Concurrent TCRA and TCRB sequencing of TCRs expressed in a single sorted cell was performed by the hTEC10 protocol. Using these samples, massive parallel sequencing of TCRA and TCRB V-D-J segments was performed after unbiased amplification of the target sequences by adaptor-ligation PCR. In some experiments, sorted NLV/A2-positive cells underwent one or two rounds of expansion with autologous PB mononuclear cells depleted of CD8+ and CD4+ T cells in the presence of NLV peptide. CMV-specific CD8+ T-cells were isolated and flow-sorted from peripheral blood (PB) mononuclear cells using an HLA-tetramer, NLV/A2, specific for HLA-A2-restricted CMV pp65-derived epitope (NLV peptide: NLVPMVATV). All donors were screened for CMV serostatus and typed for HLA-A. Methods: 20 ml peripheral blood samples were collected from healthy adult volunteers who gave written informed consent for the study. In this study, we attempted to elucidate the comprehensive TCR repertoire of cytomegalovirus (CMV)-reactive cytotoxic T-cells (CTLs) by combining quantitative deep sequencing and hTEC10. "Human TCR efficient cloning within 10 days" (hTEC10) is a powerful novel technology that enables concurrent sequencing of paired TCRA and TCRB gene segments at a single cell level (Nat Med. However, this method alone does not yield data for correct pairs of TCRA and TCRB sequences required for the structural determination of TCRαβ heterodimers expressed in a single T cell. Deep sequencing of rearranged complementarity-determining region 3 (CDR3) regions of TCRA and TCRB gene segments is an emerging technology that facilitates high-throughput and semi-quantitative analysis of TCR repertoire with high resolution and accuracy. Background: Identification of functional T-cell receptors (TCRs) to their cognate antigens is the key to the development of effective anti-viral or anti-tumor T-cell therapy.
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