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

development and application of a predictive controller to a mini district heating network fed by a biomass boiler

Abstract Energy saving is actually recognized as one of the most significant ways to reduce primary energy consumption and pollutant emissions. Due to the remarkable importance of heating systems and heat distribution grids, Siram by Veolia and the University of Parma have developed an optimal control system for District Heating Networks. Usually building control systems are designed to manage plants relying on past experience: the optimal control system described in the paper defines plant management strategy on the basis of the future behavior of the systems and the external environment. The proposed control system has been applied to the heating system and the distribution network of a school complex in Podenzano (Emilia-Romagna region). The district heating supplies heat to three different buildings (primary school, secondary school and sports hall). The heating plant is composed of three generators (two fed by natural gas and one by wooden biomass), a Thermal Energy Storage, two main distribution manifolds (supply and return) and three secondary circuits, which distribute heat to the buildings. In the first part of the paper the control algorithm is described, split into plant simulation models and the optimization algorithm. In the second part, the real application and the new communication architecture applied on site are outlined and, finally, the obtained results are reported highlighting the management strategies of generators and pumps. The optimal control strategy application gave important results in terms of energy saving, in particular the energy supplied to the buildings dropped significantly, this is the result of knowing the building behavior in advance

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors