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POSTER W01 ZOLEDRONIC ACID BOOSTS T-FUNCTIONS IN PATIENTS RECEIVING + T CELL AND CD19+ B LYMPHOCYTES DEPLETED GRAFTS FROM HAPLO-IDENTICAL DONORS Alice Bertaina, Andrea Petretto, Barbarella Lucarelli, Alessia Zorzoli, Pietro Merli, Gino Tripodi, Chiara Lavarello, Giulia Barbarito, Letizia Pomponia Brescia, Valentina Bertaina, Elvira Inglese, Francesca Antonini, Giuseppe Maria Milano, Franco Locatelli and Irma Airoldi Istituto Giannina Gaslini A new method of graft manipulation based on physical removal of + T cells and CD19+ B cells, leaving mature NK cells and T cells in the graft, was recently employed for HLA-haploidentical HSCT. We demonstrated that T cells from transplanted patients are endowed with killing capacity of leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in transplanted patients, receiving this type of graft, may boost T cell cytotoxic activity. Thirty-three transplanted patients with acute leukemias received HSCT and were treated with ZOL within 1 month after HSCT and then every 28 days for at least 2 times. T cells before and after ZOL treatments were studied by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. Proteomic analysis of T cells showed that, starting from the first infusion, ZOL caused an up-regulation of proteins involved in activation processes and immune responses, paralleled by a down-regulation of proteins involved in proliferation. These findings are consistent with an induction of V 2 differentiation paralleled by increased cytotoxicity of both V 1 and V 2 cells against primary leukemia blasts. Finally, a proteomic signature of each ZOL treatment was identified. POSTER W02 FASTING AS A STRATEGY TO DELAY TUMOR GROWTH BY MODULATING CANCER METABOLISM, ANGIOGENESIS AND ANTITUMOR IMMUNITY Giovanna Bianchi, Danilo Marimpietri, Roberto Martella, Chiara Traverso, Silvia Ravera, Laura Emionite, Chiara Lavarello, Andrea Petretto, Francesca Antonini, Genny Del Zotto, Vito Pistoia, Lizzia Raffaghello. Laboratorio di Oncologia, Istituto G. Gaslini. Background Fasting or Short term Starvation (STS) represents a novel therapeutic strategy which appears to: i) protect normal but not tumor cells against the chemotherapy-mediated cytotoxicity, ii) induce a potent chemosensitizing effect in a wide range of experimental tumor model, and iii) be feasible, safe and able to reduce common side effects induced by chemotherapy in cancer patients. However, the molecular mechanisms coupling STS with antitumor activity remain only partially understood. Results In vitro experiments showed that STS reduced melanoma cell viability by inducing apoptosis and abrogating ATP production through the inhibition of ATP-synthase activity. Accordingly, fasting delayed the growth of B16 xenograft developed in C57BL/6 mice by reducing the tumorassociated micro vascular density and by reprogramming tumor metabolism. In addition, fasting modulated the percentage of mature dendritic cells, T lymphocytes and myeloid cells isolated from the spleen and peripheral blood of B16-bearing mice. The expression of costimulatory molecules, differentiation markers and cytokines was also regulated by fasting. Conclusions Taken together, these results demonstrated that fasting reprograms tumor metabolism and modulated the antitumor activity leading to a significant decrease of tumor growth. These findings open a novel scenario in cancer treatment. 1 POSTER W03 TUMOR-IMMUNE CELLS CROSS TALK IN OSTEOSARCOMA: SEARCHING FOR NEW THERAPEUTIC TARGETS Claudia Chiodoni1, Ilaria Torselli1, Claudio Tripodo2, Lucia Bongiovanni2, Katia Scotlandi3 and Mario P. Colombo1. 1Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy. 2. Tumor Immunology Unit, Department of Health Sciences, University of Palermo, Palermo, Italy. 3.CRS Development of Biomolecular Therapies, Laboratory of Experimental Oncology, Rizzoli Orthopedic Institute, Bologna, Italy. Osteosarcoma (OS) is the most common primary bone tumor in childhood and adolescence, with poor prognosis, because of the high rate of metastases, particularly to the lungs. Therapy of OS is still firmly entrenched in conventional cytotoxic drugs and prognosis for patients with metastasis remains grim. For effective implementation of current therapies, improved understanding of the disease biology is mandatory. Few mouse models recapitulating the human disease are currently available and xenografts of human OS lines in immune-deficient mice cannot reproduce the complex interaction of tumor cells with the surrounding bone marrow stroma. To fill such a gap, we developed novel immunocompetent mouse OS models growing orthotopically in the bone cavity and spontaneously metastasizing to the lungs, phenocopying the human disease. We took advantage of these models to test the therapeutic efficacy in OS treatment of Trabectedin, a chemotherapeutic agent of marine origin, which mechanisms of action are only partially defined, Recently it has been reported that cytotoxicity on mononuclear cells is a key component of its antitumor activity. Experiments in OS models show significant inhibition of tumor growth and spontaneous metastasis and preliminary analysis suggests a double mechanism of action, affecting both the neoplastic clone and the immune microenvironment. POSTER W04 DYSREGULATED ESTROGEN RECEPTOR-β EXPRESSION IN INFLAMMATORY BOWEL DISEASES Marina Pierdominici*, Angela Maselli*, Barbara Varano, Cristiana Barbati*, Maria Rosaria Limiti°, Marco Rosati°, Paola Cesaro^, Cristiano Spada^, Angelo Zullo§, Sandra Gessani, Lucia Conti Department of Hematology, Oncology and Molecular Medicine and *Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy; °Histopathology Unit, S. Spirito Hospital, Rome, Italy; ^Digestive Endoscopy Unit, General Surgery Department, Agostino Gemelli Hospital, Catholic University, Rome, Italy; §Digestive Endoscopy Unit, Nuovo Regina Margherita Hospital, Rome, Italy. Crohn disease (CD) and ulcerative colitis (UC) are chronic forms of inflammatory bowel disease (IBD) whose pathogenesis is poorly understood. Estrogens have a complex role in inflammation and growing evidence suggests their possible impact on IBD pathogenesis. Notably, immunohistochemistry analysis revealed a markedly decreased ERβ expression in colonic mucosa of active CD/UC patients, reflecting the alterations observed in peripheral blood T cells. ERβ expression inversely correlated with IL-6 serum levels and exogenous exposure of T lymphocytes to this cytokine resulted in ERβ downregulation.We demonstrate a significant reduction of intracellular estrogen receptor (ER)β expression, as assessed by flow cytometry, in blood T lymphocytes from CD/UC patients with active disease as compared to those with inactive disease and healthy controls. In a subgroup of patients undergoing anti-TNF therapy and responsive to treatment, ERβ levels were higher than those observed in unresponsive patients and comparable to those of control subjects. These results demonstrate an altered ER profile in active IBD patients and highlight the potential exploitation of T cell-associated ERβ as a biomarker of endoscopic disease activity. As intestinal ERβ expression inversely correlates with colon cancer development/progression, ERβ down-regulation in active IBD patients might link chronic intestinal inflammation to neoplastic transformation. 2 POSTER W05 ROLE OF CX3CL1/FRACTALKINE IN MULTIPLE MYELOMA Anna Corcione Laboratory of Oncology, Istituto Giannina Gaslini, Genova Background. Multiple myeloma (MM) is characterized by accumulation and proliferation of malignant plasma cells in the bone marrow (BM). CX3CL1/fractalkine is synthesized both as membrane-bound and soluble form. Its function is mediated through CX3C chemokine receptor 1 (CX3CR1). The CX3CL1/CX3CR1 complex has been detected in many tumors where it contributes through different mechanism to the crosstalk between malignant cells and microenvironment. Aim. We investigated the expression and activity of CX3CL1/CX3CR1 axis in MM cells and the interactions between these cells and microenvironment. Methods. CX3CL1/CX3CR1 expression was analyzed by flow cytometry; soluble CX3CL1 by ELISA; chemotaxis using a transwell assay. Results and conclusions. Increased levels of soluble CX3CL1 were detected in BM plasma from MM (P=0.0005) and MGUS (P=0.03) patients compared to those from healthy donors. In contrast, CX3CL1 levels were similar in BM from smoldering MM patients and controls. CD138+ cells from the infiltrated BM of MM patients were found to express the CX3CL1/CX3CR1 complex.Five human MM cell lines also expressed CX3CL1 and CX3CR1. CX3CL1 increased significantly the spontaneous migration of JJN3 and U266 cells that have been selected for further in vivo experiments.These preliminary results demonstrate that elevated levels of CX3CL1 correlate with progression of the disease. POSTER W06 ACHIEVEMENT AND CHARACTERISATION OF A TUMOR EXTRACELLULAR MATRIX AS A 3D SCAFFOLD FOR CANCER RESEARCH. Edoardo D'Angelo Department of Surgical, Oncological and Gastroenterological Sciences, Section of Surgery, University of Padova, Via Giustiniani 2 – 35128, Padua, Italy Extracellular matrix (ECM) is a dynamic compartment that provides biochemical and biomechanical cues influencing cell behaviour. New insights on cancer research can arise from an intimate understanding of the ECM and cancer cell crosstalk. AIMS: To establish a decellularization protocol to isolate ECM from healthy mucosa and colorectal cancer. To characterize ECM protein composition comparing normal and tumor matched samples. METHODS: Normal colon mucosa and matched tumor tissue were collected by 7 surgically resected patients. Decellularization method consists of a detergent-enzymatic treatment (DET). DNA amount from fresh and decellularized tissues was quantified with Nanodrop, and nuclei counted on tissue sections with DAPI. Collagen and glycosaminoglycan were quantified with a spectrophotometric assay (Biocolor) and validated with histological staining. ECM 3D organization was investigated with SEM. RESULTS AND CONCLUSIONS: We established that 2 DET cycles are a good compromise between DNA content reduction and ECM preservation. DNA quantification and DAPI confirmed over 95% of nuclei depletion. Quantification of the total amount of collagen did not show significant differences between normal and tumor mucosa but rather a different composition and solubility underlining specific isoform type content. Glycosaminoglycan are significantly increased in tumor samples compared with normal mucosa both in fresh and decellularized tissue. 3 POSTER W07 INVOLVEMENT OF INKT CELLS IN THE CONTROL AND PROGNOSIS OF CLL Francesca Gorini1, Laura Azzimonti2, Lydia Scarfò3, Maurilio Ponzoni4, Cristina Scielzo3, Maria Teresa Bertilaccio3, Federico Caligaris Cappio3, Paolo Ghia3, Matteo Bellone1, Paolo Dellabona1, Giulia Casorati1, Claudia de Lalla1. 1Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy; 2Moxoff SRL, Milano, Italy; 3Division of Experimental Oncology, San Raffaele Scientific Institute, Milano, Italy. Chronic lymphocytic leukemia (CLL) is characterized by the progression of CD5+ B lymphocytes in bone marrow, lymphoid organs and blood. CD1d-restricted iNKT cells are a T cell subset strongly implicated in tumor immune-surveillance. CLL cells express CD1d and are recognized by iNKT cells in vitro; however, how iNKT and CLL cells interact in the natural history of the disease is unknown. To this aim, we investigated: 1. CD1d expression on leukemia cells and autologous iNKT cells in 72 CLL patients; 2. iNKT cell role in CLL progression in the Tcl1 transgenic mouse model. In patients, high CD1d expression on CLL cells correlated with low iNKT cell counts and disease progression. iNKT cells were functional in stable CD1d-low CLL patients, but hypo-responsive in progressive CD1d-high ones. In multivariate analysis including classic prognostic factors (RAI, ZAP-70, Chromosomal aberrations, IGVH mutations, CD38), iNKT cell counts and CD38 expression were the only independent parameters predicting disease progression. CLL progression was faster in iNKT cell deficient than WT TCL1 animals. Tcl1 iNKT cells decreased and became hypofunctional upon disease progression, consistent with the high CD1d expression on murine CLL cells. iNKT cells emerge as both relevant players in CLL immune-surveillance and new prognostic marker. POSTER W08 THE IL-25/IL-25R AXIS IN B CELL LYMPHOMAS OF GERMINAL CENTRE ORIGIN Elisa Ferretti, Emma Di Carlo, Claudio Tripodo, Maurilio Ponzoni and Vito Pistoia Istituto Giannina Gaslini Background. Interleukin (IL)-25, is structurally related to IL-17A but is the most divergent member of the IL17 family, enhancing Th2 cell immune response. The heterodimeric receptor for IL-25 (IL-25R) is composed of IL-17RA and IL-17RB subunits. Aim. So far, no information is available about the relationships between IL-25 and B-cell malignancies. Methods. We have investigated: i) by flow cytometry and immunohistochemistry the expression of IL-25/IL25R in primary neoplastic B cells from lymph-node biopsies of patients with Germinal Center (GC)-derived NHL; ii) the function of IL-25, in term of proliferation (tritiated thymidine incorporation) and cell signaling (flow cytometry); iii) the in vivo effects of IL-25 injecting two cell lines of GC-derived B-NHL in SCID/NOD mice; iv) the gene modulated by PCR-array. Results. We demonstrated that: i) lymphoma cells expressed IL-25R and IL-25 inhibited their proliferation; ii) IL-25 was expressed in tumor microenvironment; iii) IL-25 activated NF-kB signalling, and iv) exerted antitumor activity in vivo. Tumor masses from IL-25 treated mice displayed necrotic-haemorragic areas associated with defective microvascular supply and reduced neoplastic cell proliferation. These histopathological findings were paralleled by the blunted expression of different pro-angiogenic genes. Conclusions. These data delineate a new anti-tumor role of IL-25 in GC-derived B cell lymphomas. 4 POSTER W09 ZOLEDRONIC ACID REVERSES IMMUNOSUPPRESSION IN HUMAN MALIGNANT MESOTHELIOMA Elena Gazzano (1), Iris Chiara Salaroglio (1), Ivana Campia (1), Joanna Kopecka (1), Sara Orecchia (2), Dario Ghigo (1), Chiara Riganti (1). (1)Department of Oncology, University of Torino, Italy. (2) S.C. Anatomia Patologica, Azienda Ospedaliera S.S. Antonio e Biagio, Alessandria, Italy. Background. Human malignant mesothelioma (HMM) is an asbestos-related tumor characterized by a chemoresistant and immunosuppressive phenotype. HMM has a poor prognosis and a median survival lower than 1 year. The molecular basis of such tumor-induced immunosuppression are poorly known. Hence, an effective immunotherapy for HMM is still lacking. Aim. We investigated: 1) how HMM evade the immunosurveillance; 2) whether zoledronic acid, an aminobisphosphonate that restores the immune system recognition in specific immunoresistant tumors, is an effective immunosensitizing strategy in HMM. Methods. We compared primary HMM samples with non-transformed mesothelial cells. Results. HMM cells produced higher amounts of kynurenine (one of the strongest mediator of tumor-induced immunosuppression) and have a higher expression of indoleamine 1,2-dioxygenase (IDO, the enzyme responsible for kynurenine synthesis). This is associated with a decreased proliferation of T-lymphocytes and an expanded number of immunosuppressive T-regulatory (Treg) cells. Zoledronic acid, by down-regulating the Ras/ERK1/2/STAT3 axis, lowered IDO expression and kynurenine synthesis. In parallel it reduced the expansion of Treg cells and increased the proliferation of T-lymphocytes. Conclusions. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for IDO-mediated immunosuppression, zoledronic acid is an effective immunosensitizing agent in HMM. POSTER W10 TUMOR- ELICITED INFLAMMATION Sergei Grivennikov Fox Chase Cancer Center, Philadelphia, PA, USA ‘Tumor- elicited inflammation’(TEI) is represented by immune infiltrates and enhanced expression of inflammatory mediators in many solid tumor, including colorectal cancer (CRC) . We study the role and the mechanisms of TEI and how cytokine IL-23-IL-17 pathway drives CRC growth and progression. Analysis of IL-17A “reporter” and conditional receptor knockout mice revealed that not only conventional \"Th17\" cells but also innate lymphocytes (ILC3) integrate IL-23 and IL-1 driven signals in CRC to maintain inflammatory response and promote CRC tumor growth. Two photon imaging of IL-17A producing cells revealed their distinct behavior in the normal tissue and within the tumor microenvironment. Furthermore, TEI in sporadic and inducible models of CRC was in part controlled by distinct components of host microbiota, which influenced initial and subsequent inflammatory responses. On the other hand, distinct host derived “danger signals” linked to oncogenic process, are also implicated in TEI induction and maintenance. Therefore, TEI is essential for CRC growth and progression. 5 POSTER W11 THERAPEUTIC RIBOLOGICAL® RNA VACCINE AGAINST HPV16+ TUMOR ENTITIES Christian Grunwitz1,2 , Abderaouf Selmi3, Catharina Worm1 , Ute Schmitt2, Jan Diekmann1, Mustafa Diken3, Sebastian Kreiter3, Heinrich Haas1, Fulvia Vascotto3, Özlem Türeci4 and Ugur Sahin1,2,3 1 BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany.; 2 Research Center for Immunotherapy (FZI), University Medical Center, Johannes Gutenberg University, Mainz, Germany.; 3 TRON – Translational Oncology gGMbH, Mainz, Germany.; 4 Ganymed Pharmaceuticals AG. Mainz, Germany. The incidence of HPV16+ Head Neck squamous cell carcinoma (HNSCC) is on the rise and to date treatment is not only associated with facial disfigurement but also with a high rate of long-term morbidity. Since HPV16 E6/E7 are neo-antigens, these oncoproteins are excellent targets for therapeutic vaccinations. Here we investigated the effect of E6/E7 RNA [LIP], a RNA-based liposomal formulated vaccine, in wellestablished HPV16+ E6/E7 expressing tumours, namely TC-1/luc and C3. Repetitive immunizations with E6/E7 RNA [LIP], led to substantial antigen specific CD8+ T cell expansion, which displayed potent effector function assessed in C57BL/6 and HLA-A2 +/–/HLA-DR1+/– transgenic mice. Analysis of RNA [LIP] antitumoural activity in TC-1/luc and C3 revealed strong tumour rejection, prolonged survival and profound changes of the tumor-environment. In TC-1/luc we observed an increased frequency of tumor infiltrating E7reactive CD8+ T cells, NK cells and beneficial CD8+ T/ Treg ratio as well as polarization of macrophages towards a CD206- iNOS+ phenotype. To further characterize the modulation of the tumour environment upon RNA [LIP] therapy, we analysed the expression of receptor/ligand pairs involved in T-cell inhibition, providing a rational approach for combination therapies to achieve the ultimate goal of therapeutic vaccination sustained and complete tumor rejection. POSTER W12 CELL ADHESION AND MIGRATION REGULATION BY CRK ADAPTOR PROTEINS Pulak Ranjan Nath, Guangyu Dong, Alex Braiman and Noah Isakov The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva, Israel Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. They act as major convergence points of tyrosine kinase signaling pathways and integrate molecular information obtained from a variety of sources including growth factors, extracellular matrix, antigens and pathogens. Crk proteins also play a role in the regulation of immune cell adhesion and migration, as well as tumor cell invasion and metastasis. Thus, understanding the mechanism of regulation of Crk proteins is of clinical importance. Recent in vitro studies demonstrated that the conformation of chicken CrkII is regulated by the immunophilin, cyclophilin A. We found that a combination of immunophilin inhibitory drugs, which includes cyclosporin A and FK506, interferes with C3G binding to CrkII, an essential step in a signaling pathway regulating integrindependent cell adhesion. Overexpression of either CrkI or CrkII increased Jurkat T cell adhesion and invasion. In contrast, immunophilin inhibitors suppressed the ability of CrkII-, but not CrkI-overexpressing Jurkat T cells to adhere to fibronectin-coated surfaces and migrate toward SDF-1α. Our studies suggest that the conformation and activity of human CrkII is subjected to a regulation by immunophilins, which may impact on leukocyte as well as cancer cell adhesion and migration. 6 POSTER W13 MAST CELLS CONTRIBUTE TO T CELL TOLERANCE AGAINST PROSTATE CANCER- ASSOCIATED ANTIGENS FAVORING TUMOR GROWTH Elena Jachetti, Rigoni A , Bongiovanni L, Casalini P, Sangaletti S, Chiodoni C, Tripodo C and Colombo M P Molecular Immunology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo, 42, 20133 Milan, Italy Immunotherapy for patients with advanced prostate cancer (PC) provided limited results, due to mechanisms of tolerance adopted by the tumor. An immunosuppressive environment is established in PC patients as well as in the TRAMP mouse model, in which peripheral T cell tolerance to the tumor-associated antigen Tag is acquired. Mast cells (MCs) can mediate immunological tolerance and can foster PC development. Thus we investigated whether immunosuppression is part of their supporting adenocarcinoma outgrowth. We crossed TRAMP mice with KitWsh mice, lacking MCs. Tumor development was restrained in about 70% of KitWsh-TRAMP mice, showing at 30 weeks of age only intraepithelial neoplasia or local in situ adenocarcinoma. Conversely all MCs-sufficient age-matched TRAMP developed multifocal invasive carcinoma. Reduced tumor growth correlated with improved immune surveillance. Indeed, 16 weeks old KitWsh-TRAMP mice matched for the size of prostatic lesions and Tag expression, still mounted a Tagspecific CTL response, whereas their TRAMP counterparts, and KitWsh-TRAMP reconstituted with MCs, were tolerant. Inhibition of MCs degranulation in TRAMP mice had no effect on immune response while partially repressed tumor growth. This suggests that independent mechanisms may regulate local immunosuppression and tumor promotion by MCs, and hints that surface receptors are involved in MCsmediated T cell tolerance. POSTER W14 TAILORED CHEMOKINE RECEPTOR MODIFICATION FOR ADOPTIVE T CELL THERAPY IN SPONTANEOUS TUMOR MODEL A Stefano Garetto, Claudia Sardi, Roberta Angioni, Beatrice Claudia Cianciotti, Diego Morone, Elisa Martini, Giuliana Roselli, Anna Elisa Trovato, Davide Giuseppe Franchina, Cristiano Rumio and Marinos Kallikourdis Humanitas Clinical and Research Center Adoptive Cell Therapy(ACT) for tumors is becoming a promising therapeutic strategy. However, efficient homing of anti-tumoral T cells to the tumor/metastasis remains a substantial hurdle. The tumor site attracts both pro- and anti-tumoral immune cells through secretion of chemokines. We attempted to identify these chemokines in a spontaneous metastasis model, so as to “hijack” their function by expressing matching chemokine receptors on the T cells used in ACT, thus enabling selective enhancement of their recruitment. We show that this enabled the modified T cells to preferentially home into metastatic lymph nodes in the TRAMP spontaneous prostate tumor model. As a result of improved homing, the T cells led to a significant delay in tumor growth. These results offer a proof-of-principle for the tailored application of chemokine receptor modification to improve ACT efficacy. Surprisingly, we also uncover that the peri-tumoral capsule, which impedes T cell access to tumor, is partially dependent on host T cell presence for its formation. This highlights possible conflicting roles that T cells may play in tumor therapy; it also argues for therapeutic strategies that address not only the anti-tumoral activitity of the administered cells, but also how the treatment itself may affect the tumor microenvironment. 