Cross-Recognition of Two Middle T Protein Epitopes by Immunodominant Polyoma Virus-Specific CTL

Abstract

AbstractWe recently identified the immunodominant epitope for polyoma virus-specific CTL as the Dk-associated peptide MT389–397 derived from the middle T (MT) viral oncoprotein. Another Dk-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 Dk-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 Dk 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 Dk anchor position induced polyoma virus-specific CTL recognizing neither MT389–397 nor MT236–244 epitopes. Despite predominant usage of the Vβ6 gene segment, MT389–397/MT236–244 cross-reactive CTL clones possess diverse complementarity-determining region 3β domains; this is functionally reflected in their heterogeneous recognition patterns of alanine-monosubstituted MT389–397 peptides. Using Dk/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.