Epstein-Barr virus (EBV), an oncogenic γ-herpes virus infecting >90 % of adults worldwide, is associated with malignancies of B-cell and epithelial origins [1]. EBV has three patterns of latent viral antigens [2], some of which have been exploited to generate EBV-specific T cells against EBV-associated malignancies [[3], [4], [5], [6]]. EBV latency III (e.g., post-transplant lymphoproliferative disorder) is characterized by expression of EBV nuclear antigens (EBNAs) (EBNA1–3 and EBNA-LP) and latent membrane proteins (LMP1/2) [2]. EBV latency II (e.g. Hodgkin's lymphoma, peripheral NK/T lymphoma, and nasopharyngeal carcinoma) is characterized by expression of EBNA1, LMP1 and LMP2 [2], while EBV latency I (e.g., Burkitt's lymphoma and EBV-associated gastric carcinoma) typically expresses EBNA1 antigen [2]. Adoptive T cell therapy has succeeded in preventing/treating post-transplant lymphoproliferative disorder caused by EBV [3,7], largely attributable to the patient's being in a state of immunosuppression after transplantation and broad antigen expression (enhancing immunogenicity). LMPs-targeted T cells also show promise for latency II tumors [5,8]. However, clinical benefits from EBV-specific T cells targeting latency I typically expressing EBNA1 antigen have so far not been reported.

EBNA1 is uniquely expressed across all EBV latency types. Importantly, EBNA1 is essential for maintenance of viral episomes and is the only viral protein required for latent EBV replication [2,9]. Previous studies reported that EBNA1 could not be effectively presented by MHC I molecules due to the inhibition of cellular immunity by the glycine-alanine repeat (GAr) domain [10,11]. However, recent research has demonstrated that some immunodominant epitopes of EBNA1 can induce EBNA1-specific CD4+ and CD8+ T cell immune responses in healthy individuals [[12], [13], [14], [15], [16], [17]], highlighting its potential in the development of therapeutic strategies against EBV-associated malignancies. Thus, EBNA1 antigen as an attractive target to generate EBV-specific T cells is worth re-evaluating.

Previous studies indicated that the clinical effectiveness of EBV-specific T cell therapy for EBV-associated malignancies is still low, mainly due to the small number of effector T cells and limited replicative capacity [5,8,18,19]. Based on TCR gene transfer, large quantities of potential effector T cells for immunotherapy can be rapidly generated, that is, EBV-specific TCR-engineered T cells can be obtained. Attention has focused on the well-characterized LMP-specific TCR-engineered T cells as potentially important effector cells [[20], [21], [22]], which suit EBV latency II/III. EBNA1-specific TCR-engineered T cells, in contrast, have been rarely evaluated for a role in EBV immunity. Thus, preparing EBNA1-specific TCR-engineered T cells, which can be suitable for all the three EBV latency types (latency I/II/III), especially for latency I, is at the exploratory stage.

Here, we established a method to generate functional EBNA1-specific TCR-T cells. Specifically, EBNA1-specific T cells were stimulated using autologous dendritic cells (DCs) pulsed with peptides synthesized from the complete sequence (except the GAr region) of the EBNA1 of EBV strain B95–8, and were assessed using IFN-γ-ELISA and ELISPOT assays. IFN-γ as one effector cytokine of EBNA1-specific T cells after reactivated with EBNA1 peptide-pulsed DCs would presumably be increased. Using high-throughput single-cell TCR V(D)J sequencing of pre-stimulated and post-stimulated T cells, candidate EBNA1-specific TCRs with significantly increased frequencies could be identified. Then, EBNA1-specific TCR sequences were introduced into the peripheral blood T cells to generate EBNA1-specific TCR-engineered T cells. The functionality of EBNA1-specific TCR-engineered T cells using lymphoblastoid cell lines (LCLs) and EBNA1 peptide-pulsed DCs as targets in vitro were measured by IFN-γ-ELISA.