7 POSTER W15 A LIPOSOMAL MRNA VACCINE WITH STRONG IMMUNOSTIMULATORY PROPERTIES TARGETING LYMPHOID COMPARTMENTS FOR T CELL-MEDIATED CANCER IMMUNOTHERAPY Lena M. Kranz1,2, Mustafa Diken1, Kerstin C. Reuter2, Marc Holzmann1, Abderraouf Selmi1, Daniel Fritz3, Martin Meng3, Heinrich Haas3, Sebastian Kreiter1, Özlem Türeci4, Ugur Sahin1,2,3 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany 2 ; Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University, Mainz, Germany 3 ; BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany 4 ; Ganymed Pharmaceuticals AG, Mainz, Germany Utilizing antigen-encoding RNA for tumor immunotherapy has been proven to be a potent tool for inducing T cell-mediated antitumoral immunity in preclinical mouse models, and local application is currently being employed in clinical trials. In order to prime antigen-specific T cells on the systemic level, we developed a liposomal carrier system designed to preserve RNA integrity and physicochemically optimized for specific delivery to lymphoid compartment-resident APCs upon intravenous administration. In addition to selective translation by cDCs, pDCs and macrophages, immunization with RNA-lipoplexes led to profound proliferation of antigen-specific CD4+ and CD8+ T cells to exogenous as well as endogenous antigens. Importantly, RNA-lipoplexes conferred complete tumor protection and were able to eliminate progressing tumors in CT26 and B16-OVA models. Due to their intrinsic immunostimulatory properties, RNA-lipoplexes were also capable of inducing a strong but transient inflammatory milieu in the spleen affecting APCs and lymphocytes, which was dependent on high levels of IFNα produced by splenic pDCs and macrophages. Interestingly, T cells signaling displayed severely impaired effector functions and were not able to prevent tumor growth. These results demonstrate that antigen-encoding RNA-lipoplexes delivered intravenously and exhibiting immunostimulatory properties represent a novel, potent class of anti-cancer immunotherapeutics.primed in the absence of IFN. POSTER W16 CHRONIC RESTRAINT STRESS INDUCES INFLAMMATION IN THE SPLEEN AND PROSTATE OF C56BL7 MICE Li Ma, Heikki Rauvala, Li Tian. Neuroscience Center, University of Helsinki, Helsinki, Finland The brain connects to the somatic organs to regulate their immune response towards stress through two major trajectory arms: the hypothalamic-pituitary gland-adrenal gland (HPA) axis and the autonomic parasympathetic or sympathetic nervous system (SNS). Various kinds of stress have been shown to affect the HPA- and SNS-mediated humoral and cellular immune responses. Aim: To better understand related mechanism on inflammatory microenvironment by chronic stress. Methods: After chronic restraint stress (CRS) paradigm, body weights and body temperatures were measured, spleens and prostates were dissected for quantification of several genes by qPCR, and immune cells were analyzed by flow cytometry. Results: After 10-days CRS, there was a significantly increased body weight loss in CRS group mice, however, body temperature was no difference between control and CRS. Catecholamine-related enzyme Maob was downregulated by CRS. Inflammatory genes Cx3cl1 and Tnf in the spleen were decreased after CRS, however, neutrophils, macrophages and monocytes were no difference after CRS. Interestingly, both total and monocyte-derived macrophages were significantly increased in the prostate after CRS. Furthermore, inflammatory gene Ptgs1 was increased in the prostate after CRS. Conclusions: Chronic restraint stress induced dysfunctional immune response in the prostate, which could provide supportive inflammatory microenvironment for prostate cancer progression. 8 POSTER W17 HLA CLASS I AND KIR ASSOCIATIONS WITH RISK OF CLASSIC KAPOSI SARCOMA Maureen P. Martin, Y. Qi, X. Gao, D. Whitby, C. Lauria, F. Vitale, J. Goedert, M. Carrington Cancer and Inflammation Program, CCR, NCI, NIH and Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA Background: Kaposi sarcoma (KS) is a complication of KS-associated herpesvirus (KSHV) infection. Other oncogenic viral infections and malignancies are associated with certain HLA alleles and their natural killer (NK)-cell immunoglobulin-like receptor (KIR) ligands. We tested whether HLA-KIR influences the risk of KSHV or KS. Methods: In population-based case-control studies, we compared HLA class I and KIR gene frequencies in 250 classic (non-AIDS) KS cases, 280 KSHV-seropositive controls, and 576 KSHV-seronegative controls comprising discovery and validation cohorts. Logistic regression was used to calculate sex- and ageadjusted odds ratios and 95% confidence intervals. Results: In both the discovery and validation cohorts, KS was associated with HLA-A*11:01 (combined cohorts OR 0.4, P=0.002) and HLA-C*07:01 (OR 1.6, P=0.002). Consistent associations across cohorts were also observed with activating KIR3DS1 plus HLA-B Bw4-80I and homozygosity for HLA-C group 1. With KIR3DS1 plus HLA-B Bw4-80I, KSHV seroprevalence was 40% lower (combined cohorts, OR 0.6, P=0.01), but KS was 2-fold higher (OR 2.1, P=0.002). Similarly, KSHV seroprevalence was 40% lower (OR 0.6, P=0.01), but KS risk was 80% higher with HLA-C group 1 homozygosity (OR 1.8, P=0.005). Conclusions: KIR-mediated NK-cell activation may decrease KSHV infection, but enhance KSHV dissemination and progression to KS if infection occurs. Funded by NCI Contract No. HHSN261200800001E. POSTER W18 A NON-CANONICAL ADENOSINERGIC PATHWAY LED BY CD38 IN HUMAN MELANOMA CELLS INDUCES SUPPRESSION OF T CELL PROLIFERATION Fabio Morandi, Barbara Morandi, Alberto L. Horenstein, Antonella Chillemi, Valeria Quarona, Gianluca Zaccarello, Paolo Carrega, Guido Ferlazzo, Maria Cristina Mingari, Lorenzo Moretta, Vito Pistoia and Fabio Malavasi Laboratory of Oncology, "G. Gaslini" Scientific Institute, Largo G. Gaslini, 5 , 16147 Genoa – ITALY Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain and regulate extracellular nucleotide/nucleoside balance. Tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. Aim This work investigates ectoenzymes expression and function in primary human melanoma cells. Methods Ectoenzyme expression was evaluated by flow cytometry. T cell proliferation was evaluated by CFSE dilution. ADO production was assessed by HPLC. Intracellular signaling was analyzed using antibody arrays. Results Primary melanoma cells i) expressed CD38, CD39, CD73, and CD203a/PC-1, ii) produced ADO from AMP and NAD+ and iii) inhibited T cell proliferation through an ADO-dependent mechanism, since such inhibition was reverted using CD38/CD73 inhibitors.Melanoma cells inhibited effector memory, central memory and (only partially) naïve CD4+ T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory than in naïve CD4+ T lymphocytes. Melanoma cells also inhibited naïve, memory and (partially) effector CD8+ T cell proliferation. These differences correlated with distinct ADO receptor A2a and A2b expression patterns. Conclusions We demonstrated that primary human melanoma cells suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38/CD73 play a prominent role. 9 POSTER W19 IDENTIFICATION OF ANTIGENS RECOGNIZED BY AUTOANTIBODIES IN CHEMOTHERAPY-TREATED PANCREATIC CANCER PATIENTS Giorgia Mandili, Emanuela Mazza, Michela Capello, Paola Cappello, Daniele Giordano, and Francesco Novelli Department of Molecular Biotechnology and Healthy Sciences, Center for Experimental Research and Medical Science, AUO Città della Salute e della Scienza di Torino, Torino , Italy Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult cancer to treat, both for lack of effective screening method and for resistance phenomenon. However, chemotherapy is able to enhance tumor immunogenicity. Thus more immunogenic neo-antigens can be induced by chemotherapy and targeted by passive or active immunotherapy. To discover antigens that might be selected for immunotherapy, we have analysed antibody response in PDAC patients’ sera before and after chemotherapy. The reactivity of PDAC patients’ sera obtained before and after two rounds of chemotherapy on protein lysates from CF-PAC1 pancreatic cancer cell line was analysed by serological proteome analysis and mass spectrometry. A number of antigens, mainly metabolic enzymes, proteins involved in transcription and structural proteins, were recognized by autoantibody only after one or two rounds of chemotherapy. The identification of common and individual antigens from chemotherapy treated-patients formed the platform for ongoing immunological studies aimed to assess the ability of these antigens, compared to that identified before chemotherapy treatment, to induce specific helper and cytotoxic response to PDAC. A stronger and sustained immune response correlated to survival is expected to be induced by neo-antigens. We are validating these antigens to develop specific immunotherapy combined with chemotherapy. POSTER W20 A NOVEL LIPOSOMAL CLODRONATE DEPLETES TUMOR-ASSOCIATED MACROPHAGES IN PRIMARY AND METASTATIC MELANOMA: ANTIANGIOGENIC AND ANTITUMOR EFFECTS Piaggio F1$, Kondylis V2$, Pastorino Fabio1, Di Paolo D1, Perri P1, Cossu I1, Schorn F2, Marinaccio C3, Murgia D4, Daga A5, Loi M1#, Emionite L6, Ognio E6, Pasparakis M2, Ribatti D3,7, Ponzoni M1§, Brignole C1§*. 1Laboratory of Oncology, Istituto Giannina Gaslini, 16147,Genoa, Italy; 2Institute for Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in AgingAssociated Diseases (CECAD), University of Cologne, 50674, Cologne, Germany; 3Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, 70124, Bari, Italy; 4Department of Pathology, Istituto Giannina Gaslini, 16147,Genoa, Italy; 5Laboratorio di Trasferimento Genico, IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, 16132, Genoa, Italy; 6Animal Facility, IRCCS Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, 16132, Genoa, Italy; 7 National Cancer Institute "Giovanni Paolo II", 70124, Bari, Italy; #Present address: Department of Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, 8057 Zürich, Switzerland. Background. The depletion of tumor-associated macrophages (TAMs) is an appealing strategy in cancer therapy. Aim. The antitumor activity of novel Clodronate-containing liposomes (Clo-Lipo-DOTAP) was evaluated in murine melanoma models. Methods. Proof of functionality experiments of Clo-Lipo-DOTAP were conducted in vitro against the macrophage-like cell line RAW 264.7, and in vivo in a genetic mouse model of hepatocellular carcinoma (HCC). The antitumor effectiveness of Clo-Lipo-DOTAP was assayed in primary and metastatic melanoma models. Primary tumor volumes, number of pulmonary nodules, F4/80, αSMA, Ki-67 expression and plasma levels of macrophage-related cytokines and growth factors were determined. Results. Clo-Lipo-DOTAP inhibited proliferation, reduced viability and induced apoptosis of RAW 264.7 cells. In the HCC model, Clo-Lipo-DOTAP significantly reduced F4/80-positive cells in liver and spleen of treated mice. In B16/F10 subcutaneous melanoma-bearing mice, the reduction of F4/80-positive cells, mediated by Clo-Lipo-DOTAP, was accompanied by a decrease of microvessel density (MVD) leading to reduction of primary tumor volumes. Plasma levels of IL-10, Mo KC, TNF-α, VEGF and PDGF-bb were decreased. In B16/F10 metastatic melanoma model, Clo-Lipo-DOTAP decreased F4/80-positive cells and MVD resulting in reduction of pulmonary nodules. Conclusions. TAMs depletion in melanoma brings antitumor efficacy via inhibition of angiogenesis and modulation of inflammation related cytokines. 10 POSTER W21 REGULATORY T CELLS SHOWING PHENOTYPICAL, EPIGENETIC AND FUNCTIONAL FEATURES OF IMMUNE SUPPRESSION ARE ENRICHED IN HUMAN COLORECTAL CANCER E. Timperi1, I. Pacella1, V. Schinzari1, L. Sacco2, F. Farelli2, F. Longo3, A. Ciardi4, P. Chirletti2, V. Barnaba1,5, S. Piconese1,5 1Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy; 2Sezione di Chirurgia Interdisciplinare “F. Durante”, Sapienza Università di Roma, Rome, Italy; 3Dipartimento di Medicina Molecolare, Sapienza Università di Roma, Rome, Italy; 4Dipartimento di Scienze Radiologiche, Oncologiche e Anatomo-Patologiche, Sapienza Università di Roma, Rome, Italy; 5Istituto PasteurFondazione Cenci Bolognetti, Rome, Italy Contrary to most cancer types, in human colorectal cancer (CRC) Treg infiltration has been associated to better prognosis, leading to hypothesize that they do not crucially suppress anti-tumor immunity in this context. The goal of our study was to characterize Tregs at multiple levels (phenotypical, molecular and functional), from the tumor site (T), compared to normal mucosa (N) and peripheral blood (PB) of CRC patients. The frequency of FOXP3+CD127low/CD4+ Tregs was significantly higher in T. A differential compartmentalization was detected between Helios+ and Helios- Treg subsets (considered thymically- or peripherally-induced, respectively): while Helios- Tregs were enriched in both N and T (possibly as a result of mucosal Treg induction), only Helios+ Tregs accumulated in T, displayed highly demethylated TSDR and contained high proportions of cells expressing CD39 and OX40, markers of activation and suppression. Importantly, Treg depletion ex vivo rescued tumor-infiltrating T cell proliferation. However, Tregs may contribute to CRC progression not only by suppressing T cells but also by releasing IL-17, or by differentiating into Tfr cells potentially antagonizing a protective Tfh response, events that were both detected in T-Tregs. Our data indicate that Treg accumulation may contribute through multiple mechanisms to CRC establishment and progression. POSTER W22 THE P50 NF-ΚB SUBUNIT SHAPES INFLAMMATION ASSOCIATED WITH COLORECTAL CANCER DEVELOPMENT Chiara Porta, Alessandro Ippolito, Lorenzo Carraro, Francesca Maria Consonni, Giuseppe Celesti, Fabio Grizzi, Silvia Tartari, Fabio Pasqualini, Luigi Laghi, Antonio Sica Dep. of Pharmaceutical Sciences, University of Piemonte Orientale "A. Avogadro" Physiologic levels of inflammation are necessary for intestinal tissue homeostasis and immune tolerance, whereas excessive inflammation is deleterious and is at the basis of inflammatory bowel disease and inflammation-promoted colorectal cancer (CRC). Strikingly, the present study demonstrates that, in a murine model of colitis-associated CRC, p50 NF-κB promotes divergent clinical outcomes in colitis versus CRC. Whereas progression from colitis to cancer was associated with up-regulation of M2-related genes, ablation of p50 NF-κB exacerbated the colitis score and reduced CRC development. This latter event was associated with reduced number of tumor-associated macrophages together with increased number of NK, NKT, CD8+ T cells and apoptotic cancer cells. Colons from p50-/- tumor bearers expressed enhanced levels of M1/Th1 cytokines, including IL-12 and CXCL10, whose administration in vivo restrained colitis-associated CRC development. Finally, analysis of tumor tissues in a cohort of CRC patients indicated that high levels of M1/Th1 transcripts correlate with favorable clinical outcome, whereas high nuclear p50 in TAMs correlates with poor prognosis. Our study identifies p50 as key regulator of intestinal inflammation and provides first evidence that M1/Th1 cytokines may represent both prognostic indicators and immunotherapeutic agents in CRC. 11 POSTER W23 PRECLINICAL INVESTIGATION OF THERAPEUTIC STRATEGIES COMBINING TUMOR VASCULATURE REMODELLING AND GD2-CAR T CELL BASED IMMUNOTHERAPY FOR HUMAN NEUROBLASTOMA 1Paola 1Fabio Bocca, 2Michele Cilli, 2Laura Emionite, 3Ignazio Caruana, 3Biagio De Angelis, Morandi, 1Fabio Pastorino, 1Vito Pistoia, 1Ignazia Prigione 4Emma Di Carlo, 1Laboratory of Oncology, Istituto G.Gaslini, Genova, Italy; 2Animal Model Facility, IRCCS Azienda Ospedaliera Universitaria San Martino, IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 3 Laboratory of Tumour Immunotherapies, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy; 4 Anatomic Pathology and Molecular Medicine, Department of Medicine and Science of Aging, “G.d’Annunzio” University, Chieti, Italy. Immunotherapy represents a new therapeutic strategy for human neuroblastoma. Chimeric antigen receptor (CAR)-redirected T lymphocytes produce antitumor effects overcoming tumor escape mechanisms. However, adoptive transfer of CAR-T cells has shown less clinical efficacy in solid than in hematologic malignancies. Antiangiogenic agents by remodeling and normalization of tumor vasculature and microenvironment improve T cell penetration and persistence in tumor tissues . Aim. To investigate the activity of GD2-CAR T cells combined with anti-angiogenic therapy in GD2+ neuroblastoma preclinical models. Methods. Activated T cells were transduced with retroviral particles encoding for a third generation of CAR-GD2 expressing costimulatory molecules CD28 and OX40 and expanded with IL7/IL15. Scid/Beige mice were xenografted in the left adrenal gland with human GILIN luciferase-transduced neuroblastoma cells. Two weeks later, mice were iv treated with GD2-CAR- or Not Transfected-T cells(CTR) given alone or 48 hours after Bevacizumab (5mg/Kg, iv). Treatments were performed once a week for two weeks. For immunoistochemical studies tumors were explanted 48/72 hrs after treatments. Results. A significantly improved survival was observed in NB carrying mice receiving GD2-CAR T cells plus Bevacizumab, but not GD2-CAR Tcells alone, compared with controls. Conclusion. Anti-angiogenic agents can improve GD2-CAR T cell based immunotherapy of human neuroblastoma. POSTER W24 REGULATION OF THE TLR SIGNALING PATHWAY BY THE MIR-125A~99B~LET-7E CLUSTER AND MIR-146B Graziella Curtale a,b,1, Tiziana Ada Renzi a,b,2, Massimiliano Mirolo Luca a, Marzia Rossato c,4, Flavia Bazzoni c, Massimo Locati a,b a,b,3, Manuel Albanese a, Mariacristina De aDepartment of Medical Biotechnologies and Translational Medicine, University of Milan, Italy; bHumanitas Clinical and Research Center, Italy; cDepartment of Pathology, Division of General Pathology, University of Verona, Italy.Present address: 1Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; An appropriate immune response and maintenance of the immunological balance involve a number of negative regulatory mechanisms modulating Toll-like receptors (TLRs) activity. We identified miR125a~99b~let-7e cluster and miR-146b as microRNAs that, after LPS engagement on human monocytes, are induced by the anti-inflammatory IL-10 and TGFβ, but are inhibited by the pro-inflammatory IFNγ. Bioinformatics analysis predicted and experimental evidence demonstrated that miR-125a-5p, let-7e-5p and miR-146b directly target the TLR pathway at multiple levels, including receptors (TLR4, CD14), signaling molecules (IRAK1, MyD88, TRAF6), and effectors (TNFα, IL-6, CCL3, CCL7, CXCL8). Over-expression or inhibition of these microRNAs with lentiviral vectors had a significant impact on the production of proinflammatory cytokines in response to LPS. In particular, we identified a role for miR-125a-5p and miR-146b in mediating the LPS hyporensponsiveness observed after IL-10 or TGFβ priming or during the endotoxin tolerance, the phenomenon of reduced sensitivity to subsequent challenge of LPS. In an in vivo model of acute inflammatory response, we obtained that miR-125a-5p, miR-99b-5p and miR-146b were induced in macrophages recruited at the site of inflammation during the resolution process, and this was impaired in macrophages of IL-10 KO mice. Our studies indicated that microRNA cluster and miR-146b represent a new negative feedback mechanism of the TLR signaling pathway. 12 POSTER W25 ENHANCING TUMOR IMMUNOGENICITY USING NOD-LIKE RECEPTOR FAMILY CARD DOMAIN CONTAINING PROTEIN 5 (NLRC5) Galaxia M. Rodriguez,1 Diwakar Bobbala, 1 Marian Mayhue,1 Viktor Steimle,2 Thomas Kufer,3 Sheela Ramanathan,1 Subburaj Ilangumaran1 1 Immunology division, Department of Pediatrics, Faculty of Medicine and Health Sciences, University de Sherbrooke, Sherbrooke, Quebec, Canada. 2 Department of Biology, Faculty of Sciences, University de Sherbrooke, Sherbrooke, Quebec, Canada. 3 Institute for Medical Microbiology, Immunology and Hygiene, UniversityofCologne,Germany Background: Cancer cells often escape immune destruction by diminishing the expression of MHC-I and Ag processing machinery (APM). Reversal of these defects is considered a promising approach in cancer immunotherapy. Recent findings show that expression of MHC-I and APM is regulated by NLRC5. Aim: To investigate whether NLRC5 could be exploited to restore tumor immunogenicity. Methodology: We evaluated NLRC5 gene expression in cancer databases. We established B16-F10 melanoma cells expressing NLRC5 (B16-5), CD80 (B16-80) or both (B16-5/80), and evaluated their immunogenicity. Results: Cancer data mining showed that a large proportion of cancer cells express low or negligible NLRC5. Forced expression of NLRC5 in B16 cells induced MHC-I and APM genes, and efficiently presented the melanoma peptide gp10025-33 to naïve Pmel-1 TCR transgenic CD8+ T cells. However, B16-5/80 cells were more efficient in activating Pmel-1 cells without the addition of exogenous peptide. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 mice but not in Rag1-/- hosts, indicating elicitation of anti-tumor immune response. Accordingly, immunization of C57Bl/6 mice with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Conclusion: NLRC5 promotes antitumor immunity and can be exploited to stimulate protective anti-tumor immune response. POSTER W26 REDISTRIBUTION, HYPERPROLIFERATION, ACTIVATION OF NATURAL KILLER CELLS AND CD8 T CELLS, AND CYTOKINE PRODUCTION DURING FIRST-IN-HUMAN CLINICAL TRIAL OF RECOMBINANT HUMAN INTERLEUKIN-15 IN PATIENTS WITH CANCER Enrico Lugli, Kevin C. Conlon, Mario Roederer and Thomas A. Waldmann Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano (MI), Italy IL-15 has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. A first-in-man phase I trial of IL-15 administered i.v. for 12 consecutive days at 3.0, 1.0 and 0.3 μg/kg/day in patients with metastatic malignancy revealed dramatic efflux of NK and memory CD8 T cells within minutes of IL-15 administration, followed by influx and hyperproliferation yielding expansion of NK and memory CD8+ T cells. Hypoproliferation was observed after treatment, suggesting a peripheral compensatory mechanism to reestablish homeostasis. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities were grade 3 hypotension, thrombocytopenia, and elevations of ALT/AST, resulting in 0.3 μg/kg as the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. IL-15 could be safely administered to patients but alternative dosing strategies are required to reduce toxicity and increase efficacy. 13 POSTER TH01 IL-21-BASED COMBINATION STRATEGIES NEUROBLASTOMA IMMUNOTHERAPY TARGETING IMMUNE SUPPRESSION FOR Valentina Rigo1, Maria Valeria Corrias2, Laura Emionite1, Silvano Ferrini1, Michela Croce1*. 1IRCCS A. O. U. San Martino-IST, National Institute for Cancer Research, Largo R. Benzi 10, 16132, Genoa, Italy; 2IRCCS Gaslini Institute, L.go G. Gaslini 5, 16147, Genoa, Italy; *supported by Fondazione Italiana per la Lotta al Neuroblastoma. Background: CD4+ regulatory T cells are increased both in a syngeneic murine model and in human stage 4 neuroblastoma (NB) and may have a role NB-mediated immune suppression. IL-21 is a helper cytokine, which demonstrated anti-tumor activity in different tumor models. Aim: to study the cell populations and mechanisms involved in NB-mediated immune suppression and to specifically target these mechanisms in order to establish new IL-21-based combination strategies for NB immunotherapy (IT). Methods: rIL-21 therapy at 1 μg/dose was administered in combination with small molecules or antibodies that block Treg cell activity or immune regulatory pathways. Results: rIL-21 administration with a cell-depleting anti-CD4 mAb cured 70% of mice through a strong CTL response, and induced a long-lasting immunity. CD4+ T cells repopulating mice after IT were essential for long lasting immunity and showed reduced percentages of Treg cells, relative to NB-bearing mice. Targeting of CD39, a Treg-related enzyme, alone or in association with rIL-21 had only a limited impact on mice survival. Conclusion: Our data support a role for regulatory CD4+ T cells in NB. We are currently investigating the immune regulatory pathways involved in the search of new target for the development of combinational immunotherapeutic strategies for NB. POSTER TH02 RENAL TUMOR AND CANCER STEM CELLS EXPRESS A TRANSMEMBRANE IL-15 ISOFORM DISPLAYING DIFFERENT FUNCTIONS Cristina Romei, Rosaria Gangemi, Yanhong Gu, Silvano Ferrini, Julien Giron-Michel, Bruno Azzarone and Grazia Maria Spaggiari Department of Clinical and Experimental Immunology Istituto G. Gaslini, Genoa, Italy Intra-renal IL-15 participates in renal pathophysiology but the role of the membrane-bound isoforms remains to be elucidated. Herein, we show that primary “apparently normal” peritumoral (ptumTEC), tumoral (RCC) and renal cancer stem cells (CSC/CD105+) express a transmembrane IL-15 (tmb-IL-15) isoform of 25-27 kDa that delivers in response to its soluble receptor (sIL-15Ralpha) a reverse signal that favors, through the AKT pathway, the epithelial-mesenchymal transition (EMT) in ptumTEC and RCC but not in CSC/CD105+ cells. Indeed, in these cells it causes protection from the non-programmed cell death induced by serum starvation. Finally, tmb-IL-15 is sensitive to metalloproteases and the cleaved tmb-IL-15 (25kDa) displays a powerful anti-apoptotic effect. Overall, our data indicate that membrane bound and cleaved tmb-IL-15 isoforms play a complex role in renal tumor microenvironment acting on EMT process and cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, may contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favour tumour progression. 14 POSTER TH03 SPARC ACTIVATES TUMOR-MYELOID CELLS CROSS-COMMUNICATION TOWARD EMT IN MURINE AND HUMAN BREAST CANCERS Sangaletti Sabina1, Santangelo Alessandra1, Castioni Nadia1, Tripodo Claudio2, Chiodoni Claudia1, Orlandi Rosaria3, Tagliabue Elda3, and Mario Paolo Colombo1. 1Molecular Immunology Unit, Dept. Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori; 2Dept. of Human Pathology, University of Palermo; 3Molecular Targeting Unit Dept. Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori Epithelial-mesenchymal transition (EMT) has been associated with increased drug resistance. In addition to cell-intrinsic molecular events, myeloid cells present in the tumor stroma can contribute to EMT. The matricellular protein SPARC is a master stromal regulator with key roles in orchestrating fibrotic responses, and somewhat unexpected, in regulating the immune response. Extracellular matrix (ECM) gene expression profile of human breast carcinomas correlates SPARC expression with EMT, prognosis and response to therapy. The aim of this study was to create an experimental model suitable to test whether SPARC has a true role in resistance to therapy related to EMT and immune suppression. By using Sparc-deficient or –overexpressing mammary carcinoma cell lines we demonstrate a direct correlation between SPARC expression, resistance to chemotherapy and EMT feature. We have also provide evidence that myeloid cells are functional for EMT and that SPARC determines the phenotype of recruited myeloid cells by regulating COX2 activity and tilting the balance between GM-CSF and G-CSF. The role of myeloid cells recruited in a milieu rich in GM-CSF in chemo-resistance and EMT has been proven adding bisphosphonates or COX-2 inhibitors to chemotherapy. Indeed, their addition reverts EMT, myeloid cell phenotype and renders SPARC-producing tumors sensitive to chemotherapy. POSTER TH04 IL-33 RESTRICTS TUMOR GROWTH AND INHIBITS PULMONARY METASTASIS IN MELANOMA BEARING MICE THROUGH ACTIVE RECRUITMENT OF EOSINOPHILS IN THE LUNG Valeria Lucarini, Valentina La Sorsa, Iole Macchia, Francesca Peschiaroli, Giovanna Ziccheddu, Carla Buccione, Antonella Sistigu, Fabrizio Mattei, Claudia Afferni and Giovanna Schiavoni Istituto Superiore di Sanità, Rome The alarmin IL-33 is an IL-1 family-member that exerts pleiotropic activities through binding to its specific receptor ST2. Originally described as an inducer of Th2-type immunity in allergy, asthma and parasitic infections, IL-33 is also implied in Th1-type of immune reactions. We have investigated the role of IL-33/ST2 axis in anti-tumor response to melanoma. Injection of IL-33 in mice bearing subcutaneous B16.F10 melanoma resulted in significant tumor growth delay. This effect was associated with intratumoral accumulation of CD8 T cells and eosinophils, decrease of myeloid cell populations, and systemic activation of CD8 T and NK cells. Intranasal administration of IL-33 determined eosinophil recruitment and a mixed Th1/Th2 cytokine expression pattern in the lung that prevented the onset of pulmonary metastasis after intravenous injection of melanoma cells. This effect was ST2-dependent, since ST2-deficient mice failed to respond to IL-33 and developed pulmonary metastasis at even higher extent than wild-type counterparts. Of note, depletion of eosinophils by in vivo treatment with anti-Siglec-F antibody abolished the ability of IL-33 to condition the pulmonary microenvironment resulting in melanoma metastasis formation. Our results underscore a novel function of IL-33 as an anti-tumoral effector against melanoma and an active role of eosinophils in contrasting pulmonary metastasis. 15 POSTER TH05 TARGETING DYSFUNCTIONAL MYELOID CELLS DELAYS DISEASE DEVELOPMENT AND IMPROVES IMMUNE FUNCTION IN A MOUSE MODEL FOR CHRONIC LYMPHOCYTIC LEUKEMIA Bola Hanna, Fabienne McClanahan, Alexander Egle, John Gribben, Peter Lichter, Martina Seiffert German Cancer Research Center, Molecular Genetics Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is stringently associated with a tumorsupportive microenvironment and defective anti-tumor immunity. T cells from CLL patients show features of exhaustion, including expression of PD-1, and are highly impaired in immune synapse formation, which is mediated by aberrant expression of several inhibitory receptors like PD-L1 on CLL cells. Our previous work showed that immune checkpoint blockade using anti-PD-L1 effectively prevents disease and restores T-cell activity in a CLL mouse model. To investigate the role of myeloid cells in the CLL microenvironment, we analyzed their composition and function in the Eµ-TCL1 mouse model and observed a severe skewing of monocytes and macrophages along with CLL development. This included an accumulation of M2-like macrophages and Ly6Clow patrolling monocytes and a drop of MHC-IIhigh dendritic cells. Associated with that, several inflammatory serum factors like IL-10, TNFα and CXCL9 were upregulated in leukemic mice. Myeloid cell depletion using liposomal clodronate resulted in significant control of CLL development, repair of innate and adaptive immune cell phenotypes and partial resolution of systemic inflammation. As we observed aberrantly high PD-L1 expression on CLL-associated monocytes and dendritic cells, their contribution to defective T-cell responses and treatment success of PD-L1 blockade seems very likely. Therefore, targeting myeloid cell survival and immunosuppressive activity can serve as a novel strategy for CLL immunotherapy. POSTER TH06 BONE MARROW RESPONSE TO CANCER ONSET Andrea Tomirotti1, Claudia Chiodoni1, Matteo Dugo2, Claudio Tripodo3, Mario Paolo Colombo1 1Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2Functional Genomics and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 3Department of Human Pathology, University of Palermo, Italy Cancer is often associated with the expansion and recruitment of BM-derived immune cells harboring immunosuppressive functions. We hypothesized that relevant modifications in the BM hematopoietic environment functional to the establishment of a tumor-adapted hematopoiesis can be induced by peripheral tissue perturbation at time of cancer onset. We investigated stromal and hematopoietic modifications in the BM of tumor-bearing mice by flow cytometry, in situ histopathology and immunohistochemistry, and gene expression profiling (GEP) at the time of tumor initiation. To challenge the hypothesis that BM perceives peripheral cancerogenesis, we first analyzed the changes occurring in the BM hematopoietic and stromal environment in BALB/NeuT transgenic mice (developing spontaneous mammary tumors) at 24 weeks of age, when invasive tumors are present. At this stage of cancer progression, the hematopoietic marrow showed an expansion of myeloid cells (CD11b + and CD11b+Gr1high cells) and a contraction of B cells (B220+) in comparison to wild type mice. The analysis of precursor cells in NeuT mice, showing higher rate of GMP (granulocyte-monocyte progenitors) in comparison to controls, confirmed a boost in myelopoiesis. These modifications were paralleled by the remodeling of the Nestin+ mesenchymal BM cells, which was associated with redistribution of CXCL-12/CXCR4 gradients. On the same BM samples GEP has been performed and results showed 208 genes significantly modulated at FDR < 0.05 and FC 1.5. Gene Ontology showed a down-modulation of genes related to immune response and an up-regulation of genes involved in inflammation and response to danger signals. We are now testing whether the same quality of modifications in hematopoietic and stromal components can be observed, though less magnified, in the BM of BALB/NeuT transgenic mice at early age age, a time point corresponding to in situ stage. Taking together the results demonstrate profound alterations in BM environment of tumor-bearing mice that confirm our hypothesis suggesting of searching the molecules that sending information to the BM might be of values as potential biomarkers in cancer development. 16 POSTER TH07 AUTOPHAGY IMPAIRMENT CAUSES DYSREGULATED LIPID HOMEOSTASIS AND INFLAMMATION IN HEPATITIS C VIRUS INFECTION Tiziana Vescovo, Marco Corazzari, Fabiola Ciccosanti, Tonino Alonzi, Mauro Piacentini and Gian Maria Fimia National Institute for Infectious Diseases IRCCS ‘L. Spallanzani’, Rome, Italy Chronic infection with Hepatitis C Virus (HCV) is one of the main leading cause of hepatocellular carcinoma (HCC). HCV-induced HCC develops in an environment of lipid accumulation and persistent inflammation, defined as steatohepatitis. Autophagy has been shown to play an important role in preventing either lipid deposits or excessive inflammation. We have recently demonstrated that HCV infection triggers a lipid-selective type of autophagy that counteracts the viral-induced accumulation of cholesterol. Notably, we also found that decreased autophagy levels correlate with the presence of microsteatosis in HCV patients. Being cholesterol a potent inducer of inflammation, we are now investigating whether autophagy impairment and cholesterol accumulation leads to increased inflammation in vitro. We observed that in HCV replicon cells the downregulation of the expression of the autophagic gene Beclin1 leads to an increased production of inflammatory citokines, such as IL1b, IL6 and TNF. We are currently characterizing if and how cholesterol accumulation is responsible for the upregulation of the inflammation state in HCV-infected cells. Altogether, our data suggest that autophagy impairment may contribute to establish chronic inflammation in the liver of HCV patients, which is an important risk factor for the development of HCC. POSTER TH08 IL-1β-RELEASING HUMAN AML BLASTS AFFECT NK/ILC3 CELL RECOVERY AND DIFFERENTIATION Chiara Vitale1,2, Paolo Ambrosini2, Romana Conte1, Filippo Ballerini Mingari1,2 1,2, Lorenzo Moretta3, and Maria Cristina 1IRCCS AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 2Dipartimento Medicina Sperimentale (DIMES) Università degli Studi di Genova, Italy; 3IRCCS Giannina Gaslini, Genova, Italy. In the haploidentical-hematopoietic stem cell transplantation setting, CD34+ donor-derived NK cells play a major role in the control of leukemic relapses. Thus, it is important to clarify whether leukemic blasts could interfere with NK cell differentiation. To analyze this issue, UCB-derived CD34+ cells were cultured in the absence or in the presence of fresh AML blasts. We show that a group of AML markedly inhibited the NK cell recovery, limiting NK precursors proliferation while accelerating their maturation towards NK cells. This effect correlated with IL-1β by leukemia cells. In vitro studies performed with recombinant IL-1β showed that IL-1β inhibited CD161+CD56+ cell proliferation, but induced the expression of LFA-1, CD94/NKG2A, KIRs, Perforin, i.e. typical markers of mature NK cells. In addition, within the CD161+CD56+IL-1RI+LFA-1- cell fraction (representing ILC3-like cells), an increase of EOMES, NKp46 and CD94/NKG2A receptors, cytolytic granules and IFNγ was detected. This increase was paralleled by a decrease of related orphan receptors (RORγt) TF and IL-22 production. Our results suggest that IL-1 β inhibits ILC3 while favoring NK cell maturation. Since transplant-associated conditioning regimen or residual leukemia cells may induce IL-1 β production, this may influence the NK/ILC3 development from donor-derived CD34+ precursors and impact on NK cell-mediated Graft versus Leukemia. 