Interferon Fundamentals 2014
From Molecular Mechanisms
to Human Diseases
Rome, February 20, 2014
Auditorium Pocchiari
Istituto Superiore di Sanità
Abstract Book
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
Over the last two decades, we have witnessed a tremendous progress in understanding the mechanisms by
which interferon (IFN) and other related cytokines act on cells and animal models. Thus, the recent progress in
the IFN research has been instrumental for acquiring new and important knowledge in various fields, such as
cytokine signaling, antiviral responses, innate and adaptive immunity, immune regulation and tumor
immunology and immunotherapy. IFNs (especially IFN-α) have been widely used in clinical practice for the
treatment of some human malignancies and certain infectious diseases with variable responses and some
types of IFN are still used in clinical practice.
The meeting is dedicated to the memory of Professor Giovanni Battista Rossi, who introduced the IFN
research at the Istituto Superiore di Sanità and promoted a national research program on AIDS in Italy, 20
years after his death.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
PROGRAMME
8.00
Registration
8.30
Welcome address
F. Oleari, President – Istituto Superiore di Sanità, Rome (Italy)
L. Frati, Rector – Università di Roma "La Sapienza", Rome (Italy)
S. Mobilio, Director – Dipartimento di Scienze, Università degli Studi “Roma Tre”, Rome (Italy)
OPENING SESSION
Chairpersons: G. Rezza, M. Pocchiari
8.50
Working with GB. Rossi at the Istituto Superiore di Sanità: a personal memory
P. Verani
9.00
GB. Rossi’s vision of Biomedical Research
S. Vella
On Intuition and the Discovery of Interferon
I. Gresser
9.10
SESSION I
Chairpersons: E. Affabris, G. Romeo
9.30
The role of interferons and other cytokines in the regulation of the immune response
G. Trinchieri
10.00 Type I interferon signalling and its stringent control
S. Pellegrini
10.30 The NK cells and the Interferons
A. Santoni
11.00 Coffee Break
SESSION II
Chairpersons: A. Battistini, EM. Coccia
11.30 Innate Immune Response to Human Retrovirus Infection
J. Hiscott
12.00 The interferon system and autoimmunity
S. Landolfo
12.30 High efficiency targeting of IFN-α activity: possible applications in fighting tumors, infections and
autoimmunity
G. Uzé
13.00 Lunch and Poster Session
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
SESSION III
Chairpersons: A. Dolei, MR. Capobianchi
14.00 Role of type I interferon in inducing a protective immune response: perspectives for clinical
applications
F. Belardelli
14.30 Interferon and antitumor immunity
TF. Gajewski
15.00 From infection to immunotherapy: the role of interferon in HCV infection.
V. Barnaba
15.30 Coffee Break
ROUND TABLE
What have we learned from the clinical use of interferon and how can we translate the recent
knowledge in future clinical applications?
Chairpersons: E. Proietti, G. Antonelli
15.45 Introductory remark
I. Gresser
15.55 The use of interferon in melanoma patients
P. Ascierto
16.10 What IFN-α has meant to hematology and what hematology has meant to IFN-α
R. Foà
16.25 Lessons from the clinical use of interferon in patients with viral hepatitis
M. Levrero
16.40 Interferon and HIV infection: an old therapy for a new opportunity
G. D’Offizi
16.55 IFN-β and Multiple Sclerosis: from etiology to therapy and back
M. Salvetti
17.10 Biological and clinical monitoring of IFN-β therapy in Multiple Sclerosis
A. Bertolotto
17.25 Open discussion
18.00 Final remarks
F. Belardelli
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
SPEAKERS AND CHAIRPERSONS
Elisabetta Affabris, Dipartimento di Scienze, Università degli Studi “Roma Tre”, Rome (Italy)
Guido Antonelli, Dipartimento di Medicina Molecolare, Università di Roma "La Sapienza", Rome (Italy)
Paolo Ascierto, Istituto Nazionale Tumori Fondazione "G. Pascale", Naples (Italy)
Vincenzo Barnaba, Dipartimento di Medicina Interna, Università di Roma "La Sapienza", Rome (Italy)
Angela Battistini, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di
Sanità, Rome (Italy)
Filippo Belardelli, Department of Haematology, Oncology and Molecular Biology, Istituto Superiore di Sanità,
Rome (Italy)
Antonio Bertolotto, Azienda Ospedaliero Universitaria “San Luigi Gonzaga”, Orbassano (Italy)
Maria Rosaria Capobianchi, Laboratorio di Virologia, National Institute for Infectious Diseases Lazzaro
Spallanzani, Rome (Italy)
Eliana Marina Coccia, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore
di Sanità, Rome (Italy)
Gianpiero D’Offizi, Immunoinfettivologia, National Institute for Infectious Diseases Lazzaro Spallanzani,
Rome (Italy)a
Antonina Dolei, Dipartimento di Scienze Biomediche, Università di Sassari, Sassari (Italy)
Robin Foà, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma "La Sapienza", Rome
(Italy)
Luigi Frati, Rector – Università di Roma "La Sapienza", Rome (Italy)
Thomas F. Gajewski, University of Chicago, Chicago, Illinois (USA)
Ion Gresser, Centre des Recherches Biomedicales des Cordeliers, Paris (France)
John Hiscott, Division of Infectious Diseases, Vaccine & Gene Therapy Institute of Florida, Forida (USA)
Santo Landolfo, Dipartimento di Sanità Pubblica e Microbiologia, Università di Torino, Turin (Italy)
Massimo Levrero, Dipartimento di Medicina Interna, Università di Roma "La Sapienza", Rome (Italy)
Settimio Mobilio, Direttore, Dipartimento di Scienze, Università degli Studi “Roma Tre”, Rome (Italy)
Fabrizio Oleari, President – Istituto Superiore di Sanità, Rome (Italy)
Sandra Pellegrini, Département d’Immunologie, Institut Pasteur, Paris (France)
Maurizio Pocchiari, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome (Italy)
Enrico Proietti, Department of Haematology, Oncology and Molecular Biology, Istituto Superiore di Sanità,
Rome (Italy)
Giovanni Rezza, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di
Sanità, Rome (Italy)
Giovanna Romeo, Dipartimento di Scienze e biotecnologie medico-chirurgiche, Università di Roma "La
Sapienza", Rome (Italy)
Marco Salvetti, Centro Neurologico di Terapia Sperimentale (CENTERS), Ospedale S. Andrea, Università di
Roma "La Sapienza", Rome (Italy)
Angela Santoni, Dipartimento di Medicina Molecolare, Università di Roma "La Sapienza", Rome (Italy)
Giorgio Trinchieri, Center for Cancer Research, National Cancer Institute, Frederick, Maryland (USA)
Gilles Uzé, Unité Mixte de Recherche 5235, Centre National de la Recherche Scientifique, University of
Montpellier II, Montpellier (France)
Stefano Vella, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità,
Rome (Italy)
Paola Verani, Istituto Superiore di Sanità, Rome (Italy)
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
SCIENTIFIC COMMITTEE
Filippo BELARDELLI
Elisabetta AFFABRIS
Guido ANTONELLI
Angela BATTISTINI
Eliana M. COCCIA
Gianna FIORUCCI
Giovanna ROMEO
SCIENTIFIC SECRETARIAT
Franca MORETTI
Phone: +39-06-49906095
TECHNICAL SECRETARIAT
Anna FERRIGNO
Phone: +39-06-49906093, Fax: +39-06-49902097
Rosario INTERNULLO
Phone: 39­‐06­‐49906096, Fax 06­‐49902097
Veronica MICHETTI
Phone: +39 06 49906094, Fax: +39 06 49902097
e-mail: ifnfundamentals2014@iss.it
GENERAL INFORMATION
Venue
Istituto Superiore di Sanità, Pocchiari Auditorium
Viale Regina Elena, 299 – 00161 – Rome
No Continuing Medical Education (CME) credits will be awarded for this event.
This event is part of the scientific and tutorial activities of the Doctoral School in Biology and PhD Program in
Biomedical Sciences and Technologies of Università degli Studi “Roma Tre”, Rome.
www.iss.it/ifnf/
Interferon Fundamentals 2014
February 20, 2014
Istituto Superiore di Sanità, Rome
From Molecular Mechanisms to Human Diseases
Organized by
Department of Hematology, Oncology and Molecular Medicine
in association with
Department of Infectious, Parasitic and Immune-mediated Diseases
Department of Sciences
Department of Medico-Surgical
Sciences and Biotechnologies
Doctoral School in Biology and PhD Program in Biomedical Sciences and
Technologies
Under the patronage of
Società Italiana
di Virologia
Associazione Italiana
Sclerosi Multipla
Società Italiana di
Neuroimmunologia
Società Italiana
di Virologia Medica
Alleanza Contro il Cancro
Società Italiana di Immunologia,
Immunologia Clinica e Allergologia
Associazione dei Microbiologi Clinici
Italiani
With the unconditional support of
Bristol-Myers Squibb –
Roma
Beckman Coulter Italia
Becton dickinson Italia
Alfa Wassermann –
Bologna
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
INDEX
PROGRAMME.....................................................................................................................................................................3 OPENING SESSION ..................................................................................................................................................................... 3 SESSION I..................................................................................................................................................................................... 3 SESSION II.................................................................................................................................................................................... 3 SESSION III................................................................................................................................................................................... 4 ROUND TABLE ............................................................................................................................................................................. 4 SPEAKERS AND CHAIRPERSONS ............................................................................................................................................. 5 SCIENTIFIC COMMITTEE ............................................................................................................................................................ 6 SCIENTIFIC SECRETARIAT......................................................................................................................................................... 6 TECHNICAL SECRETARIAT ........................................................................................................................................................ 6 GENERAL INFORMATION............................................................................................................................................................ 6 INDEX ..................................................................................................................................................................................8 ORAL PRESENTATIONS .................................................................................................................................................10 ON INTUITION AND THE DISCOVERY OF INTERFERON............................................................................................................ 11 TYPE I IFN SIGNALING AND ITS STRINGENT CONTROL ........................................................................................................... 12 INNATE IMMUNE RESPONSE TO HUMAN RETROVIRUS INFECTION ...................................................................................... 13 THE INTERFERON SYSTEM AND AUTOIMMUNITY..................................................................................................................... 14 HIGH EFFICIENCY TARGETING OF IFN-α ACTIVITY: POSSIBLE APPLICATIONS IN FIGHTING TUMORS, INFECTIONS AND
AUTOIMMUNITY.............................................................................................................................................................................. 15 ROLE OF TYPE I INTERFERON IN INDUCING A PROTECTIVE IMMUNE RESPONSE: PERSPECTIVES FOR CLINICAL
APPLICATIONS ............................................................................................................................................................................... 16 TYPE I IFNS AND ANTI-TUMOR IMMUNITY.................................................................................................................................. 17 FROM INFECTION TO IMMUNOPATHOLOGY: THE ROLE OF INTERFERON IN HCV INFECTION .......................................... 18 THE JANUS FACES OF INTERFERON REVISITED ...................................................................................................................... 19 THE USE OF INTERFERON IN MELANOMA PATIENTS............................................................................................................... 20 INTERFERON AND HIV INFECTION: AN OLD THERAPY FOR A NEW OPPORTUNITY ............................................................ 21 IFN-β AND MULTIPLE SCLEROSIS: FROM ETIOLOGY TO THERAPY AND BACK.................................................................... 22 NABS TESTING IN IFN-β TREATMENT ......................................................................................................................................... 23 POSTER PRESENTATIONS.............................................................................................................................................24 TYPE III INTERFERON EXPRESSION DIFFER BETWEEN LOW-RISK AND HIGH-RISK HUMAN PAPILLOMAVIRUS POSITIVE
PATIENT CERVICAL CELLS........................................................................................................................................................... 25 IFN-λ1-3 EXPRESSION IN INFANTS HOSPITALIZED FOR RSV OR HRV ASSOCIATED BRONCHIOLITIS ............................. 26 DENDRITIC CELL ENGINEERING WITH IRF-1 AND IRF-8 FOR NEW IMMUNOTHERAPY INTERVENTIONS.......................... 27 IKAPPAB KINASE EPSILON TARGETS INTERFERON REGULATORY FACTOR 1 IN ACTIVATED T LYMPHOCYTES ........... 28 CANCER CHEMOTHERAPY AND VIRAL MIMICRY ...................................................................................................................... 29 TYPE I IFN IN TUBERCULOSIS: FRIEND OR FOE ....................................................................................................................... 30 www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
EXOGENOUS HIV-1 NEF INDUCES PROINFLAMMATORY SIGNALING EVENTS IN MURINE MACROPHAGES AND
MICROGLIAL CELLS....................................................................................................................................................................... 31 TYPE I IFN AND 5-AZA-2’-DEOXYCITIDINE SYNERGIZE FOR THE INHIBITION OF SOLID CANCER PROGRESSION.......... 32 IMMUNOMODULATORY AND ANTIVIRAL EFFECTS OF IFN-β THERAPY IN MULTIPLE SCLEROSIS: TWO FACES OF THE
SAME COIN. FOCUS ON B LYMPHOCYTES................................................................................................................................. 33 IFNAR-1 UPREGULATION IN PBMC FROM HCV NAÏVE PATIENTS CARRYING CC GENOTYPE. POSSIBLE ROLE OF IFN-λ
......................................................................................................................................................................................................... 34 HIV-1 NEF INDUCES PROINFLAMMATORY STATE IN HUMAN MACROPHAGES THROUGH ITS ACIDIC CLUSTER DOMAIN:
INVOLVEMENT OF TNF-α RECEPTOR ASSOCIATED FACTORS .............................................................................................. 35 IFN-α ANTAGONIZES REGULATORY T CELL-MEDIATED SUPPRESSION AND FAVORS THEIR PLASTICITY INTO TH1-LIKE
CELLS.............................................................................................................................................................................................. 36 NOVEL INSIGHTS INTO THE DAMP-LIKE PRO-INFLAMMATORY BEHAVIOUR OF MISLOCALIZED NUCLEAR IFI16 IN THE
EXTRACELLULAR SPACE.............................................................................................................................................................. 37 TYPE III INTERFERON (IFN-λ) ANTAGONIZES THE ANTIVIRAL ACTIVITY OF INTERFERON-ALPHA IN VITRO ................... 38 CHARACTERIZATION OF THE ROLE OF TYPE I AND III INTERFERON RESPONSE DURING WEST NILE VIRUS AND
USUTU VIRUS INFECTION............................................................................................................................................................. 39 PRO-INFLAMMATORY CYTOKINE ANALYSIS IN HPV-POSITIVE CANCER CELLS .................................................................. 40 MICRORNA PROFILING IN E6/E7 HPV-TRANSFORMED HUMAN KERATINOCYTES. THE EFFECT OF IFN-β. .................... 41 ROLE OF IFN-I IN THE SPONTANEOUS DEVELOPMENT OF MAMMARY TUMORS IN MICE KNOCK-OUT FOR TYPE I IFN
RECEPTOR AND TRANSGENIC FOR THE HER2/NEU ONCOGENE .......................................................................................... 42 www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
ORAL PRESENTATIONS
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
ON INTUITION AND THE DISCOVERY OF INTERFERON
Ion GRESSER1
1. Centre de Recherches Biomedicales des Cordeliers, Paris (France)
Abstract
Intuition is often an essential component of any discovery.
Although the solution to a problem seems to appear suddenly, it is often preceded by a very long period of thought, work
and gestation. This was so for the discovery of Interferon, which stemmed from studies of “viral interference” a subject of
intense research in the preceding 20 years. The discovery in 1957 by Alick Isaacs and Jean Lindenmann that viral
infected tissues released a soluble anti-viral factor named “Interferon” was however just the the beginning of fifty more
years of other on going discoveries of its multiple actions and it’s role in physiology and pathology. Furthermore, the
discovery of interferon led to the discovery of many other pleotropic substances of a family named “Cytokines”.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE I IFN SIGNALING AND ITS STRINGENT CONTROL
Sandra PELLEGRINI1
1. Institut Pasteur - Paris (France)
Abstract
Since the early 90’s when the Jak/Stat signaling pathway was first described, our molecular understanding of
type I IFNs action at the cellular level has considerably advanced. For each Jak/Stat player studies have
moved from biochemical to structural analyses, from mRNA and protein to description of isoforms and posttranslational modifications, from gene targeting in mouse models to identification of human gene variants and
of patients with inborn genetic errors. Today’s general picture remains that of a relatively simple information
flow from the ligand-engaged receptor at the plasma membrane to the Tyk2/Jak1 enzymatic switch that
activate Stat1 and Stat2 as main effectors of the ISG transcriptional response. Cell-specific crosstalks with
other pathways as well as positive/negative regulatory networks have been described that contribute to
diversify and balance the IFN response. Here I will focus on two ISGs (the isopeptidase USP18 and the
ubiquitin-like ISG15) whose functional interplay tunes down signaling by IFN. Consistently, patients with
inherited ISG15-deficiency exhibit enhanced type I IFN signaling at the cellular level and show clinical
symptoms of the rare Mendelian autoinflammatory interferonopathies associated with upregulation of ISGs.
Thus, the USP18/ISG15 feedback loop protects from inappropriate inflammation and autoimmune pathologies
caused by dysregulation of type I IFN.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
INNATE IMMUNE RESPONSE TO HUMAN RETROVIRUS INFECTION
John HISCOTT1
1. Vaccine & Gene Therapy Institute of Florida, Port Saint Lucie, Florida (USA)
Abstract
Research of the past decade has provided tremendous advances in our understanding of the innate immune response
to many pathogens, including RNA viruses such as influenza and dengue. However, the host mechanisms that sense
and respond to the human retroviruses - human immunodeficiency virus (HIV) and human T-cell leukemia virus type 1
(HTLV-1) - have remained poorly understood. Recent studies have begun to reveal the complex regulation of the innate
immune response to human retroviral infection. We have used de novo HTLV-1 infection of primary myeloid lineage cells
to investigate the mechanisms underlying monocyte depletion by HTLV-1 infection. We demonstrate that HTLV-1
infection rapidly induced an innate antiviral response and triggered apoptosis in a manner dependent on the host
restriction factor SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase that limits retroviral replication by
reducing the availability of deoxynucleoside triphosphates (dNTPs) required for reverse transcription. Silencing of
SAMHD1 by RNA interference, addition of exogenous dNTPs to the infected monocyte culture, or pre-treatment with
azidothymidine (AZT) to block reverse transcription also inhibited the caspase 3 mediated apoptosis. To investigate a
role for reverse transcription intermediates (RTI) in triggering apoptosis, a biotinylated 90 nucleotide RTI from the U5
region of HTLV-1 was introduced into monocytes; strikingly, the biotinylated HTLV-1 DNA intermediate induced
apoptosis in monocytes and bound to the endoplasmic reticulum resident cGAS-STING sensor - which mediates
activation of the host antiviral response via IRF3. Furthermore, STING-mediated apoptosis in HTLV-1 infected
monocytes required the generation of pro-apoptotic complex between IRF3 and the Bcl-2 family member Bax. These
studies provide a mechanistic explanation for HTLV-1 abortive infection of monocytes and report a link between
SAMHD1 restriction of reverse transcription, sensing of retroviral reverse transcription intermediates by STING, and the
initiation of IRF3-Bax driven apoptosis.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
THE INTERFERON SYSTEM AND AUTOIMMUNITY
Santo LANDOLFO1
1. University of Turin (Italy)
Abstract
IFI16, a nuclear pathogenic DNA sensor induced by Interferons (IFNs), is a multifaceted protein with various
functions, including suppression of transcription, induction of inflammation, and restriction of virus replication. It
is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients
affected by systemic autoimmune diseases. Various research groups, including ours, have shown that
following transfection of virus-derived DNA, IFI16 delocalizes from the nucleus to the cytoplasm and is then
eventually released into the extracellular milieu. In this study, using an in-house capture ELISA we
demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of patients
affected by different autoimmune diseases. We also show that the recombinant IFI16 protein, when added in
vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell
migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal
antibodies, indicating that its extracellular activity resides within the N-terminus. Immunofluorescence assays
revealed the existence of high-affinity membrane receptor(s) on the plasma membrane of endothelial cells.
Free recombinant IFI16 binds to these receptors with a dissociation constant of 2.7nM, and radioiodinated and
unlabeled IFI16 compete for binding sites, with an inhibition constant (Ki) of 14.43nM and a half maximal
inhibitory concentration (IC50) of 67.88nM; these data allow us to estimate the presence of 250,000 to
450,000 specific binding sites per cell. Corroborating the results from the functional assays, this binding could
be completely inhibited using the anti-IFI16 N-terminal antibody, but not with an antibody raised against the
IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of
autoimmune patients suggesting a new pathogenic mechanism through which IFNs and IFN-inducible genes
trigger the development of autoimmunity.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
HIGH EFFICIENCY TARGETING OF IFN-α ACTIVITY: POSSIBLE
APPLICATIONS IN FIGHTING TUMORS, INFECTIONS AND AUTOIMMUNITY
Gilles UZÉ1
1. CNRS UMR 5235, Montpellier (France)
Abstract
Dose-related severe side effects currently prevents full clinical application of type I interferons (IFNs) for the treatment of
cancers and viral infections. The large cellular specificity of IFN is believed to be responsible of its systemic toxicity.
Although the use of IFNs fused to antibodies resulted in a 10-fold increased activity towards targeted cells, off-target side
effects experienced by the patients are expected to remain a major obstacle. We present a novel strategy to engineer
IFN-α with high in vitro and in vivo activity towards cells expressing a specific surface marker while having an up to
1000-fold reduced activity towards non-relevant cells. The concept lies in the use of immunocytokines based on IFN
mutants with strongly reduced binding affinity for the IFN receptor. Such a targeted IFN recovers almost maximally its
antiviral and antiproliferative activities uniquely on the targeted cells. IFN molecules have been designed to specifically
act on various tumor cells or on the CD8α+ dendritic cells subset when injected in mice. This novel concept holds
promise to revitalize the clinical potential of IFN-α and other cytokines.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
ROLE OF TYPE I INTERFERON IN INDUCING A PROTECTIVE IMMUNE
RESPONSE: PERSPECTIVES FOR CLINICAL APPLICATIONS
Filippo BELARDELLI1
1. Department of Haematology, Oncology and Molecular Biology, Istituto Superiore di Sanità, Rome (Italy)
Abstract
Type I IFNs are currently recognized as crucial factors regulating the induction of protective immune responses. Such
knowledge mostly stems from early in vivo studies carried out at the Istituto Superiore di Sanità, in collaboration with Ion
Gresser in Paris, based on the use of IFN-resistant Friend virus-transformed leukemic cells (FLC), originally isolated by
Elisabetta Affabris in Giovanni Rossi’s laboratory in 1982. By using these cells, we could subsequently demonstrate the
crucial importance of antibodies to tumor associated antigens and CD4+ T cells in the IFN-induced antitumor response.
Additional in vivo studies with genetically modified tumor cells expressing IFN-α revealed the importance of other cells of
the immune system, including CD8+ T lymphocytes, in the cytokine-induced antitumor response. Further studies
revealed that type I IFN can also act as an effective adjuvant of the influenza virus vaccine in mice. Notably, the activity
of some potent adjuvants (i.e., CpG oligonucleotides) turned out to be mostly mediated by endogenous type I IFNs and
low levels of these cytokines proved to play a role in the innate control of tumor growth in a variety of mouse models. Of
interest, type I IFN does also play a critical role in the antitumor response to some chemotherapeutic agents. Today,
many studies published by our group as well as by others demonstrate that type I IFNs act as efficient factors in
promoting the differentiation and activation of both mouse and human dendritic cells (DC). Thus, treatment of human
monocytes with IFN-α in the presence of GM-CSF resulted in the rapid generation of highly activated DC expressing
factors potentially involved in a protective immune response (i.e., CXCL10, IL-23 and IL-15). This DC population (“IFNDC”) exerts direct cytotoxic activity and is capable of generating a potent antibody response and a clear-cut CD8+ T cell
response against both viral and cancer antigens both in vitro and in humanized mouse models. Further studies allowed
us to characterize the mechanisms by which these IFN-DC can take up apoptotic bodies and efficiently process complex
antigens, inducing cross-priming of CD8+ T cells. Thus, we are currently testing these IFN-DC preparations as new cell
drugs in clinical trials with cancer patients. These data, together with the results of pilot clinical trials based on the local
co-injection of IFN with a given antigen, support the candidacy of IFN-α as cancer vaccine adjuvant and as a critical
factor for the development of more effective DC-based therapeutic vaccines. Finally, additional opportunities of
combination of IFN-α with other drugs (including monoclonal antibodies, chemotherapy and target therapies) can be
envisaged today, as a result of the new knowledge of the mechanisms by which these cytokines can regulate the
generation of a protective immune response under different conditions.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE I IFNS AND ANTI-TUMOR IMMUNITY
Thomas F. GAJEWSKI1
1. University of Chicago, Illinois (USA)
Abstract
Unexpectedly, many cancers appear to induce a spontaneous adaptive T cell response. The presence of a T cell
infiltrate has been linked to favorable clinical outcome in multiple cancer types, and also may predict response to specific
immunotherapies. However, the innate immune pathways that bridge to an adaptive immune response under sterile
conditions have, until recently, remained poorly understood. Human melanoma metastases containing a CD8+ T cell
infiltrate were associated with a type I IFN transcriptional signature. Using mouse models for mechanistic experiments,
we have observed that tumors can induce type I IFN production by host antigen-presenting cells which is required for
induction of spontaneous T cell responses in vivo. This process requires action of type I IFNs directly on host DCs,
particularly the CD8α+ DC subset in the mouse. The innate immune sensing pathways that trigger type I IFN production
are being elucidated, and our data suggest that the host SING pathway which senses cytosolic DNA is critical. In fact,
direct engagement of the STING pathway with specific agonists is potently therapeutic in vivo, inducing robust anti-tumor
T cell responses. Targeting low levels of type I IFNs to the tumor microenvironment through conjugation to tumortargeting mAbs also is therapeutic via host immunity and is attractive for clinical translation. In contrast to the
immunologic role for low levels of endogenous type I IFNs induced in the cancer context, a different mechanism may be
operational for the therapeutic efficacy of high doses of exogenous type I IFNs. In that instance we have found that type
IFN signaling is required on non-hematopoietic stromal cells, and is linked to a direct anti-angiogenic effect of IFNs. This
notion was confirmed using conditional deletion of the type I IFNR on Tie2-expressing vascular endothelial cells. Thus,
transient low levels and high doses of type I IFNs may have distinct biologic effects and mechanisms of tumor control.
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Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
FROM INFECTION TO IMMUNOPATHOLOGY: THE ROLE OF INTERFERON IN
HCV INFECTION
Vincenzo BARNABA1, Helene MARTINI1, Silvia PICONESE1, Alessandra CITRO1, Ylenia PACELLA1, Eleonora
TIMPERI1
1. Dipartimento di Medicina Interna e Specialita Mediche, Università di Roma "La Sapienza", Rome (Italy)
Abstract
Interferon type I (IFN) provides pleiotropic effects both in physiology and pathology. We determined its effects on both
chronic immune activation and regulatory T cell (Treg) function in patients with HCV infection undergoing peg-IFNα/Ribavirin (IFN/RBV) therapy. First, we explored the effect on CD8+ T cells specific to caspase-cleaved peptides
derived from T cell apoptosis. We previously showed that T cell apoptosis determines the generation of caspase-cleaved
peptides (apoptotic epitopes [AE]), which can be cross- presented to specific autoreactive CD8+ T cells. This event
participates to immune activation in chronic infections. At the T0, no difference was observed in HCV- or AE- specific
CD8+ T cell frequencies between Relapsers (R) and Sustained Virological Responders (SVR). However, the latter
significantly increased only in R since T1 (after 1 month of therapy). At T0, IFN-γ production by autoreactive CD8+ T
cells was lower in R and, conversely, PD1 expression higher. This data suggests that higher AE-specific CD8+ T cell
frequencies predict an unfavorable response to IFN/RBV therapy. The second purpose of these studies was to
characterize the functional changes induced in Treg upon exposure to IFN/RBV therapy. Our data demonstrates that
IFN-α amplifies Th1-type inflammation by limiting the pool of fully suppressive Tregs and concomitantly fostering Treg
de-programming into IFN-γ-producing plastic Tregs (defined as “Th1-like” Tregs).
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
THE JANUS FACES OF INTERFERON REVISITED
Ion GRESSER1
1. Centre de Recherches Biomedicales des Cordeliers, Paris (France)
Abstract
Two different faces of Interferon’s actions are discussed.
The first face concerns the beneficial antitumor effects of type 1 interferon which appears to be the “king-pin” of the
host’s antitumor resistance.
Its presence increases the therapeutic effectiveness of conventional tumor therapy; its neutralization or absence not only
renders some of these therapies ineffective, but also decreases the innate defenses of the host to tumor growth.
The second face concerns the adverse effects of type 1 interferon. It is known that administration of type 1 interferon to
suckling mice or rats induces disease
as does injection of LCM virus which induces large amounts of type 1 IFN.
Administration of antibody to type 1 IFN to LCM infected mice neutralizes the interferon and attenuates the disease.
Several diseases in man have been named “interferonopathies” because they are associated with the continuous
production of alpha interferon. It is not known for the present whether the presence of interferon is the result of the
disease process or is part of the pathogenesis.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
THE USE OF INTERFERON IN MELANOMA PATIENTS
Paolo A. ASCIERTO1
1. Melanoma, Cancer Immunotherapy and Innovative Therapy Unit – Istituto Nazionale Tumori Fondazione “G. Pascale”, Naples
(Italy)
Abstract
The use of interferon α-2b (IFN) in the adjuvant therapy of melanoma at high risk of recurrence, is still
debated. Discussions about the optimal treatment scheme and concerns over toxicity, have limited its clinical
use. However, IFN remains the only currently available adjuvant option for melanoma. In fact, based on metaanalyses of clinical trials, there is no doubt that treatment with IFN is beneficial with regard to overall and
recurrence-free survival. Anyway, predictive factors to identify patients who most likely could benefit from
adjuvant IFN therapy are required.
For the future the efforts should be focused in increasing the activity of IFN with possible combination. It was
recently found that IFN receptor 1 (IFNAR1) expression is affected by ERK activation. As a result IFNAR1 is
downregulated in cells with ERK activation. IFNAR1 is upregulated by BRAF-inhibitor in melanoma cells with
an active BRAF mutation, and BRAF inhibitors enhance the functional properties of IFN-α in melanoma cells
with V600E BRAF mutation, showing in vitro anti-proliferative activity, pro-apoptotic activity, HLA Class I
upregulation, enhancement of melanoma cell sensitivity to T cell recognition and In vivo anti-tumor activity.
The association of BRAF-I and INF-α in melanoma cells and preclinical model resulted in a strong antiproliferative activity. This combination will be explored in a phase I-II clinical study. Another possible
combination it’s based on the evidence that IFN is able to induce the PD-L1 expression on the surface of
cancer cells. This coulb be the basis for a possible combination of IFN with the anti-PD-1/PD-L1 compounds.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
INTERFERON AND HIV INFECTION: AN OLD THERAPY FOR A NEW
OPPORTUNITY
Gianpiero D'OFFIZI1
1. National Institute for Infectious Diseases Lazzaro Spallanzani, Rome (Italy)
Abstract
At the beginning of AIDS, an antiretroviral activity of IFN-α was demonstrated in the early stages of HIV infection and
Kaposi’s sarcoma.
This therapeutic approach was abandoned, when chemotherapeutic agents became available for single or combined
administration leading to very successful regimens. It was evident, however, that maintaining these regimens
continuously and for long periods of time was more problematic because of the development of serious side effects.
Structured interruptions of therapy (STI) were applied for predefined drug-free periods of time, with resumption of
treatment when scheduled or when a worrying rise in viral load occurred. Most of these studies gave controversial
results, mainly because of the frequent relapse of progression during the drug-free phases, thus posing serious
questions about the risk-benefit ratio of this procedure.
That IFN has promise in this role, can be deduced from several studies showing that IFN-α leads to a control of viral
replication in absence of HAART. In addition it was demonstrated that it is possible to subject participants to an STI
without increasing the reservoir of HIV DNA in the peripheral blood.
IFN-α administration, expecially before and during STIs, could provide a safer way to evaluate viral control in the
absence of HAART.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IFN-β AND MULTIPLE SCLEROSIS: FROM ETIOLOGY TO THERAPY AND
BACK
Marco SALVETTI1
1. Centre for Experimental Neurological Therapies (CENTERS) - Neurology and Department of Neuroscience, Rome (Italy)
Abstract
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by
progressive neurological dysfunction due to demyelination and axonal lesion. Various immunomodulatory and
immunosuppressive therapies are available for this disease. However, Interferon-Beta (IFN-β) represents a
first-line treatment that is widely used. Interferons (IFNs) are proteins secreted by cells and are involved in self
defense to viral infections, in the regulation of cell growth and in the modulation of immune responses. IFN
signaling is complex, from receptor binding events to the generation of effector mechanisms that dampen
inflammation. IFN-β has pleiotropic effects, including antiviral, antitumor, and anti-inflammation. Although the
precise mechanism of IFN-β is unclear, many studies indicate some potential mechanisms including blocking
T cells activation, controlling pro- and anti-inflammatory cytokine secretion, preventing activated immune cell
migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells
into oligodendrocytes.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
NABS TESTING IN IFN-β TREATMENT
Antonio BERTOLOTTO1
1. Neurologia 2 - Centro Riferimento Regionale Sclerosi Multipla (CRESM), Azienda Ospedaliera Universitaria San Luigi,
Orbassano, Turin (Italy)
Abstract
Although IFN-β has improved treatment options of MS, the long-term efficacy of IFN-β can be compromised due to
development of neutralising antibodies (NAbs).
High titre Nabs develop in about 15% of patients, they abolish biological activity, and therapeutic action of IFN-β
It is important to find bio-marker of efficacy of IFN-β therapies, based on the physiologic response to IFN-β.
The most popular IFN-β biomarker is MxA, a reliable bio-marker for detection of IFN-β responders, as:
1) MxA level in treated patients is higher than in non-treated;
2) MxA is not influenced by disease activity;
3) MxA level correlates with molecules involved in IFN-β therapeutic actions;
4) Absence of MxA induction by IFN-β in patients reflects complete loss of bioactivity;
5) patients with NABs do not present an increase of MxA;
6) MxA levels predicts IFN-β response.
The quantification of MxA and/or Nabs identifies a subset of patients treated with IFN-β without clinical efficacy, that
should discontinue IFN-β treatment.
Conclusion:
i) reliable biological methods identify a significant subset of patients that are non-responders to IFN-β;
ii) MxA and/or Nabs quantification should be implemented in clinical practice
iii) Nabs+ve patients must be switched to alternative treatment.
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
POSTER PRESENTATIONS
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE III INTERFERON EXPRESSION DIFFER BETWEEN LOW-RISK AND
HIGH-RISK HUMAN PAPILLOMAVIRUS POSITIVE PATIENT CERVICAL
CELLS
Fabiana CANNELLA1, Giulia TRANQUILLI1, Claudia ZAMPETTI1, Carla SELVAGGI1, Carolina
SCAGNOLARI1, Nadia RECINE2, Patrizia STENTELLA2, Guido ANTONELLI1, Alessandra PIERANGELI1
1. Department of Molecular Medicine, Laboratory of Virology, Università di Roma "La Sapienza", Rome (Italy)
2. Department of Obstetric and Gynaecological Sciences and Urologic Sciences, Università di Roma "La Sapienza", Rome (Italy)
Abstract
A persistent infection by high-risk (HR) Human Papillomavirus (HPV) types is a pre-requisite for progression to
cancer. HPV persistence could be due to an impaired innate immunity; in particular, it has been recognized
that HR HPV inhibit the type I Interferon (IFN) response. In the mucosal innate immune response, an important
component is the recently described type III IFN, but very little is known on its role during HPV infection. To
clarify the question, we evaluated, for the first time, the activation of type III IFNs pathways (IFN-λ1, IFN-λ2/3,
the IFN-λ receptor complex, and the IFN induced genes ISG15, MxA and ISG56) during in vivo HPV infection
from about 150 patients. Results were analyzed and correlated with the HPV genotype risk, and with the
cytological grade of the lesions.
