Advances in next-generation sequencing (NGS) have improved the resolution of T-cell receptor (TCR) repertoire analysis, and recent single-cell sequencing has made it possible to obtain information about TCR pairs. In our previous study, cytomegalovirus (CMV) pp65-specific T-cell response restricted by a single human leukocyte antigen (HLA) class I allotype was observed in an individual. Therefore, to effectively clone an antigen-specific TCR from these T cells, we developed a TCR cloning system that does not require a single cell level. First, we established the improved Jurkat reporter cell line, which was TCRαβ double knock-out and expressed CD8αβ molecules. Furthermore, functional TCRs were directly obtained by reverse TCR cloning using unique CDR3-specific PCR primers after bulk TCR sequencing of activation marker-positive CD8 T cells by NGS. A total of 15 TCRα and 14 TCRβ strands were successfully amplified by PCR from cDNA of 4-1BB-positive CD8 T cells restricted by HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. The panels with combinations of TCRα and TCRβ genes were investigated using Jurkat reporter cell line and artificial antigen-presenting cells (APCs). In two TCR pairs restricted by HLA-A*02:01, one TCR pair by HLA-A*02:06, four TCR pairs by HLA-B*07:02, and one TCR pair by HLA-B*40:06, their specificity and affinity were confirmed. The TCR pair of A*02:01/1-1 showed alloreactivity to HLA-A*02:06. The one TCR pair showed a higher response to the naturally processed antigen than that of the peptide pool. This reverse TCR cloning system will not only provide functional information to TCR repertoire analysis by NGS but also help in the development of TCR-T therapy.
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.
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