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

Exploiting the Interplay between Innate and Adaptive Immunity to Improve Immunotherapeutic Strategies for Epstein-Barr-Virus-Driven Disorders

The recent demonstration that immunotherapeutic approaches may be clinically effective for cancer patients has renewed the interest for this strategy of intervention. In particular, clinical trials using adoptive T-cell therapies disclosed encouraging results, particularly in the context of Epstein-Barr-virus- (EBV-) related tumors. Nevertheless, the rate of complete clinical responses is still limited, thus stimulating the development of more effective therapeutic protocols. Considering the relevance of innate immunity in controlling both infections and cancers, innovative immunotherapeutic approaches should take into account also this compartment to improve clinical efficacy. Evidence accumulated so far indicates that innate immunity effectors, particularly NK cells, can be exploited with therapeutic purposes and new targets have been recently identified. We herein review the complex interactions between EBV and innate immunity and summarize the therapeutic strategies involving both adaptive and innate immune system, in the light of a fruitful integration between these immunotherapeutic modalities for a better control of EBV-driven tumors

Integration of miRNA:mRNA Co-Expression Revealed Crucial Mechanisms Modulated in Immunogenic Cancer Cell Death

Immunogenic cell death (ICD) in cancer represents a functionally unique therapeutic response that can induce tumor-targeting immune responses. ICD is characterized by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which confer adjuvanticity to dying cancer cells. The spatiotemporally defined emission of DAMPs during ICD has been well described, whereas the epigenetic mechanisms that regulate ICD hallmarks have not yet been deeply elucidated. Here, we aimed to examine the involvement of miRNAs and their putative targets using well-established in vitro models of ICD. To this end, B cell lymphoma (Mino) and breast cancer (MDA-MB-231) cell lines were exposed to two different ICD inducers, the combination of retinoic acid (RA) and interferon-alpha (IFN-alpha) and doxorubicin, and to non ICD inducers such as gamma irradiation. Then, miRNA and mRNA profiles were studied by next generation sequencing. Co-expression analysis identified 16 miRNAs differentially modulated in cells undergoing ICD. Integrated miRNA-mRNA functional analysis revealed candidate miRNAs, mRNAs, and modulated pathways associated with Immune System Process (GO Term). Specifically, ICD induced a distinctive transcriptional signature hallmarked by regulation of antigen presentation, a crucial step for proper activation of immune system antitumor response. Interestingly, the major histocompatibility complex class I (MHC-I) pathway was upregulated whereas class II (MHC-II) was downregulated. Analysis of MHC-II associated transcripts and HLA-DR surface expression confirmed inhibition of this pathway by ICD on lymphoma cells. miR-4284 and miR-212-3p were the strongest miRNAs upregulated by ICD associated with this event and miR-212-3p overexpression was able to downregulate surface expression of HLA-DR. It is well known that MHC-II expression on tumor cells facilitates the recruitment of CD4+ T cells. However, the interaction between tumor MHC-II and inhibitory coreceptors on tumor-associated lymphocytes could provide an immunosuppressive signal that directly represses effector cytotoxic activity. In this context, MHC-II downregulation by ICD could enhance antitumor immunity. Overall, we found that the miRNA profile was significantly altered during ICD. Several miRNAs are predicted to be involved in the regulation of MHC-I and II pathways, whose implication in ICD is demonstrated herein for the first time, which could eventually modulate tumor recognition and attack by the immune system