Screening and functional identification of HLA-A*24:02-restricted HBsAg-specific TCR based on single-cell TCRαβ double-stranded amplification pairing
10.3760/cma.j.cn501113-20240307-00117
- VernacularTitle:基于单细胞TCR αβ双链扩增配对的HLA-A*24：02限制性HBsAg特异性TCR筛选及功能鉴定
- Author:
Guojun SHEN
1
;
Anqi ZHENG
;
Mengfen SHI
;
Xueying LI
;
Baolin LIAO
;
Zhanhui WANG
;
Yuecheng YU
Author Information
1. 九江市第三人民医院肝病科，九江 332000
- Publication Type:Journal Article
- Keywords:
T cell receptor;
Hepatitis B virus;
Hepatitis B surface antigen;
HLA restriction;
Therapeutic
- From:
Chinese Journal of Hepatology
2025;33(1):41-47
- CountryChina
- Language:Chinese
-
Abstract:
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. The α and β chains in TCRs of single cells were amplified by PCR. TCR double-chain pairing and lentiviral packaging were performed through high-throughput sequencing. Re-infected Jurkat-76-NFAT-GFP 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 TCR-expressing cell lines were successfully prepared based on single-cell TCRαβ double-chain amplification and 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 was successfully obtained based on the new experimental technology system, laying an important foundation for further exploration of antiviral immunotherapy based on HBV-specific TCR.
