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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.
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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.
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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.
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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+IFNlow 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
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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).
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