Nasopharyngeal carcinoma (NPC) is a diverse cancer with no well-defined tumor antigen, associated with oncogenic Epstein-Barr Virus (EBV), and with usually late-stage diagnosis and survival <40%. Current radiotherapy and chemotherapy have low effectiveness and cause adverse effects, which calls for the need of new therapy. In this regard, adoptive immunotherapy using γδ T cells has potential, but needs to be coupled with butyrophilin 2A1 and 3A1 protein expression to achieve tumoricidal effect. Methods: Human γδ T cells were expanded (with Zol or PTA) and used for cytotoxicity assay against NPC cells, which were treated with the EBV EBNA1-targeting peptide (L₂)P₄. Effect of (L₂)P₄ on BTN2A1/BTN3A1 expression in NPC cells was examined by flow cytometry and Western blot. An NPC-bearing NSG mice model was established to test the effectiveness of P₄ and adoptive γδ T cells. Immunofluorescence was performed on NPC tissue sections to examine the presence of γδ T cells and expression of BTN2A1 and BTN3A1. EBV gene expression post-(L₂)P₄ treatment was assessed by qRT-PCR, and the relationship of LMP1, NLRC5 and BTN2A1/BTN3A1 was examined by transfection, reporter assay, Western blot, and inhibition experiments. Results: Zol- or PTA-expanded the Vδ2 subset of γδ T cells that exerted killing against certain NPC cells. (L₂)P₄ reactivates latent EBV, which increased BTN2A1 and BTN3A1 expression and conferred higher susceptibility towards Vδ2 T cells cytotoxicity in vitro, as well as enhanced tumor regression in vivo by adoptive transfer of Vδ2 T cells. Mechanistically, (L₂)P₄ induced EBV LMP1, leading to IFN-γ/p-JNK and NLRC5 activation, and subsequently stimulated the expression of BTN2A1 and BTN3A1. Conclusions: This study demonstrated the effectiveness of using the EBV-targeting probe (L₂)P₄ and adoptive γδ T cells as a promising combinatorial immunotherapy against NPC. The identification of the LMP1-IFN-γ/p-JNK-NLRC5-BTN2A1/BTN3A1 axis may lead to new insight and therapeutic targets against NPC and other EBV⁺ tumors.

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