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Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data

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...

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Autores principales: Hong, Cheol-Hwa, Pyo, Hong-Seon, Baek, In-Cheol, Kim, Tai-Gyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9716090/
https://www.ncbi.nlm.nih.gov/pubmed/36466875
http://dx.doi.org/10.3389/fimmu.2022.1021067
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author Hong, Cheol-Hwa
Pyo, Hong-Seon
Baek, In-Cheol
Kim, Tai-Gyu
author_facet Hong, Cheol-Hwa
Pyo, Hong-Seon
Baek, In-Cheol
Kim, Tai-Gyu
author_sort Hong, Cheol-Hwa
collection PubMed
description 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.
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spelling pubmed-97160902022-12-03 Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data Hong, Cheol-Hwa Pyo, Hong-Seon Baek, In-Cheol Kim, Tai-Gyu Front Immunol Immunology 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. Frontiers Media S.A. 2022-11-18 /pmc/articles/PMC9716090/ /pubmed/36466875 http://dx.doi.org/10.3389/fimmu.2022.1021067 Text en Copyright © 2022 Hong, Pyo, Baek and Kim https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Hong, Cheol-Hwa
Pyo, Hong-Seon
Baek, In-Cheol
Kim, Tai-Gyu
Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title_full Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title_fullStr Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title_full_unstemmed Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title_short Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
title_sort rapid identification of cmv-specific tcrs via reverse tcr cloning system based on bulk tcr repertoire data
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9716090/
https://www.ncbi.nlm.nih.gov/pubmed/36466875
http://dx.doi.org/10.3389/fimmu.2022.1021067
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