The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, 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 nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical 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 immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.

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.