Broadening specificity and enhancing cytotoxicity of adoptive T cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxorubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-κB activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable.