CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249–59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes

Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249–59 and Tp1214–224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide–MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298–106), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249–59-reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250–59 represents a distinct epitope and that tetramers produced with Tp50–59 and Tp49–59 show no cross-reactivity. The Tp249–59 and Tp250–59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide–MHC I complexes adopt structurally different conformations and Tcell receptor β sequence analysis showed that Tp249–59 and Tp250–59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249–59 and Tp250–59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214–224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249–59 epitope response was generally more dominant than the Tp250–59 response and confirmed that the Tp298–106 response was subdominant.