actually induced an immune response. No peptides outside of the motifs were recognized. Tolerization to β-gal96–103 significantly prolonged β-gal transgenic skin graft survival, confirming its immune dominance. Therefore, single-determinant dominance characterized this CD8 cell response. The data demonstrate the feasibility of large-scale, comprehensive, class I determinant mapping, an approach that should be indispensable in measuring CD8 cell immunity in humans.