Identification of Naturally Processed Epitope Region Using Artificial APC Expressing a Single HLA Class I Allotype and mRNA of HCMV pp65 Antigen Fragments

Recently, long synthetic peptides or in silico-predicted epitope peptides have been used to identify T cell epitopes, but these approaches may not be suitable for investigating naturally processed epitopes. Here, mRNAs, including fragments or predicted epitope sequences of HCMV pp65 antigen, were generated by in vitro transcription following transcriptionally active PCR. Then, artificial antigen-presenting cells (aAPCs) expressing a single HLA allotype were transfected with mRNAs to identify epitopes in donors with T cell responses that recognize pp65 antigen restricted to HLA-A*02:01, -A*02:06, or -B*07:02. T cells restricted to a particular HLA allotype showed positive responses in some of the 10 fragment antigens. Among predicted epitopes within these positive fragments, three epitopes of HLA-A*02:01, -A*02:06, and -B*07:02 were confirmed. In addition, T cells expanded by anti-CD3 stimulation for two weeks could also be effectively used for the identification of these T cell epitopes, although there were individual differences. These results demonstrated that fragment antigens and epitopes can be rapidly generated using mRNA, and naturally processed antigenic regions can be detected using aAPCs without a T cell cloning procedure. This method will help to identify novel T cell epitopes for developing immunotherapy and vaccines against infectious diseases and cancer.