17 POSTER TH09 MECHANISMS OF GENERATION OF LONG-LIVED T MEMORY STEM CELLS DURING LYMPHOPENIA Alessandra Roberto*, Veronica Zanon, Karolina Pilipow, Luca Castagna, Stefania Bramanti, Roberto Crocchiolo, Paolo Tentorio, Armando Santoro, Emma Gostick, Kristin Ladell, James McLaren, David A. Price, Mario Roederer, Domenico Mavilio and Enrico Lugli Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy *EFIS FELLOWSHIP AWARDEE Recent evidence indicated that T memory stem cells (T SCM), the earliest differentiated memory T cell subset in humans, display superior persistence in vivo as well as effector functions; however, their mechanisms of generation in vivo remain elusive. In the context of T-replete haploidentical bone marrow transplantation, we show that TSCM cells of donor origin dominate the T cell compartment of lymphopenic recipients early after T cell transfer and precede the expansion of effector cells. Importantly, transferred naïve T cells (T N), but not TSCM or memory cells (TMEM), preferentially survive post-transplant cyclophosphamide, used as GVHD prophylaxis, thus suggesting that post-transplant TSCM originate from TN cells. In this regard, TN-derived TSCM cells specific for exogenous and self/tumor antigens contribute to peripheral reconstitution of lymphopenic patients by differentiating into effector T cells. Likewise, pathogen-specific TMEM cells generate detectable recall responses, but only in the presence of the cognate antigen. The genome-wide expression analysis on sorted TN, TSCM and TMEM cells under different stimulatory conditions revealed gene products specifically expressed by self-renewing TSCM cells that are shared with somatic stem cells. These genes are currently being used to induce stem cell-like properties in tumor-specific T cells to be used in adoptive transfer experiments. POSTER TH10 PTX3 IS AN EXTRINSIC INFLAMMATION IN CANCER ONCOSUPPRESSOR REGULATING COMPLEMENT DEPENDENT Euardo Bonavita1, E. Magrini1, S. Gentile1, M. Rubino1, V. Maina1, R. Papait1, 2, P. Kunderfranco1, C. Greco1, F. Feruglio1, M. Molgora1, I. Laface1, S. Tartari1, A. Doni1, F. Pasqualini1, E. Barbati1, G. Basso1, M. R. Galdiero1, M. Nebuloni3, M. Roncalli1, P. Colombo1, L. Laghi1, J. D. Lambris4, S. Jaillon1, C. Garlanda1, A. Mantovani1,5 1Humanitas Clinical and Research Center, Rozzano (Milan), 20089, Italy; 2Institute of Genetics and Biomedical Research, National Research Council, Rozzano (Milan), 20089, Italy; 3Pathology Unit, L. Sacco Department of Clinical Sciences, University of Milan, Milan, 20157, Italy; 4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; 5Humanitas University, Rozzano (Milan), 20089, Italy. *EFIS FELLOWSHIP AWARDEE Inflammation is a key component of tumor microenvironment. However, no dominant or suppressor oncogene encodes for an effector molecule of the humoral arm of the innate immune system. The long pentraxin PTX3 is a prime member of the humoral branch of innate immunity playing non-redundant roles in resistance against selected microbes and in the regulation of inflammation. The deficiency of PTX3 was associated with increased susceptibility to carcinogenesis. Higher tumor incidence in Ptx3-/- mice was associated with enhanced macrophage infiltration, cytokine production, Trp53 mutations and DDR activation. Gene targeted mice and pharmacological blocking experiments demonstrated that PTX3deficiency resulted in amplification of Complement activation, CCL2 production and tumor-promoting macrophage (TAM) recruitment. Treatment with recombinant PTX3 significantly reduced tumor incidence, TAM infiltration and C3 deposition. In an effort to translate preclinical evidence to human, we analysed the epigenetic modifications of PTX3 gene and observed that the expression of PTX3 was epigenetically silenced in selected human tumors (e.g. leiomyosarcomas and colorectal cancer) by methylation of the promoter region and of a putative enhancer. Thus, PTX3 acts as an extrinsic oncosuppressor gene in mouse and man by regulating Complement-dependent, macrophage sustained tumor-promoting inflammation. 18 POSTER TH11 TUMOR INFILTRATING (TINKS) AND TUMOR ASSOCIATED (TANKS) NATURAL KILLER CELLS (TINKS): A NEW HALLMARK IN COLORECTAL CANCER 1, 2Antonino Cassinotti, Bruno*, 2Barbara Bassani, 1Silvia Zanellato, 2Davide G. D’Urso, Boni, 3Lorenzo Dominioni, 2Adriana Albini, 1, 2Douglas M. Noonan 1Elisabetta Gini 3Elisa 3Luigi 1Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; and Technology Pole (PST), IRCCS MultiMedica, Milano, Italy; 3Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy *EFIS FELLOWSHIP AWARDEE 2Science Natural Killer (NK) cells are effectors lymphocytes of innate immunity primarily involved in immunosurveillance against tumors through their cytotoxic activity. We reported that NKs from Non Small Cell Lung Cancer (NSCLC) show a decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNlow phenotype. Aim: Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NKs undergo an angiogenic-switch in colorectal cancer (CRC). Methods: NK subset distribution and cytokine profiling were performed by flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used for secretomic profiling using antibody membrane arrays or functional angiogenesis assays on human umbilical endothelial vein cells (HUVECs). Results: We found that CD56brightCD16- NK cells predominate in CRC tumor and adjacent tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Secretomic analysisis on CRC peripheral blood NKs revealed up regulation of several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Finally, CM from CRC peripheral blood and tumor tissue FACS sorted NKs induced HUVEC proliferation, migration and morphogenesis. Conclusions: Taken together, our data show that TINK/TANK phenotype/function has implications in the immune response against tumors, placing NKs as a new player in the orchestration of tumor development and progression. POSTER TH12 ABATACEPT INHIBITS RECALL ANTIGEN PROLIFERATION AND TNF-Α PRODUCTION IN T HELPER LYMPHOCYTES FROM JUVENILE IDIOPATHIC ARTHRITIS PATIENTS Manuela Capone1*, L. Maggi1, R. Cimaz2, V. Santarlasci1, M. Rossi1, A. Mazzoni1, G. Montaini1, F. Liotta1, E. Maggi1, G. Simonini2, F. Annunziato1, L. Cosmi1; 1Department of Experimental and Clinical Medicine and DENOTHE Center, University of Florence, Florence, Italy, 2Anna Meyer Children's Hospital and University of Florence, Florence, Italy. *EFIS FELLOWSHIP AWARDEE Objective: to evaluate the impact of abatacept in terms of both therapeutic efficacy and interference with T helper functions in juvenile idiopathic arthritis (JIA) patients. Methods: the ability of abatacept to in vitro modulate the cytokines production profile and the proliferative response to both recall antigens and polyclonal stimulation, was firstly assessed on peripheral blood mononuclear cells (PBMC) of healthy donors. Then, 10 JIA patients who were going to start abatacept treatment, have been recruited and longitudinally evaluated during the first 90 days of therapy for clinical response and other immunological parameters. Results: abatacept in vitro was able to reduce the proliferative response to recall antigens and the antigendriven production of TNF-α in healthy donors. Abatacept was efficient in improving symptoms and in reducing parameters of inflammation in JIA patients. The long-term effects, in terms of T cell subsets, consist in a reduction of the frequencies of circulating Treg cells. Finally, we observed a reduction of the proliferative response to recall antigens, and of TNF-α production in PBMC derived from abatacept-treated JIA patients, soon after (two days) drug infusion. Conclusions: abatacept in vitro inhibits TNF-α production and recall antigens proliferation in healthy donors, and in vivo reduces parameters of inflammation in JIA patients. The reduction of both the proliferative response to recall antigens and of TNF-α production in PBMC derived from abatacept-treated JIA patients, was evident only soon after drug administration. 19 POSTER TH13 NCR+ILC3 CONCENTRATE IN HUMAN LUNG CANCER AND ASSOCIATE WITH INTRATUMORAL LYMPHOID STRUCTURES Paolo Carrega1*, Fabrizio Loiacono1, Emma Di Carlo2,3, Angelo Scaramuccia4, Romana Conte4, Barbara Morandi5, Maria Cristina Mingari4,5, Lorenzo Moretta1 and Guido Ferlazzo6,7 1Istituto G. Gaslini, Genova - 16148, Italy; 2Department of Medicine and Sciences of Aging, "G. d'Annunzio" University, Chieti - 66013, Italy; 3Centro di Scienze dell’Invecchiamento, Fondazione Universita` “G. d’Annunzio,” Chieti - 66013, Italy; 4Istituto di Ricovero e Cura a Carattere Scientifico, Azienda Ospedaliera Universitaria San Martino/IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa - 16132, Italy; 5Department of Experimental Medicine (DIMES) and Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genoa - 16132, Italy; 6Laboratory of Immunology and Biotherapy, Department of Human Pathology, University of Messina, Messina - 98125, Italy; 7Cell Therapy Program, Azienda Ospedaliera Universitaria Policlinico “Gaetano Martino”, Messina - 98125, Italy *EFIS FELLOWSHIP AWARDEE Tertiary lymphoid structures (TLS) are a common finding in non-small-cell lung cancer (NSCLC) and are predictors of favorable clinical outcome. Here we show that NCR+ Innate Lymphoid Cells (ILC)3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumor-associated TLS. