CTG-initiated cryptic peptide translation up and downstream of a canonical ATG start codon is enhanced by TLR stimulation and induces tumor regression in mice

Abstract Cytotoxic T-lymphocytes (CTLs) screen cells for signs of infection and transformation by recognizing peptides displayed on major histocompatibility complex (MHC) class I molecules. Next to canonical ATG-initiated open reading frames (ORFs), non-canonical translation can result in synthesis of non-conventional or `cryptic´ polypeptides. These can originate from translation initiation at non-canonical start codons, a process previously associated with inflammation and oncogenic transformation. Cryptic translation products are efficiently presented on MHC class I molecules and therefore increasingly recognized as potential targets for cancer immunotherapy. Here, we studied how localization of a CTG-initiated ORF relative to a canonical ATG start codon can influence cryptic expression after innate immune stimulation. We generated immortalized C57BL/6J mouse-derived bone marrow progenitor cells (HoxB8) expressing tandem minigene constructs, which encoded a CTG-driven chicken ovalbumin-derived SIINFEKL (S8L) epitope (CTG-S8L; H-2Kb-restriced) either up or downstream of a canonical ATG-initiated UTY-derived peptide WI9 (ATG-WI9; H-2Db-restriced). Treatment of HoxB8-derived macrophages with Toll-like receptor agonists enhanced position-independent CTG-S8L translation, without affecting ATG-driven expression. Downstream CTG-S8L translation was driven by leaky scanning or ribosome re-initiation rather than read-through translation. Mouse AE17 mesothelioma and B16F10 melanoma cells expressing cryptic S8L either up or downstream of a canonical ORF were efficiently killed by H-2Kb/S8L-restriced OT-I T-cells in vitro, even though their antigen expression levels were extremely low. Mice implanted with tumors expressing cryptic S8L showed delayed tumor progression in vivo. In summary, our study contributes to the characterization of non-canonical start codon-driven cryptic antigen translation and highlights its potential for cancer immunotherapy.