Improved Adoptive T cell Therapy Protocols for EBV-driven Malignancies.

BACKGROUND: Although adoptive infusion of EBV-specific T cell lines constitutes a promising strategy for nasopharyngeal carcinoma (NPC) therapy, however, the clinical benefit of current protocols is still poor. One major limitation is constituted by the restricted number of EBV antigens that can be targeted and their poor immunogenicity. The oncogenic EBV protein BARF1 is expressed in the majority of NPC cases and may constitute an optimal target. We have previously demonstrated that NPC patients have strong spontaneous CD4 and CD8 T cell responses against BARF1 protein and derived epitopes. Moreover, BARF1-specific cytotoxic T lymphocytes (CTLs) can be easily generated from healthy donors. Nevertheless, T cell lines obtained with current protocols for adoptive immunotherapy of NPC include only negligible numbers of BARF1-specific CTLs, pointing to the need to implement these T-cell cultures in BARF1 specificities. AIM: To develop optimized, GMP-upgradable protocols for adoptive immunotherapy of NPC based on the generation of T-cell lines enriched in BARF1-specific effectors METHODS: Lymphoblastoid cell lines (LCLs) were treated with different EBV lytic cycle inducers at doses able to induce abortive or limited EBV replication while preserving cell viability. Expression of EBNA2, BARF1, LMP1, ZEBRA and EA mRNA was quantified by qRT-PCR. EBV-specific donor- and patients-derived CTLs were generated with LCLs treated with the different drugs from PBMCs. Standard cytotocxicity assays were used to assess the specificity of CTLs. Content in Granzyme B granules was assessed by multispectral imaging flow cytometry. RESULTS: Treatment with low doses of doxorubicin (DX) proved to be the most suitable and simple protocol to enhance BARF1 expression (3.3 fold increase), without down-regulating other viral antigens that are targeted by EBV-specific CTLs. By contrast, TPA/Na-butyrate (TPA+NaB) or cisplatin (CSP) were less effective in up-regulating BARF-1 and induced higher levels of cell apoptosis. CTLs induced with DX-treated LCLs (DX-CTLs) showed high levels of specific cytotoxicity against NPC cells endogenously expressing BARF1 (c666.1-A2, >90% of specific lysis) or T2A2 cells loaded with BARF1 or LMP1 HLA-A2 peptides (30% specific lysis). CTLs generated with LCLs either untreated or exposed to TPA+NaB of CSP induced only low levels of BARF1 or LMP1 specific cytotoxicity. Notably, the extent of specific lysis induced by DX-CTLs was higher against the BARF-1 peptides. In addition, qRT-PCR and Western blot showed up-regulation of HLA class I and immunogenic cell death markers activation in DX-LCLs. DX-CTLs have also a higher content in Granzyme B granules. The identified protocol were also verified using patients-derived LCLs. CONCLUSIONS: These findings provide the rationale for a rapid up-grading at the GMP level of the use of DX-treated LCLs for the generation of CTL lines enriched in BARF1 specificities for adoptive immunotherapy of resistant or relapsing NPC.