Despite an elevated individual variability, IFN-λ1 and IFN-λ2/3 levels were correlated with their receptor IFNλR1 values, and ISG15 values were correlated with IFN-λ1 and IFN-λR1 levels, in the same patient.
IFN-λ1 and IFN-λR1 were expressed at significantly higher level in patients with Low Risk (LR) HPV infection
compared to HR HPV and to HPV-negative cells. Furthermore, we observed that the expression of IFN-λ1
decreased significantly with the increase of the cytological grade of lesions. Besides, ISG15 was expressed at
a significantly higher level in patients with LR HPV infection compared to HR HPV and to HPV-negative cells.
In conclusion, our results suggests that LR-HPV infection stimulate and activate IFN-λ pathways in cervical
cells and that ISG15 could be a relevant ISG activated during this infection. Differently, HR-HPVs seemed to
interfere with type III IFN signaling in a biological relevant way, since we found lower levels of expression of
IFN-λ1 in patients with HR HPV infection and in patients with SIL. Further studies measuring IFN-related gene
expression in cervical cells from patients at baseline and at follow-up visits might be of value to clarify the
relative role of IFN-λ in determining HPV clearance or persistence. If the role of IFN-λ were confirmed, the
possibility of a therapeutic use of type III IFN in the treatment of HPV-associated lesions should be evaluated.
Poster 1
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IFN-λ1-3 EXPRESSION IN INFANTS HOSPITALIZED FOR RSV OR HRV
ASSOCIATED BRONCHIOLITIS
Carla SELVAGGI1, Alessandra PIERANGELI1, Maria CURTOSI1, Giulia CACCIOTTI1, Giulia ERRICO1,
Isabella CALICCHIA1, Massimo GENTILE1, Fabio MIDULLA2, Guido ANTONELLI1, Carolina SCAGNOLARI1
1. Department of Molecular Medicine, Laboratory of Virology, Università di Roma "La Sapienza", Rome (Italy)
2. Pediatric Department, Università di Roma "La Sapienza", Rome (Italy)
Abstract
The airway expression of type III interferons (IFNs) was evaluated in infants hospitalized for respiratory
syncytial virus (RSV) or rhinovirus (HRV) bronchiolitis. As an additional objective we sought to determine
whether a different expression of IFN-λ1-3 was associated with different harboring viruses, the clinical course
of bronchiolitis or with the levels of well established IFN stimulated genes (ISGs), such as mixovirus resistance
A (MxA) and ISG56.
The analysis was undertaken in 118 infants with RSV or HRV bronchiolitis. Nasopharyngeal washes were
collected for virological studies and molecular analysis of type III IFN responses.
RSV elicited higher levels of IFN-λ subtypes when compared with HRV. A similar expression of type III IFN
was found in RSVA or RSVB infected infants and in those infected with HRVA or HRVC infections. Results
also indicate that IFN-λ1 and IFN-λ2-3 levels were correlated each other and with MxA-mRNA. In addition, a
positive correlation exists between the IFN-λ1 levels and the respiratory rate during RSV infection.
These findings show that differences in the IFN-λ1-3 levels in infants with RSV or HRV infections does exist
and that the expression of IFN-λ1 correlates with the severity of RSV bronchiolitis.
Poster 2
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
DENDRITIC CELL ENGINEERING WITH IRF-1 AND IRF-8 FOR NEW
IMMUNOTHERAPY INTERVENTIONS
Alessandra FRAGALE1, Immacolata PIETRAFORTE2, Giulia ROMAGNOLI2, Elena TOSCHI2, Massimo
SANCHEZ3, Roberto ORSATTI1, Anna L. REMOLI1, Caterina GIULIANI1, Marco SGARBANTI1, Maurizio
FEDERICO4, Lucia GABRIELE*2, Angela BATTISTINI*1
*These authors contributed equally to this work
1.
2.
3.
4.
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
Department of Haematology, Oncology and Molecular Biology, Istituto Superiore di Sanità, Rome (Italy)
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome (Italy)
National AIDS Center, Istituto Superiore di Sanità, Rome (Italy)
Abstract
IFNs are drugs of choice in treatment of cancer and their antitumor effects are mediated by their potent
immunomodulatory ability. Two members of IFN Regulatory Factor (IRF) transcription factor family, IRF-1 and
IRF-8 that are known tumor suppressors involved IFN signaling, play a crucial role in the development and
maturation of both pDCs and mDCs, and, at least for IRF-1, in Treg development and function. To date,
several DC-based cancer vaccines showed modest efficacy in activating tumor specific immune responses.
This poses the need of enhancing efficacy of specific cancer immunotherapy by genetic DC engineering for
expression of immunostimulatory molecules. In this study we show that constitutive expression of IRF-1 and
IRF-8 in DCs by lentiviral vector transduction resulted in altered antigen-presentation and evocation of an
antitumor response. IRF-DCs indeed expressed increased levels of CD123 (IL3Rα), BDCA3, and showed a
parallel downregulation of markers of myeloid differentiation, such as BDCA1. We also identified IFN
stimulated responsive Elements (ISRE) on CD123 human promoter and found strong binding ability of IRF-1
and IRF-8 to this element in activated DCs. Overall these data indicate a direct transcriptional role of IRF-1
and IRF-8 in stably skewing DCs toward anti-tumor phenotype.
Poster 3
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IKAPPAB KINASE EPSILON TARGETS INTERFERON REGULATORY
FACTOR 1 IN ACTIVATED T LYMPHOCYTES
Marco SGARBANTI1, Giulia MARSILI1, Anna L. REMOLI1, Emilia STELLACCI1, Antonello MAI2, Dante
ROTILI2, Edvige PERROTTI1, Chiara ACCHIONI1, Roberto ORSATTI1, Nunzio IRACI3, Mathieu FERRARI4,
Alessandra BORSETTI4, John HISCOTT5, Angela BATTISTINI1
1.
2.
3.
4.
5.
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
Department of Drug Chemistry and Technologies, Università di Roma "La Sapienza", Rome (Italy)
Dipartimento di Chimica e Tecnologia del Farmaco, University of Perugia, Perugia (Italy)
National AIDS Center, Istituto Superiore di Sanità, Rome (Italy)
Vaccine & Gene Therapy Institute of Florida, Port Saint Lucie, Florida (USA)
Abstract
IkappaB kinase epsilon (IKK-ε) has an essential role as a regulator of innate immunity, functioning
downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present
study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4+ T
cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28
stimulation of primary CD4+ T cells; however, in T cells treated with poly(I:C), TCR/CD28 costimulation
blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated the transcription factor
IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4+ T cells and blocked its
transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction
between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA.
These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in
turn contributes to suppression of IFN-β production.
Poster 4
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
CANCER CHEMOTHERAPY AND VIRAL MIMICRY
Antonella SISTIGU1, Takahiro YAMAZAKI2, Erika VACCHELLI2, Kariman CHABA2, Catarina REMEDIOS2,
Dalil HANNANI2, Laetitia AYMERIC2, Mireia NISO-SANTANO2, Oliver KEPP2, Joachim SCHULTZE3, Thomas
TUTING4, Filippo BELARDELLI1, Valentina LA SORSA1, Laura BRACCI1, Giovanna ZICCHEDDU1, Paola
SESTILI1, Francesca URBANI1, Mauro DELORENZI5, Aicha GOUBAR2, Magali LACROIX-TRIKI6, Virginie
QUIDVILLE2, Rosa CONFORTI2, Jean-Philippe SPANO7, Lajos PUSZTAI8, Vichnou POIRIER-COLAME2,
Alexander EGGERMONT2, Marco BIANCHI9, Robert SCHREIBER10, Mark SMYTH11, Melvyn CHOW11, Gilles
UZÉ12, Enrico PROIETTI1, Fabrice ANDRÉ2, Guido KROEMER2, Laurence ZITVOGEL
1. Istituto Superiore di Sanità - Rome (Italy)
2. Institut Gustave Roussy - Villejuif (France)
3. University of Bonn - Bonn (Germany)
4. University Hospital Bonn - Bonn (Germany)
5. Swiss Institut of Bioinformatics - Lausanne (Switzerland)
6. Institut Claudius Regaud - Toulouse (France)
7. Hopital Pitie Salpetriere - Paris (France)
8. Yale School of Medicine - New Haven
9. Università Vita Salute San Raffaele - Milan (Italy)
10. Washington University School of Medicine - St. Louis (USA)
11. Queensland Institute of Medical Research - Herston
12. University Montpellier II - Montpellier (France)
Abstract
Distinct cell death-associated molecular patterns might define cancers proned to respond to a cytotoxic
therapy by mounting a protective T cell-based anticancer immunity. Here, we show that immunogenic
chemotherapy phenocopies viral infection leading to autocrine IFN-αβ/IFNAR1/2 signalling in tumor cells
initiated by recognition of self dsRNA by endosomal pattern recognition receptors (PRRs) that usually
recognize virus-encoded nucleic acids, namely TLR3/TRIF. In detail, TLR3/TRIF (endosomal dsRNA sensors)
and IFNAR1/2 (Type I IFN receptors) must signal within the tumor cells so that chemotherapy can induce
downstream CXCL10/CXCR3 axis and elicit therapeutic responsiveness in vivo. Moreover, the IFN fingerprint
of human breast cancers allowed to predict tumors proned to benefit from adjuvant anthracyclines.
