The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can in some cases be directed against peptides presented by non-classical MHC-Ib, for example the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of non-classical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of “cryptic” viral epitopes, defined as those undetectable by conventional mapping techniques. Here, we used an immunopeptidomics approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza A virus (IAV). We identify a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two ways: first, it is presented by Qa-1, and second, it maps to an alternative reading frame of the influenza matrix protein 1 (M1) mRNA and appears to derive mainly from an unannotated 16-residue peptide that is dispensable for viral replication. Presentation and immunogenicity of M-SL9 were dependent on the second AUG codon of the positive sense matrix RNA segment, supporting translation initiation by leaky ribosomal scanning. This work suggests that non-canonical translation products must be examined in order to fully reveal the T cell repertoire and adds to a growing body of evidence that MHC-E-restricted T cells may have significant therapeutic value.
The extent to which non-canonical translation products drive antiviral T cell responses is incompletely understood. We explored this question using an immunopeptidomic approach in an influenza virus model. C57Bl/6 mouse-derived cells were infected with influenza virus A/Puerto Rico/8/1934 (PR8), and MHC-bound peptides were eluted and analyzed by tandem mass spectrometry. Mass spectra were searched against a database of all viral gene segments translated in all possible reading frames, irrespective of the presence of start codons. We identified a 9-mer peptide, here termed M-SL9, that maps to an alternative reading frame of the sequence encoding matrix protein 1 (M1). To validate M-SL9 as a T cell epitope, we infected C57Bl/6 mice with PR8, collected tissues, and measured T cell reactivation by synthetic M-SL9 peptide. Remarkably, 10% of all CD8 T cells in the lung were specifically reactivated by M-SL9 peptide, and the vast majority of these were polyfunctional, producing two or more Th1 cytokines. An analysis of M-SL9 processing and presentation revealed that M-SL9 is restricted to the non-classical MHC class Ib molecule, Qa-1, and that presentation is dependent on the second 5′-proximal AUG codon within the M1-coding sequence, suggesting that its translation results from leaky ribosomal scanning. These data contribute to a growing body of work showing the importance of non-canonical MHC presentation in protective immune responses.
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