Rapid single-cell identification of Epstein-Barr virus-specific T-cell receptors for cellular therapy

Background and aims: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. Methods and Results: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA- B*35:01-or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRab-sequenced. To confirm specificity, 11 HLA-B*35:01-and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To con-firm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA- B*35:01-and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. Conclusions: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies. (C) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc.

Automated summary

The study presents a highly efficient method for identifying EBV-specific T-cell receptor (TCR) that can be expressed on human T cells and recognize EBV-infected cells. The researchers expanded mononuclear cells from stem cell grafts in vitro and sorted and sequenced the TCRs. The identified TCRs exhibited specificity and recognized their target peptides. The expression of these TCRs on human peripheral blood lymphocytes enabled recognition of EBV-infected cells. This approach could help address the clinical needs in the treatment and prevention of EBV reactivation and associated malignancies.