From an evolutionary viewpoint, while tumors (like viruses) have evolved mechanisms to evade an IFN
response, chemotherapy-induced viral mimicry might contribute to bypass such as immunoediting.
Poster 5
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE I IFN IN TUBERCULOSIS: FRIEND OR FOE
Marilena P. ETNA1, Martina SEVERA1, Maria E. REMOLI1, Valérie GAFA1, Melania CRUCIANI1, Sandra
PELLEGRINI2, Manuela PARDINI1, Daria BOTTAI3, Roland BROSCH2, Eliana M. COCCIA1, Elena
GIACOMINI1
1. Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
2. Institute Pasteur - Paris (France)
3. Università di Pisa - Pisa (Italy)
Abstract
Efforts to develop strategies for containing Tuberculosis (TB) remain a priority. Accordingly, the currently used
bacillus Calmette-Guérin (BCG) vaccine partially protects against pulmonary TB and needs to be improved or
replaced. Given the crucial role played by dendritic cells (DC) in controlling protective T-cell response against
Mycobacterium tuberculosis (Mtb), we characterized DC reaction to the infection with BCG and Mtb.
BCG partially stimulated DC maturation and conversely to Mtb, did not induce IFN-β and IL-12 expression.
Interestingly, when BCG-infected DC were exposed to IFN-β, a fully mature phenotype was displayed and, in
turn, translated into an enhanced capacity to expand Th1 response suggesting IFN-β as a possible adjuvant.
To better understand the basis of BCG ineffectiveness, we analyzed the role of the RD1 locus, which is absent
in BCG and underlines Mtb virulence. The comparative analysis of maturation profile and released cytokines
obtained from DC infected with Mtb and BCG recombinant strains, indicated that RD1 is necessary but not
sufficient to stimulate type I IFN and, consequently, to promote IFN-γ producing T cells. Although IFN role in
TB immunopathogenesis is not clearly established, IFN-β release by DC participates to the creation of an
inflammatory milieu critical for the induction of effector immune responses.
Poster 6
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
EXOGENOUS HIV-1 NEF INDUCES PROINFLAMMATORY SIGNALING
EVENTS IN MURINE MACROPHAGES AND MICROGLIAL CELLS
Giorgio MANGINO1, Marylinda FAMIGLIETTI2, Valeria SERRA2, Zulema A. PERCARIO2, Caterina CAPONE2,
Stefano LEONE2, Gianna FIORUCCI3, Sebastian LÜLF4, Giovanna ROMEO1, Cristina AGRESTI5, Tiziana
PERSICHINI2, Matthias GEYER4, Elisabetta AFFABRIS2
1. Dept. Science, Università degli Studi “Roma Tre”, Dept.Medico-Surgical Sciences Biotechnologies, Università di Roma "La
Sapienza", Rome (Italy)
2. Dept. of Science, Università degli Studi “Roma Tre”, Rome (Italy)
3. Inst. of Molecular Biology and Pathology, CNR, Rome (Italy)
4. Center of Advanced European Studies and Research, Group Physical Biochemistry, Bonn (Germany)
5. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome (Italy)
Abstract
Murine (mu) cells are not permissive for Human Immunodeficiency Virus-1 (HIV-1), but transgenic mice
expressing selected HIV-1 genes revealed that Nef is a major disease determinant. Nef is a molecular adapter
that induces regulation of cell surface receptors expression and activation signals interacting with cellular
partners. In hu macrophages Nef expression induces the production of extracellular factors that recruit T cell
favouring their infection. In addition Nef transfers itself to uninfected cells via exosomes, cellular protusions or
cell to cell contacts and Nef treatment of hu macrophages induces its internalization and production of proinflammatory molecules. Here we describe that mu macrophages respond to Nef treatment activating IKK and
specific MAPKs. Activation of NF-κB is mandatory to produce inflammatory molecules inducing tyrosinephosphorylation of STAT-1, -2, and -3. Therefore mu macrophages respond to Nef similarly to hu ones. On the
other hand Nef treatment of mu microglial cells leads to nitric oxide (NO) production and upregulates the
expression of inducible NO synthase (iNOS). Nef regulates iNOS through NF-κB and IFN-β release. Both
myristoylation and the conserved acidic cluster of the viral protein are required for all those effects. Finally Neftreated mu microglial cells appears to produce neurotoxic factors.
Poster 7
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE I IFN AND 5-AZA-2’-DEOXYCITIDINE SYNERGIZE FOR THE INHIBITION
OF SOLID CANCER PROGRESSION
Fabrizio MATTEI1, Maria BUONCERVELLO1, Valeria LUCARINI1, Giulia ROMAGNOLI1, Elena TOSCHI1,
Alessandra FRAGALE1, Caterina GIULIANI1, Martina MUSELLA1, Massimo SPADA1, Filippo BELARDELLI1,
Giovanna SCHIAVONI1, Lucia GABRIELE1
1. Istituto Superiore di Sanità - Rome (Italy)
Abstract
Type I Interferons (IFN-I) exert anti-tumor activities through both direct tumor cell growth inhibition and
stimulation of host immune responses. Resistance to IFN-I-induced anti-neoplastic effects has been reported
in many hematologic and solid malignancies, and arise in part from epigenetic silencing by DNA methylation of
IFN-stimulated genes that limit IFN-I responsiveness in cancer cells. 5-Aza-2’-Deoxycitidine (Aza) is an antimethylating drug that reactivates epigenetically silenced tumor-suppressor genes. Therefore, Aza-induced
epigenetic restoration of IFN-stimulated genes may enhance the susceptibility to IFN-I-induced anti-tumoral
effects. In this study, we evidenced that a combined administration of IFN-I and Aza significantly inhibits the
growth of melanoma and colon carcinoma. IFN-I/Aza-treated tumor cells exhibit a higher apoptotic rate and
undergo cell cycle arrest, with respect to control cells and to cells exposed to single treatments. When
transplanted into recipient mice, IFN-I/Aza-treated cancer cells display a decreased tumorigenic potential, with
respect to controls. Overall, our findings suggest that a combined synergy between Aza and IFN-I may be
essential for a more effective inhibition of tumor growth and provide insights on the possible use of therapeutic
strategies involving the combined use of epigenetic drugs with immunotherapy for improving cancer
management.
Poster 8
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IMMUNOMODULATORY AND ANTIVIRAL EFFECTS OF IFN-β THERAPY IN
MULTIPLE SCLEROSIS: TWO FACES OF THE SAME COIN. FOCUS ON B
LYMPHOCYTES
Fabiana RIZZO1, Elena GIACOMINI1, Rosella MECHELLI2, Viviana ANNIBALI2, Silvia ROMANO2, Marco
SALVETTI2 , Eliana M. COCCIA1 , Martina SEVERA1
1. Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
2. S. Andrea Hospital-site, Università di Roma "La Sapienza", Rome (Italy)
Abstract
Type I IFNs are inducible cytokines with potent antiviral, anti-proliferative and immunomodulatory effects.
Among them, IFN-β is commonly used for treatment of Relapsing-Remitting Multiple Sclerosis (MS)
decreasing relapse rate and slowing progression of disability.
MS was always considered a T cell-driven disorder, however implication of B lymphocytes in MS
immunopathology is increasingly recognized. Here, impact of IFN-β therapy on B cell biological functions was
evaluated.
Our studies revealed a specific defect in TLR7-mediated B cell response. Frequency of TLR7-induced Igsecreting cells was significantly lower in MS patients than in healthy donors (HD), but that was replenished
upon IFN-β therapy. These results perfectly mirrored the impaired expression of TLR7 mRNA observed in
PBMCs from MS-affected individuals, and specifically in monocytes, a key cell type for B cell maturation and
differentiation.
IFN-β also reduces the number of circulating memory B lymphocytes. Interestingly, these cells are also the
natural reservoir of Epstein-Barr virus (EBV) infection, which growing evidences associate with MS
pathogenesis. In line with these data, we observed higher EBV gene expression in MS patients than in HD,
level restored by IFN-β.
Thus, we can conclude that IFN-β treatment in MS may play a dual role by exerting both immunomodulatory
and anti-viral activities.
