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Knowledge of viral targets is crucial to improve monitoring of high\u2010risk patients and optimise antiviral T\u2010cell therapy. To expand the epitope spectrum, genetically\u2010engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble <jats:italic>(s)HLA\u2010A*11:01<\/jats:italic> and infected with an HCMV mutant lacking immune evasion molecules (US2\u20106\u2009+\u200911). More than 700 <jats:italic>HLA\u2010A*11:01<\/jats:italic>\u2010restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open\u2010reading\u2010frames (ORFs) were identified by mass spectrometry and screened for <jats:italic>HLA\u2010A*11:01<\/jats:italic>\u2010binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy <jats:italic>HLA\u2010A*11:01<\/jats:italic><jats:sup><jats:italic>+<\/jats:italic><\/jats:sup>\/HCMV<jats:sup>+<\/jats:sup> donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti\u2010apoptotic protein UL36 and ORFL101C (A11<jats:sub>SAL<\/jats:sub>), were identified. Their <jats:italic>HLA\u2010A*11:01<\/jats:italic> complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T\u2010cell responses against A11<jats:sub>SAL<\/jats:sub>, with T\u2010cell responses comparable to the immunodominant <jats:italic>HLA\u2010A*02:01<\/jats:italic>\u2010restricted HCMVpp65<jats:sub>NLV<\/jats:sub> epitope. A11<jats:sub>SAL<\/jats:sub>\u2010specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV\u2010infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically\u2010engineered DCs to identify naturally presented immunodominant HCMV\u2010derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.<\/jats:p>","DOI":"10.1111\/tan.15541","type":"journal-article","created":{"date-parts":[[2024,6,25]],"date-time":"2024-06-25T05:12:45Z","timestamp":1719292365000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Engineered <scp>HCMV<\/scp>\u2010infected <scp>APCs<\/scp> enable the identification of new immunodominant <scp>HLA<\/scp>\u2010restricted epitopes of anti\u2010<scp>HCMV<\/scp> T\u2010cell immunity"],"prefix":"10.1111","volume":"103","author":[{"given":"Maria Michela","family":"Santamorena","sequence":"first","affiliation":[{"name":"Institute of Transfusion Medicine and Transplant Engineering Hannover Medical School (MHH)  Hannover 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