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue inducing properties and, upon activation, produces IL-22, TNF, IL-8, IL-2 and activates endothelial cells. Tumor-NCR+ILC3 may interact with both lung tumor cells and tumor-associated fibroblasts, resulting in the release of cytokines primarily upon engagement of the NKp44 activating receptor. In patients, NCR+ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumor stages and their presence correlate with the density of intratumoral TLS. Our results indicate that NCR+ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumor-associated TLS. POSTER TH14 B CELLS MODULATE THE ANTITUMOR IMMUNE RESPONSE ACCORDING TO THEIR SPATIAL DISTRIBUTION WITHIN THE MICROENVIRONMENT OF PANCREATIC ADENOCARCINOMA Giovanni Francesco Castino*, Nina Cortese, Giovanni Capretti, Simone Serio, Giuseppe Di Caro, Rossana Mineri, Elena Magrini, Fabio Grizzi, Paola Cappello, Francesco Novelli, Cristina Ridolfi, Francesca Gavazzi, Alessandro Zerbi, Paola Allavena, Federica Marchesi. Humanitas Clinical and Research Center, Rozzano (Milan), 20089, Italy *EFIS FELLOWSHIP AWARDEE Recent studies revealed a positive effect of PDAC stromal reaction to immunotherapeutic strategies. A better definition of PDAC-associated immune contexture would help identifying patients more likely to benefit from immunotherapeutic approaches. In the effort of investigating the spontaneous immune response in human PDAC, we have focused on B cells and analyzed their clinical relevance, in relation to their spatial organization in the tumor microenvironment. B cell relevance has been evaluated in a retrospective study involving 104 PDAC patients, by correlating computer-quantified immunohistochemical stainings to clinical outcomes. The role of B cells has been further investigated in PDAC preclinical models. B cells distributed in the PDAC stromal region either as scatter tumor infiltrating B cells (B-TILs), or within organized tertiary lymphoid structures (B-TLS). Low density of B-TILs and high density of B-TLS associated to longer survival. Notably, the occurrence of B-TLS correlated with higher infiltration of CD8+ T cells and germinal center-related signature. Accordingly, in PDAC preclinical models, B-TILs selective targeting of by anti-CD20 antibody treatment increased the expression of genes related to infiltration of cytotoxic cells. B cells predict PDAC patient survival according to their distribution in the microenvironment, and their selective targeting could support an effective antitumor immune response. 20 POSTER TH15 REMODELING OF LAMINA PROPRIA IN THE UNINVOLVED HUMAN RECTAL MUCOSA 10 CM AND 20 CM AWAY FROM THE MALIGNANT TUMOR Sanja Z. Despotović1*, Lalić IM1, Milićević NM1, Milićević Ž1 1 Institute of Histology and Embryology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia *EFIS FELLOWSHIP AWARDEE We demonstrated the structural alterations (reduced cellularity and tissue edema accompanied by disorganized extracellular matrix) of the lamina propria of the mucosa 10 cm and 20 cm away from the rectal adenocarcinoma. The aim of this study was to investigate and quantify the changes of reticular fibers in the lamina propria of the uninvolved human rectal mucosa 10 cm and 20 cm away from the malignant tumor. Morphometric study of rectal mucosa was performed in samples taken during endoscopic examination 10 cm and 20 cm away from the malignant tumor of 15 patents and those obtained from 6 healthy controls. Tissue sections were stained with Gomori's silver impregnation technique. The density of reticular fibers in the lamina propria was determined using Color Picker Threshold plugin, Icy. Measurements of the spaces between reticular fibers were performed using plugin BoneJ, Fiji. The density of reticular fibers was significantly lower and the diameter of spaces between the reticular fibers was significantly increased 10 cm away from the adenocarcinoma, compared with both healthy controls and samples taken 20 cm away from the tumor. Tumor induces changes of connective tissue in the uninvolved lamina propria of rectal mucosa 10 cm away from the malignant lesion. POSTER TH16 CX3CR1 AND HEMEOXYGENASE: A PROTECTIVE LINK AGAINST CANCER DEVELOPMENT? Giulia Marelli 1 1,2* 1 1 1,2 1 , Erreni M. , Belgiovine C. , Mantovani M. and Allavena P. , Clinical and Research Institute Humanitas, Milano, Italy; Translational Medicine, University of Milano, Italy. 2 Department of Medical Biotechnology and *EFIS FELLOWSHIP AWARDEE + CX3CR1 cells in the gut are considered resident macrophages able to maintain homeostasis by the production of IL-10. However, their role in colon carcinogenesis is unknown. GFP/GFP We used mice lacking the functional receptor (CX3CR1 ) and we compared them with wild type mice. Using the AOM/DSS model of colitis-induced carcinogenesis, we found significantly higher signs of inflammation in KO-mice in term of both cytokine production and immune cells infiltration as well as a higher score of tissue damage and number of polyps. However, KO-mice try to switch off inflammation; indeed, the levels of IL-10 family-cytokines are over expressed in comparison to WT-mice. We found that only heme-oxygenase –an anti-inflammatory enzyme- was under-expressed in KO-mice. In vitro studies demonstrated that the CX3CL1-CX3CR1 axis is able to induce the production of hemeoxygenase in + CX3CR1 macrophages while in vivo experiments revealed that treatment with coPP, a drug stimulating the production of heme-oxygenase, is able to revert the phenotype in KO-mice, resulting in lower inflammation and reduced tumor load. Taken together our results show that the production of heme-oxygenase is under the control of the CX3CL1-CX3CR1 axis. These experiments unveiled a previously unidentified role of the chemokine receptor CX3CR1 in the regulation of colon inflammation. 21 POSTER TH17 POTENTIATION OF NK CELLS ANTI-TUMORAL POTENTIAL BY COMPLEX BIDIRECTIONAL INTERACTIONS WITH POLARIZED MACROPHAGES Mattiola* ¥†‡, Matthieu Pesant¥, Paolo Tentorio†, Martina Marcenaro§, Enrico Lugli†, Massimo Locati¥‡ and Domenico Mavilio †‡ Irene Molgora¥‡, Emanuela ¥Leukocyte Biology Unit, Humanitas Clinical and Research Center, via Manzoni 113, I-20089 Rozzano, Milan, Italy; † Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, via Manzoni 113, I-20089 Rozzano, Milan, Italy; ‡ Department of Medical Biotechnologies and Translational Medicine, University of Milan, via Manzoni 113, I-20089 Rozzano, Milan, Italy; § Dipartimento di Medicina Sperimentale and Centro di Eccellenza per le Ricerche Biomediche, Università degli Studi di Genova, via Alberti 2, I-16132, Genova Italy. *EFIS FELLOWSHIP AWARDEE BACKGROUND: NK cells have anti-tumoral properties and participate to tumor editing. Macrophages acquire different polarization states ranging from classic (M1) to alternative (M2) activation, and in the tumor microenvironment show an M2-like pro-tumoral phenotype. AIM: We have investigated the reciprocal functional influence of these two key cell types with respect to their potential impact on tumor biology. METHODS: Autologous human NK cells and monocyte-derived macrophages were obtained from healthy donors and their cross-talk has been investigated in vitro. RESULTS: While M2 had no effect on NK cell functions, M1 increased NK cell cytotoxicity (via IL-23 and IFN-β-dependent up-regulation of NKG2D, IL-1β-dependent up-regulation of NKp44, trans-presentation of IL-15), and triggered NK cell production of IFN-γ (via induction of IFN-β- dependent cis-presentation of IL15 on NK cells and 2B4 engagement). In turn, NK cells activated by M1 macrophages repolarized M2 macrophages (down-regulation of CD206, ALOX-15, CCL18, CCL22; up-regulation of CD80, CD48, IL-1β, IL-15 IL-15Rα). CONCLUSIONS: A complex network of soluble mediators and cell-to-cell interactions allows M1 macrophages to support NK cells cytotoxic activity and conversely promotes NK cells ability to “reeducate” M2 macrophages. These results shed light on an intercellular network with the potential to interfere with the pro-tumoral microenvironment sustained by M2 macrophages. POSTER TH118 + ANALYSIS OF MEMORY LIKE NK CELLS IN HCMV-INFECTED CHILDREN UNDERGOING Α Β T- AND B-CELL DEPLETED HSCT FOR HEMATOLOGICAL MALIGNANCIES 1 2 3 4 4 Letizia Muccio* , Alice Bertaina , Michela Falco , Daniela Pende , Raffaella Meazza , Miguel Lopez5 2,6 1 1 2 Botet , Lorenzo Moretta , Franco Locatelli , Alessandro Moretta * and Mariella Della Chiesa 1 Dipartimento di Medicina Sperimentale and Centro di Eccellenza per la Ricerca Biomedica, 2 3 Università di Genova, Genova, Italy; IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy; IRCCS 4 Istituto Giannina Gaslini, Genova, Italy; Istituto di Ricovero e Cura a Carattere Scientifico, Azienda Ospedaliera Universitaria San Martino-Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 5 Universitat Pompeu Fabra and Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain; 6 Dipartimento di Scienze Pediatriche, Università di Pavia, Pavia, Italy *EFIS FELLOWSHIP AWARDEE We analyzed the impact of human cytomegalovirus (HCMV) infection on the development of NK cells in 27 pediatric patients, affected by hematological malignancies, who had received a HLA-haploidentical HSCT, + depleted of both α/β T cells and B cells. In line with previous studies in adult recipients of umbilical cord 22 blood transplantation (UCBT), we found that HCMV reactivation accelerated the generation of mature NK cells. Thus, most children displayed a progressive expansion of a memory-like NK cell subset expressing NKG2C, a putative receptor for HCMV, and CD57, a marker of terminal NK differentiation. + + NKG2C CD57 NK cells were detectable by month 3 after HSCT and expanded at least until month 12. These cells were characterized by high KIRs and LIR-1 and low Siglec-7, NKG2A and IL-18Rα expression, killed tumor targets and responded to cells expressing HLA-E (a NKG2C ligand). In addition, they were poor IFN-γ producers in response to IL12 and IL18. The impaired response to these cytokines, together with their highly differentiated profile, may reflect their skewing toward an adaptive condition specialized in controlling HCMV. In conclusion, also in pediatric patients receiving a source of HSC different from UCB, HCMV induced memory-like NK cells possibly controlling infections and reinforcing antileukemia effects. POSTER TH19 B7-H6-MEDIATED DOWNREGULATION OF NKP30 IN NK CELLS CONTRIBUTES TO OVARIAN CARCINOMA IMMUNE ESCAPE 1 2 1,3 2 2 Pesce Silvia* , Tabellini Giovanna , Cantoni Claudia , Patrizi Ornella , Coltrini Daniela , Rampinelli 4 5 5 1 2 1 Fabio , Matta Jessica , Vivier Eric , Moretta Alessandro , Parolini Silvia , Marcenaro Emanuela 1 Dipartimento di Medicina Sperimentale and Centro di Eccellenza per le Ricerche Biomediche, Università 2 degli Studi di Genova, Genova, Italy; Dipartimento di Medicina Molecolare e Traslazionale, Brescia Italy; 3 4 Istituto Giannina Gaslini, Genova, Italy; Dipartimento di Ostetricia e Ginecologia, Spedali Civili di Brescia, 5 Brescia, Italy; Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Marseille, France and Service d'Immunologie, Assistance Publique-Hôpitaux de Marseille, Hôpital de la Conception, Marseille, France *EFIS FELLOWSHIP AWARDEE Background: Papillary serous ovarian carcinoma is the gynecological cancer with the highest mortality rate, therefore it’s important to develop innovative diagnostic/therapeutic approaches. A promising field of research is to identify molecules expressed by tumor cells, as well as to improve anti-tumor immune responses, designing therapeutic protocols based on the use of molecules and/or cells of innate immunity, such as NK cells. Aim: In this study, we analyzed the molecular mechanisms that may contribute to suppress the NK cellmediated responses in neoplastic microenvironment in papillary serous ovarian carcinoma patients. We focused our attention to the interaction between the activating NK receptor NKp30 and its ligand, B7-H6. Matherials and Methods: The study will be conducted in a cohort of patients with papillary serous ovarian carcinoma. Biological material used: ovarian carcinoma cells isolated from ascitic fluid, NK cells isolated from peripheral blood (PB-NK) or from ascitic fluid (PF-NK), serum, ascitic fluid. NK cells will be characterized phenotypically by flow cytometry techniques, and functionally by cytotoxic/degranulation assays. Results: Our data indicate that in a fraction of these patients the expression of the NKp30 activating receptor is substantially reduced in PF-NK cells as compared to PB-NK cells. The impaired expression of this receptor was associated with the presence of its ligand B7-H6, which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the PF. Patients expressing low levels of NKp30 displayed a compromised NK-mediated anti-tumor cytolytic activity and low IFNgamma production in response to B7-H6+ target cells. Discussion: Our results suggest that chronic receptor-ligand interaction may cause loss of NKp30 expression on NK cells in the tumor microenvironment, thereby contributing to poor NK cell-mediated elimination of ovarian carcinoma cells. This mechanism represents a novel way by which the tumor microenvironment may contribute to the escape from NK-mediated immune surveillance. 