Poster 9
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IFNAR-1 UPREGULATION IN PBMC FROM HCV NAÏVE PATIENTS CARRYING
CC GENOTYPE. POSSIBLE ROLE OF IFN-λ
Eleonora LALLE1, Licia BORDI1, Claudia CAGIOTI1, Anna Rosa GARBUGLIA1, Concetta CASTILLETTI1,
Laura TAIBI1, Francesca CRISTOFARI1, Maria Rosaria CAPOBIANCHI1
1. National Institute for Infectious Diseases “L. Spallanzani", Rome (Italy)
Abstract
The study aim was to establish possible relationships between IL28 rs12979860 genotype and expression of
IFNAR-1 in naive HCV patients, and to explore the possible role of IFN-λ.
The ability of IFN-λ to up-regulate the expression of IFNAR-1 was established in PBMC from naive patients
with chronic hepatitis C and from healthy donors carrying different IL-28B genotypes.
IFNAR-1 mRNA levels in PBMC from naive patients were lower than in PBMC from healthy donors. While in
healthy donors IFNAR-1 mRNA levels were independent from IL28B genotype, in CHC an increasing gradient
of expression was observed in TT vs CT vs CC carriers. In the latter group, a direct correlation between
IFNAR-1 and endogenous IL28B expression was observed. IFN-λ up-regulated IFNAR-1 expression in normal
PBMC in a time-and dose-dependent manner, with a more effective response in CC vs TT carriers.
Endogenous levels of IFN-λ may be responsible for partial restoration of IFNAR-1 expression in CHC patients
with favourable IL28 genotypes. This, in turn, may confer to CC carriers a response advantage to either
endogenous or exogenous IFN-α, representing the biological basis for the observed association between CC
genotype and favourable outcome of either natural infection or IFN therapy.
Poster 10
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
HIV-1 NEF INDUCES PROINFLAMMATORY STATE IN HUMAN
MACROPHAGES THROUGH ITS ACIDIC CLUSTER DOMAIN: INVOLVEMENT
OF TNF-α RECEPTOR ASSOCIATED FACTORS
Zulema A. PERCARIO1, Giorgio MANGINO2, Gianna FIORUCCI3, Gabriele VACCARI4, Filippo ACCONCIA1,
Cristiano CHIARABELLI1, Stefano LEONE1, Alessia NOTO1, Florian A HORENKAMP5, Santiago MANRIQUE5,
Giovanna ROMEO7, Fabio POLTICELLI1, Matthias GEYER6, Elisabetta AFFABRIS1
1. Department of Science, Università degli Studi “Roma Tre”, Rome (Italy)
2. Dept Science, Università degli Studi “Roma Tre” and Dept Medical-Surgical Sc. Biotechnologies, "La Sapienza" University of
Rome, (Italy)
3. Institute of Molecular Biology and Pathology, CNR, Rome (Italy)
4. Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Rome (Italy)
5. Abteilung Physikalische Biochemie, MPI für Molekulare Physiologie, Dortmund (Germany)
6. MPI für Molekulare Physiologie, Dortmund, and Center of Advanced European Studies and Research, Group (Germany)
7. Department of Medical-Surgical Sciences and Biotechnologies, Università di Roma "La Sapienza", Rome (Italy)
Abstract
HIV-1 Nef is a major virulence factor of HIV. It intersects the CD40 signalling in macrophages leading to
modification in the pattern of secreted factors that recruit and render T lymphocytes susceptible to HIV
infection. CD40 - CD40L signalling cascade involves specific tumor necrosis factor receptor-associated factors
(TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3
in Nef-treated macrophages leading to the production of proinflammatory cyto-chemokines and IFN-β.
Searching for TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence
encompassing the conserved four-glutamate acidic cluster. The integrity of the acidic cluster is required for the
signalling effects induced in Nef-treated macrophages. In addition, Nef interacts in vitro with TRAF2, but not
TRAF6, and this interaction involves the acidic cluster. Finally silencing of both TRAF2 and, surprisingly,
TRAF6 abolishes the Nef-induced tyrosine phosphorylation of STAT1 and STAT2 in THP-1 monocytic cells.
Conclusion: we propose TRAF2 as a new possible cellular interactor of Nef and highlights that Nef is able to
manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes in monocytes/macrophages, thereby
inducing the synthesis of proinflammatory cyto-chemokines as well as IFN-β.
Poster 11
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
IFN-α ANTAGONIZES REGULATORY T CELL-MEDIATED SUPPRESSION
AND FAVORS THEIR PLASTICITY INTO TH1-LIKE CELLS
Ilenia PACELLA1, Silvia PICONESE1, Eleonora TIMPERI1, Daniele ACCAPEZZATO1, Giancarlo LABBADIA1,
Vincenzo BARNABA1
1. Università di Roma "La Sapienza", Rome (Italy)
Abstract
Regulatory T cells (Treg) are classically viewed as immune suppressive cells, tipping the balance between
host defense from pathogens and prevention of excessive immunity. However, under inflammatory conditions,
Treg can be de-programed into cytokine-producing cells. The purpose of this study was to characterize the
functional changes induced in Treg upon exposure to IFN-α.
Recombinant IFN-α exerted in vitro an anti-proliferative effect against both conventional T cells (Tconv) and
Treg, without affecting the Treg-intrinsic suppressive function in Tconv-Treg cocultures. We tested IFN-α
effects in vivo in patients with chronic HCV infection undergoing peg-interferon/ribavirin therapy, before and 2
days after the starting of therapy. In this setting, IFN-α decreased the frequency of Treg, and especially of the
most suppressive Treg subset, mostly by enhancing apoptosis. The polarization of IFN-γ-producing, so called
“Th1-like”, plastic Treg was boosted by IFN-α, both in vitro and in vivo. Of note, IFN-γ secretion by Treg and
Tconv cell subsets showed a linear correlation, suggesting that Tconv-derived IFN-γ might play a relevant role
in Treg diversion into Th1-like cells.
Our data indicate that IFN-α may amplify Th1-type inflammation by limiting the pool of fully suppressive Treg
and concomitantly fostering Treg de-programming into Th1-like cells.
Poster 12
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
NOVEL INSIGHTS INTO THE DAMP-LIKE PRO-INFLAMMATORY BEHAVIOUR
OF MISLOCALIZED NUCLEAR IFI16 IN THE EXTRACELLULAR SPACE
Mandar BAWADEKAR1, Marco DE ANDREA2, Valeria CANEPARO3, Valentina DELL'OSTE2, Santo
LANDOLFO2, Marisa GARIGLIO1
1. Department of Translational Medicine & Interdisciplinary Research Center of Autoimmune Diseases (Italy)
2. Department of Public Health and Pediatric Sciences, Medical School of Turin (Italy)
3. NoToPharm s.r.l. Colleretto Giacosa, Ivrea (Italy)
Abstract
The intracellular DNA sensor IFI16 protein has been much implicated towards its role in inflammasome
signaling and as viral restriction factor. Following transfection of virus-derived DNA, or treatment with UVB,
IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular
milieu. Recently, our group has demonstrated the occurrence of IFI16 in the sera of systemic autoimmune
patients which hampers the biological activity of endothelia through high affinity membrane binding. It seems
that during cell death/apoptosis/necrosis, endogenous nuclear IFI16 is passively released into the extracellular
space. In this study, we have addressed the pro-inflammatory activity of endotoxin free recombinant IFI16
protein on endothelial cells. We assessed m-RNA levels of a group of inflammatory cytokines/chemokines/
receptors in endothelial cells after endo-free rIFI16 treatment. HUVEC have shown to express significantly
high levels of m-RNA of inflammatory chemokines such as CCL2, CCL5 and CXCL8. A clear passage of the
p65 domain of Nf-κB into the nucleus was observed using confocal microscopy which was confirmed by
EMSA. Using luciferase assays we obtained evidence that the expression of these chemokines is regulated by
Nf-κB and AP-1 transcription factors. Moreover, using single analytical ELISA assays we demonstrate the
production of these chemokines is rIFI16 dose dependent and time dependent manner. Altogether our data
suggest that extracellular IFI16 protein acts like DAMPs, as once released from the cells, it can exert chronic
autocrine and paracrine actions to stimulate inflammation promoting environment, which might be responsible
for the development of autoimmunity.
Poster 13
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
TYPE III INTERFERON (IFN-λ) ANTAGONIZES THE ANTIVIRAL ACTIVITY OF
INTERFERON-ALPHA IN VITRO
Licia BORDI1, Eleonora LALLE1, Daniele LAPA1, Claudia CAGLIOTI1, Serena QUARTU1, Maria Rosaria
CAPOBIANCHI1, Concetta CASTILLETTI1
1. National Institute for Infectious Diseases “L. Spallanzani”, Rome (Italy)
Abstract
Type III interferons (IFN-λ) are the most recently discovered members of IFN family. Synergism between
different IFN types is well established, but for type I and type III IFNs no conclusive evidence has been
reported so far.
Possible synergism/antagonism between IFN-α and/or IFN-λ in the inhibition of virus replication (EMCV, WNV
lineage 1 and 2, CHIKV, CCHF and HSV-1), and in the activation of intracellular pathways of IFN response
(MxA and 2’-5’ OAS) was evaluated in different cell lines (Vero E6, A549, HuH-7 and Wish cells).
