Abstract: Objective To screen and identify human leukocyte antigen class Ⅱ (HLA-II) restricted hepatitis B core antigen (HBcAg) specific T cell receptor (TCR), laying the foundation for exploring CD4⁺T cell function and new potential immunotherapy in HBV-infected patients. Methods Peripheral blood mononuclear cells (PBMCs) from acute hepatitis B patient were stimulated with HBcAg peptide library, and CD4⁺CD154⁺T single cells were sorted. TCR α/β double strands were amplified and paired. The paired TCR packaged by lentiviral vectors infected Jurkat-76-NFAT-GFP cells, which further established stable TCR expression cell line. The co culture of HBcAg peptide library presented by immortalized B cell lines aimed to screen for HBcAg specific TCRs. The specific peptide recognized by TCRs was determined through the HBcAg peptide library matrix. The HLA restriction of TCRs were determined by using anti-HLA-I and K562 cell lines expressing different combinations of HLA class II molecules. Finally, the functional avidity of the TCRs was determined through peptide concentration gradient analysis. Results Based on CD4⁺CD154⁺T single-cell sorting and TCR α/β pairing, 4 HBcAg specific TCRs recognizing the same peptide (EYLVSFGVWIRTPPA) were successfully screened from 13 TCR clones. All 4 TCRs can recognize the peptide presented by DPA1*01:03/ DPB1*05:01, DPA1*01:03/DPB1*21:01and DPA1*02:02/ DPB1*05:01 expression K562 cells. Functional avidity analysis showed that 4 TCRs have high avidity, with EC50 of up to 7.817 nM. Conclusion HLA-DP restricted HBcAg specific TCRs with high functional avidity were successfully obtained, providing a new possible pipeline for the next step to study the functional changes of CD4⁺T cells in HBV infected patients and develop new immunotherapy strategies based on HBcAg specific TCRs.

Key words: T cell receptor, Hepatitis B virus, Hepatitis B core antigen, HLA restriction