23 POSTER TH20 HYPOXIA REPROGRAMS HUMAN MACROPHAGES TOWARDS A PROINFLAMMATORY DIRECTION 1 1 2 2 1 Federica Raggi* ; Simone Pelassa ; Daniele Pierobon ; Mirella Giovarelli ; Luigi Varesio ; Maria 1 Carla Bosco 2 Istituto G.Gaslini, Genova, ITA; Centro Ricerche in Medicina Sperimentale (CERMS), Torino, ITA *EFIS FELLOWSHIP AWARDEE 1 Causal relationship between chronic inflammation and tumorigenesis was reported. Tumorassociated macrophages are major components of the inflammatory circuit that promotes tumor progression. They differentiate from circulating monocytes recruited to tumor sites, where they can be polarized into M1 or M2 subsets, respectively expressing a proinflammatory or an anti- inflammatory phenotype. M1 and M2 polarization is regulated by microenvironment factors. An important signal generated in the tumor microenvironment is hypoxia. Aim: The objective of this study was to assess the impact of hypoxia on M1/M2 polarization + + Methods: M1 (CD80 ) and M2 (CD206 ) macrophages were generated by culturing human monocytes with LPS or IL4 for 24h under normoxia (20%O2) or hypoxia (1%O2). Results: We show that hypoxia amplifies M1 macrophage proinflammatory state and reprograms M2 macrophages towards a proinflammatory direction by increasing proinflammatory cytokines/chemokine production. Furthermore, we demonstrate that hypoxia upregulates the expression of TREM-1, an Iglike immunoregulatory receptor and a strong amplifier of inflammation, in both M1 and M2 macrophages. Engagement of TREM-1 by agonist Ab triggers further production of proinflammatory cytokines/chemokines in both macrophage populations. Conclusions: These results suggest the role of the hypoxic environment in skewing macrophages towards a M1-like proinflammatory phenotype, with important implications for tumor growth. POSTER TH21 INHIBITION OF MTORC2/AKT SIGNALING TO ENHANCE THE THERAPEUTIC POTENTIAL OF CD8 T CELLS Lianjun Zhang1*, Benjamin O. Tschumi1, Susanne G. Oberle2, Isabel C. Lopez-Mejia 3, Marten Meyer4, Markus A. Rüegg5, Michael N. Hall5, Lluis Fajas3, Dietmar Zehn2, Jean-Pierre Mach6, Alena Donda1, Pedro Romero1 1Translational Tumor Immunology Group, Ludwig Cancer Research, University of Lausanne, Switzerland Vaccine Research Institute, Switzerland 3Department of Physiology, University of Lausanne, Switzerland 4German Cancer Research Center (DKFZ), University of Heidelberg, Germany 5Biozentrum, University of Basel, Switzerland and 6Department of Biochemistry, University of Lausanne, Switzerland *EFIS FELLOWSHIP AWARDEE 2Swiss CD8 T cells mediate protective immune responses against infections and cancer. Upon infection, antigenspecific naïve CD8 T-cells are activated and differentiate into short-lived effector (SLEC) and memory precursor cells (MPEC). The T-cell intrinsic signaling pathways underlying this differentiation remain largely unresolved. Here we show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficient T cells form enhanced memory without dampening effector function, have increased IL-2 secretion capacity and mediate more potent recall responses. Mechanistically, enhanced memory formation in the absence of functional mTORC2 was associated with transcriptional and metabolic reprogramming by Eomes and Tcf-1 upregulation, repression of T-bet and nuclear stabilization of Foxo1. Elimination of Foxo1 reversed the increased MPECs differentiation and IL-2 production in Rictor KO mice. Effective T cell therapy against cancer depends highly on the generation of long-term persistent memory CD8 T cells. Our preliminary data show that Rictor deficient CD8 T cells show superior tumor protection effects in mouse melanoma model. Together, our study identifies mTORC2 as a central regulator of CD8 T-cell differentiation and inhibition of mTORC2 or Akt might represent an effective strategy for both adoptive cell transfer and vaccine-based cancer therapies. 24 POSTER TH22 AUTOIMMUNITY-ASSOCIATED FUNGAL INFECTION IS INVOLVED IN ESOPHAGEAL CANCER Feng Zhu,1 Jami Willette-Brown,1 Na-young Song1, Dakshayani Lomada,2,5 Sean R. Davis,3 Zhonghe Sun,4 Xiaolin Wu,4 Ellen Richie,2 and Yinling Hu1* 1Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21701, USA; 2Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Taxes 78967, USA; 3Molecular Genetics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 4Laboratory of Molecular Technology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA; 5Present address: Department of Genetics & Genomics, Yogi Vemana University, Kadapa, AP 516003, India Patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), who develop chronic mucocutaneous candidiasis due to lack of central tolerance, are susceptible to oral and esophageal carcinoma. However, it is not clear whether autoimmunity-associated fungal infection contributes to esophageal malignancy. Here, we report that mice carrying a kinase-dead IKKalpha (hereafter referred to as mutant mice) develop impaired central tolerance, systemic inflammation, fungal infection, and esophageal squamous cell carcinoma (SCC), resembling human APECED. IKKalpha plays a central role in the pathogenesis of the mutant mice, as lack of IKKalpha kinase activity results in a significantly reduced number of thymic stromal cells and the generation of autoreactive T cells. However, reintroducing IKKalpha back to the mutant mice rescued the phenotype and eliminated fungal infection. We found that mutant micederived autoreactive T cells are required for fungal infection and esophageal malignancy. Anti-fungal treatment diminished both inflammation and esophageal malignancy, suggesting autoimmunity-associated fungal infection is associated with esophageal SCC. As in the mice, IKKalpha reduction and inflammation are detected in human esophageal SCCs, suggesting that the mouse esophageal SCCs described here represents a new model for human esophageal SCCs. Thus, our findings provide strong evidence that autoimmunity-associated fungal infection contributes to the pathogenesis of esophageal cancer, shedding new light on the orchestration of autoimmunity, fungal infection and cancer. POSTER TH23 MyD88 SIGNALING IN CD11b+ MYELOID CELLS PROCTS AGAINST COLONC ADENOMA FORMATION Rosalba Salcedo1, Jonathan Badger1, Amiran Dzutsev1, Ren-Ming Dai1, Loretta Smith1, Megan Karwan 1, GiorgioTrinchieri1 1Cancer and Inflammation Program, National Cancer Institute, Bethesda, MD 20892, USA Myeloid differentiation factor 88 (MyD88) is an important signaling molecule which senses microbial products via recognition mediated by toll-like receptors. Using the AOM/DSS model of colitis, we previously described that the inability of Myd88-/- mice to heal ulcers induced upon DSS, creates an inflammatory microenvironment resulting in increase in adenoma formation. To identify the cellular compartment involved in MyD88 protection of colitis and colon cancer, we generated bone marrow chimeras and found that the hematopoietic compartments contribute to the protective role of MyD88 against colon cancer. Specific deletion of Myd88 in Cd11b+ cells (Myd88CD11b Δ/Δ ) resulted increased colonic adenoma formation in response to AOM-DSS treatment. In contrast, deletion of Myd88 in B cells, T cells and dendritic cells did not impact the formation of colonic polyps. The gene expression profile of sorted myeloid cells from inflamed colons indicated that Myd88 deficiency resulted in increased expression of acute inflammatory markers including S100a8, S100a9, Il1 and Il6. Importantly, in the absence of Myd88, and upon exposure of the colonic mucosa to microbial translocation, compensatory antimicrobial mechanisms were activated in myeloid cells including enhanced expression of microbial peptide receptor genes Fpr1 and Fpr2, and the siderophore sequestering Lipocalin-2 (Lnc2).The gene expression profile of sorted myeloid cells from inflamed colons indicated that Myd88 deficiency resulted in increased expression of acute inflammatory markers including S100a8, S100a9, Il1 Importantly, Myd88-/- and Myd88CD11b Δ/Δ animals carry a pro-tumorigenic microbiota which was transmissible to wild type animals, since post cohousing and in response to AOM/DSS treatment, wild type animals developed a remarkable increase in multiplicity and size of colonic adenomas, relative to segregated 25 controls. Cohousing resulted in evident but partially transient changes in microbiota composition in both wild type and mutant mice. The correlation of particular bacteria species with adenoma multiplicity in cohoused animals is under current investigation. This data indicates that deficiency of Myd88 in myeloid cells favors the development of a pro-tumorogenic microbiome, which is transmissible and which under the exposure to a carcinogen and under chronic inflammatory conditions results in increased tumor development. POSTER TH24 REGULATION OF THE FIBROGENIC RESPONSE OF HEPATIC STELLATE CELLS BY SOCS1 Rajani Khandi, Diwakar Bobbala, Mehdi Yeganeh, Sheela Ramanathan, Alfredo Menendez and Subburaj Ilangumaran* Department of Pediatrics, Immunology Division, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada Background & Aim: Hepatic stellate cells (HSC) play a key pathogenic role in liver fibrosis that precedes hepatocellular carcinoma. Activation pathways of HSCs are well characterized, but regulatory mechanisms of liver fibrosis are not well understood. Socs1 (Suppressor Of Cytokine Signaling 1) haplo-insufficient mice are susceptible to liver fibrosis. Our aim is to elucidate the SOCS1-dependent regulatory mechanisms of hepatic fibrogenic response. Methodology: SOCS1-deficient and control mice were treated with dimethylnitrosamine. Liver damage and fibrosis were evaluated. Hepatic fibrogenic response was assessed by qRT-PCR and western blot. Cytokine responses of HSCs were evaluated in vitro. Results: SOCS1-deficient mice showed elevated serum ALT levels and increased liver fibrosis compared to control mice, associated with increased collagen deposition and loss of hepatic lobular structure. SOCS1deficient livers showed increased expression of genes coding for smooth muscle actin, collagen alpha 1 and matrix metalloproteases, and altered expression of genes encoding tissue inhibitor of metalloproteinases (Sma, Cola1, Mmp3, Mmp9, Timp1, Timp2, Timp4). SOCS1-deficient HSCs displayed increased activation and fibrogenic gene expression following stimulation with IL-6 or TGFβ, and showed increased proliferation to EGF, HGF and PDGF. Conclusions: SOCS1 is an important regulator of hepatic fibrogenic response, and a part of this regulation occurs in HSCs POSTER TH25 ATTENUATED MUTANT STRAIN OF SALMONELLA TYPHIMURIUM (STMZNUABC) CONTRASTS TUMOR GROWTH AND PROMOTES ANTITUMOR IMMUNE PATTERNS Barbara Chirullo*, Ammendola S, Leonardi L, Falcini R, Petrucci P, Pistoia C, Vendetti S, Battistoni A and Pasquali P. Istituto Superiore di Sanità, Rome, Italy *EFIS FELLOWSHIP AWARDEE The use of bacteria in cancer therapy has been studied for more than a century. There is established evidence that solid tumors may undergo regression after bacterial infection (Coley, Clin Orthop Relat Res. 1991). We investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STMznuABC) devoid of the whole operon coding for the highaffinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens (Ammendola et al, Infect Immun 2007; Pasquali et al, patent pending 2014) We show that STMznuABC is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STMznuABC in mammary adenocarcinoma mouse model leads to both reduction of tumor growth and increase in life expectancy of STMznuABC treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STMznuABC stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors (Chirullo et al, Oncotarget 2015). 26