EBV latent membrane protein 1 augments γδ T cell cytotoxicity against nasopharyngeal carcinoma by induction of butyrophilin molecules

Yue Liu, Ka Sin Lui, Zuodong Ye, Tsz Yan Fung, Luo Chen, Ping Yiu Sit, Chin Yu Leung, Nai Ki Mak, Ka Leung Wong, Hong Lok Lung, Yoshimasa Tanaka, Allen Ka Loon Cheung*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

4 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)458-471
Number of pages14
Issue number2
Publication statusPublished - Jan 2023

Scopus Subject Areas

  • Medicine (miscellaneous)
  • Pharmacology, Toxicology and Pharmaceutics (miscellaneous)

User-Defined Keywords

  • Nasopharyngeal carcinoma
  • γδ T cells
  • butyrophilin
  • NLRC5
  • EBV latent membrane protein 1 (LMP1)


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