Objective To establish a new method and platform for screening,identifying,and exploring a new strategy for anti-hepatitis B immunotherapy based on hepatitis B virus(HBV)-specific TCR.Methods Peripheral blood mononucl...Objective To establish a new method and platform for screening,identifying,and exploring a new strategy for anti-hepatitis B immunotherapy based on hepatitis B virus(HBV)-specific TCR.Methods Peripheral blood mononuclear cells were isolated from patients with acute hepatitis B.CD3+CD8+CD137+T single cells were sorted out after stimulation with the HBsAg peptide library.Theαandβchains in TCRs of single cells were amplified by PCR.TTCR double-chain pairinggand lentiviral packaging were performed throughhighthroughput sequencing.Re-infected Jurkat-76-NFATGFP cells and the cell lines stably expressing TCR were screened.HBsAg peptide library and immortalized B lymphocytes co-cultured with J76N-TCR were used to screen HBsAg-specific TCRs.K562 cell lines stably expressing HLA-A 24:02 were established to determine epitope peptide by screening A 24:02-restricted TCR.The screened TCRs were replaced with mouse C regions and packaged with lentiviruses.Functional validation was performed on healthy human CD4^(+)T and CD8^(+)T lymphocytes following infection.Results Stable TCRexpressing cell lines were successfully prepared based on single-cellTCRαβ double-chainnamplificationand pairing technology.Twenty-one TCRs were screened using immortalized B lymphocytes,resulting in nine possible HLA-A 24:02-restricted HBsAg-specific TCRs.Further screening with K562-A2402 resulted in six A*24:02-restricted HBsAg-specific TCRs with identically recognized epitope peptide.The functional determination of the two TCR clones revealed their specific recognition function for target cells expressing HBsAg.Conclusion HLA-A 24:02-restricted HBsAg-specific TCR with recognition function for target cells expressing HBsAg is successfully obtained based on the new experimental technology system,laying an important foundation for further exploration of antiviral immunotherapy based on HBV-specific TCR.展开更多
文摘Objective To establish a new method and platform for screening,identifying,and exploring a new strategy for anti-hepatitis B immunotherapy based on hepatitis B virus(HBV)-specific TCR.Methods Peripheral blood mononuclear cells were isolated from patients with acute hepatitis B.CD3+CD8+CD137+T single cells were sorted out after stimulation with the HBsAg peptide library.Theαandβchains in TCRs of single cells were amplified by PCR.TTCR double-chain pairinggand lentiviral packaging were performed throughhighthroughput sequencing.Re-infected Jurkat-76-NFATGFP cells and the cell lines stably expressing TCR were screened.HBsAg peptide library and immortalized B lymphocytes co-cultured with J76N-TCR were used to screen HBsAg-specific TCRs.K562 cell lines stably expressing HLA-A 24:02 were established to determine epitope peptide by screening A 24:02-restricted TCR.The screened TCRs were replaced with mouse C regions and packaged with lentiviruses.Functional validation was performed on healthy human CD4^(+)T and CD8^(+)T lymphocytes following infection.Results Stable TCRexpressing cell lines were successfully prepared based on single-cellTCRαβ double-chainnamplificationand pairing technology.Twenty-one TCRs were screened using immortalized B lymphocytes,resulting in nine possible HLA-A 24:02-restricted HBsAg-specific TCRs.Further screening with K562-A2402 resulted in six A*24:02-restricted HBsAg-specific TCRs with identically recognized epitope peptide.The functional determination of the two TCR clones revealed their specific recognition function for target cells expressing HBsAg.Conclusion HLA-A 24:02-restricted HBsAg-specific TCR with recognition function for target cells expressing HBsAg is successfully obtained based on the new experimental technology system,laying an important foundation for further exploration of antiviral immunotherapy based on HBV-specific TCR.
