Cross-recognition of two middle T protein epitopes by immunodominant polyoma virus-specific CTL

We recently identified the immunodominant epitope for polyoma virus-specific CTL as the D-k-associated peptide MT389-397 derived from the middle T (MT) viral oncoprotein, Another D-k-restricted peptide corresponding to residues 236-244 of MT was recognized by nearly all MT389-397-reactive CTL clones, but required concentrations at least 2 logs higher to sensitize syngeneic target cells for lysis, Except for identity at the three putative D-k-peptide anchor residues, MT236-244 shares no homology with MT389-397.Using a novel europium-based class I MHC-peptide binding immunoassay, we determined that MT236-244 bound D-k 2-3 logs less well than MT389-397. Infection with a mutant polyoma virus whose MT is truncated just before the MT389-397 epitope or immunization with MT389-397 or MT236-244 peptides elicited CTL that recognized both MT389-397 and MT236-244. Importantly, infection with a polyoma virus lacking MT389-397 and mutated in an MT236-244 D-k anchor position induced polyoma virus specific CTL recognizing neither MT389-397 nor MT236-244 epitopes. Despite predominant usage of the V beta 6 gene segment, MT389-397/MT236-244 cross-reactive CTL clones possess diverse complementarity-determining region 3 beta domains; this is functionally reflected in their heterogeneous recognition patterns of alanine-monosubstituted MT389-397 peptides. Using D-k/MT389-397 tetramers, we directly visualized MT236-244 peptide-induced TCR down-modulation of virtually all MT389-397-specific CD8(+) T cells freshly explanted from polyoma-infected mice, suggesting that a single TCR recognizes both Dk-restricted epitopes, The availability of immunodominant epitope-specific CTL capable of recognizing a second epitope in MT, a viral protein essential for tumorigenesis, may serve to amplify the CTL response to the immunodominant epitope and prevent the emergence of immunodominant epitope-loss viruses and virus-induced tumors.