Next Generation Sequencing-Based Identification of T-Cell Receptors for Immunotherapy Against Hepatocellular...

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains a global health concern, and HBV proteins may be ideal targets for T cell-based immunotherapy for HCC. There is a need for fast and efficient identification of HBV-specific T cell receptors (TCRs) for the development of TCR-transduced T (TCR-T) cell-based immunotherapy. Two widely employed TCR identification approaches, T cell clonal expansion and single-cell sequencing, involve a TCR singularization process for the direct identification of Vα and Vβ pairs of TCR chains. Clonal expansion of T cells is well known to have tedious time and effort requirements due to the use of T cell cultures, whereas single-cell sequencing is limited by the requirements of cell sorting and the preparation of a single-cell immune-transcriptome library as well as the massive cost of the whole procedure. Here, we present a next-generation sequencing (NGS)-based HBV-specific TCR identification that does not require the TCR singularization process. Conclusion: Two pairing strategies, ranking-based strategy and α-β chain mixture-based strategy, have proved to be useful for NGS-based TCR identification, particularly for polyclonal T cells purified by a peptide-major histocompatibility complex (pMHC) multimer-based approach. Functional evaluation confirmed the specificity and avidity of two identified HBV-specific TCRs, which may potentially be used to produce TCR-T cells to treat patients with HBV-related HCC.