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 Dκ-associated peptide MT_389-397 derived from the middle T (MT) viral oncoprotein. Another Dκ-restricted peptide corresponding to residues 236-244 of MT was recognized by nearly all MT_389-397- 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 Dκ-peptide anchor residues, MT_236-244 shares no homology with MT_389-397. Using a novel curopium-based class I MHC-peptide binding immunoassay, we determined that MT_236-244 bound Dκ 2-3 logs less well than MT_389-397. Infection with a mutant polyoma virus whose MT is truncated just before the MT_389-397 epitope or immunization with MT_389-397 or MT_236-244 peptides elicited CTL that recognized both MT_389-397 and MT_236-244. Importantly, infection with a polyoma virus lacking MT_389-397 and mutated in an MT_236-244 Dκ anchor position induced polyoma virus-specific CTL recognizing neither MT_389-397 nor MT_236-244 epitopes. Despite predominant usage of the Vβ6 gene segment, MT_389-397/MT_236-244 cross-reactive CTL clones possess diverse complementarity-determining region 3β domains; this is functionally reflected in their heterogeneous recognition patterns of alanine-monosubstituted MT_389-397 peptides. Using Dκ/MT_{389- 397} tetramers, we directly visualized MT_236-244 peptide-induced TCR down-modulation of virtually all MT_389-397-specific CD8⁺ T cells freshly explanted from polyoma-infected mice, suggesting that a single TcR recognizes both Dκ-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.