The antiviral potency of IFN-λ1 and -λ2 was lower than that of IFN-α. When IFN-α and -λ were used
together, the Combination Index (CI) for virus inhibition was >1 virtually for all virus/host cell systems,
indicating antagonistic effect. Antagonism between IFN-α and -λ was also observed for the induction of
mRNA for both MxA and 2’-5’OAS.
Elucidating the interplay between IFN-α and -λ may help to better understand innate defence mechanisms
against viral infections, including the molecular mechanisms underlying the influence of IL-28B polymorphisms
in the response to HCV and other viral infections.
Poster 14
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
CHARACTERIZATION OF THE ROLE OF TYPE I AND III INTERFERON
RESPONSE DURING WEST NILE VIRUS AND USUTU VIRUS INFECTION
Giulia CACCIOTTI1, Carla SELVAGGI1, Giulia RIBELLI1, Katia MONTELEONE1, Giulia ERRICO1, Claudio
LORENZI1, Anna PAPA2, Massimo GENTILE1, Alessandra PIERANGELI1, Guido ANTONELLI1, Carolina
SCAGNOLARI1,
1. I Department of Molecular Medicine, Laboratory of Virology, Università di Roma "La Sapienza", Rome (Italy)
2. Department of Microbiology, National Reference Centre for Arboviruses, Medical School, Aristotle Uni
Abstract
Previous studies have demonstrated that interferon (IFN) and specific IFN stimulated genes (ISGs) can
restricts West Nile virus (WNV) and USUTU virus (USUV) replications. Therefore, we performed a research
aimed at evaluating the role of type I and III interferon (IFN) response during the natural history of USUV and
WNV (Lineage 1 and 2) infection. In particular, we measured, in vitro, the sensitivity of USUV (strain Vienna
2001-blackbird) and WNVs (lineage 1 strain NY 99 and lineage 2 strain Austria) to the antiviral activity of type I
and III IFNs in A549 (adenocarcinomic human alveolar basal epithelial cells) and SK-N-SH (human
neuroblastoma cells). We also evaluated the activation of IFN responses in USUV and WNVs infected human
dendritic cells (DCs). Furthermore, in order to further understand the role of IFN during WNV infection, we
measured levels of ISG15, an well known IFN induced protein, in sera collected from WNV infected patients
during the 2010 outbreak of WNV infection in Greece. Results were compared with those obtained from the
analysis of ISG15 levels in sex and aged matched healthy controls. We found that IFN-α, -β and -λ 1-3 can
reduce in vitro significantly the replication of WNV (lineage 1 and 2) and USUV. We observed also that USUV
is more sensitive to the antiviral activity of types I and III IFNs than WNVs. In addition, we observed that the
levels of IFN-α detected in DCs infected with USUV at different multiplicity of infections (MOI 0.01-0.1-1
TCID50/cell) are significantly higher than those found in DCs infected with both WNV lineage 1 and 2. As far
as the analysis of ISG15 levels during human WNV infection is concerned, the main results indicate that the
serum ISG15 levels were significantly reduced in WNV positive patients compared to healthy individuals. No
significant correlations were observed between ISG15 levels and the clinical severity in WNV infected patients.
In conclusion our data indicates that WNV can evade or control the human IFN response more efficiently than
USUV. In addition we found that there is an impaired IFN induced ISG15 response in WNV infected patients
suggesting that a defect in the innate immune system may be determinant during human WNV infection.
Poster 15
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
PRO-INFLAMMATORY CYTOKINE ANALYSIS IN HPV-POSITIVE CANCER
CELLS
Girogio MANGINO1, Maria Simona ZANGRILLO1, Maria Vincenza CHIANTORE2, Marco IULIANO1, Martina
SEVERA2, Gianna FIORUCCI3, Eliana M. COCCIA2, Giovanna ROMEO1
1. Department of Medico-Surgical Sciences and Biotechnologies, Università di Roma "La Sapienza", Rome (Italy)
2. Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
3. Inst. of Molecular Biology and Pathology, CNR, Rome (Italy)
Abstract
Recent data expanded the concept that inflammation is a critical component of tumor progression. The tumor
microenvironment is an indispensable participant in the neoplastic process, fostering proliferation, survival and
migration. Tumor cells co-opted signalling molecules of the innate immune system, such as selectins,
chemokines and their receptors for invasion, migration and metastasis. In this respect, Virus-induced tumors,
like Papillomavirus-induced Squamous Cell Carcinomas (SCC), could represent a paradigmatic example of
interplay between inflammatory responses and malignant transformation.
To establish a tumorigenic role of inflammatory mediators in HPV+ SCC, we analyzed by real time RT-PCR
the expression of inflammatory cytokines, chemokines and related molecules in HPV+ carcinoma cell lines,
HPV- SCC (C33A) and in human foreskin keratinocytes transduced by E6 and E7 derived from mucosal HPV16 or cutaneous HPV-38 genotypes. We also tested the effect of the IFN-β on the levels of these proinflammatory mediators.
In HPV+ SCC the level of both IL-1β and IL-6 mRNAs were augmented if compared to HPV- SCC.
Interestingly, levels of cytokines in supernantants, tested by CBA assay, revealed that despite huge increase
in mRNA levels, these cells didn’t secrete IL-1β, whereas IL-6 appeared to be released by HPV+ SCC.
Poster 16
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
MICRORNA PROFILING IN E6/E7 HPV-TRANSFORMED HUMAN
KERATINOCYTES. THE EFFECT OF IFN-β.
Maria Vincenza CHIANTORE1, G. MANGINO2, M. IULIANO2, S. ZANGRILLO2, Gabriele VACCARI3, R.
ACCARDI4, M. TOMMASINO4, Elisabetta AFFABRIS5, Gianna FIORUCCI1,6, Giovanna ROMEO1,2,6
1.
2.
3.
4.
5.
6.
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome (Italy)
Dept of Medico-Surgical Sciences and Biotechnologies, Università di Roma "La Sapienza", Rome (Italy)
Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Rome (Italy)
Infections and Cancer Biology Group, International Agency for Research on Cancer-WHO, Lyon (France)
Department of Biology, Università degli Studi “Roma Tre”, Rome (Italy)
Institute of Molecular Biology and Pathology, CNR, Rome (Italy)
Abstract
MicroRNAs (miRNAs) are naturally occurring noncoding RNAs that play a key role in gene regulation by
cleaving the mRNA targets or repressing their translation. This posttranscriptional gene regulation is active in
fundamental cellular processes as cell proliferation, differentiation, and death. Accumulating evidence
indicates a role of miRNAs in tumorigenesis, tumor cells bearing a specific and altered pattern of miRNA
expression. Type I IFN can modulate cellular miRNAs, indicating that they may be used by mammalian
organisms as a target of IFN system to combat viral infection and related diseases.
We performed a high throughput screening using a TaqMan MicroRNA assay for the quantitation of 384
miRNAs in primary human keratinocytes compared to keratinocytes transformed by E6 and E7 oncoproteins
from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38 cells, respectively). Our analysis identified different
miRNAs mainly deregulated in either K16 and/or K38 cells, successively tested using miRNA single assay.
Since IFN-β may interfere with HPV E6 and/or E7-dependent transformation, we studied miRNA expression in
K16 and K38 cells treated with this cytokine. Preliminary results show that also in our system IFN-β can affect
the expression of some miRNAs, according to its ability to inhibit proliferation of HPV-transformed
keratinocytes.
Poster 17
www.iss.it/ifnf/
Interferon Fundamentals 2014
From Molecular Mechanisms to Human Diseases
February 20, 2014
Istituto Superiore di Sanità, Rome
ROLE OF IFN-I IN THE SPONTANEOUS DEVELOPMENT OF MAMMARY
TUMORS IN MICE KNOCK-OUT FOR TYPE I IFN RECEPTOR AND
TRANSGENIC FOR THE HER2/NEU ONCOGENE
Eleonora ARICÒ1, Paola SESTILI1, Manuela IEZZI2, Alessia LAMOLINARA2, Fiorella CIAFFONI3, Giovanna
SCHIAVONI1, Domenica M. MONQUE1, Maria FERRANTINI1, Enrico PROIETTI1 and Filippo BELARDELLI1
1. Department of Haematology, Oncology and Molecular Biology, Istituto Superiore di Sanità, Rome (Italy)
2. Immuno-Oncology Laboratory, Aging Research Center (CeSI), G.d'Annunzio University Foundation, Department of Medicine and
Aging Sciences G. d'Annunzio University of Chieti-Pescara (Italy)
3. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome (Italy).
Abstract
Mice transgenic for the HER-2/neu oncogene and knock-out for type I IFN receptor were generated in our
laboratory to analyze the role of endogenous IFN-I in the spontaneous carcinogenesis occurring in hosts that
are tolerant to the HER-2/neu oncoantigen.
Our results indicated that the development of mammary adenocarcinomas was significantly exacerbated in
HER-2/neu+/- IFNARI-/- with respect to control HER-2/neu+/- IFNARI+/+ mice, in terms of early onset as well as
increased tumor multiplicity throughout mice life-span. The morphologic and histologic features of mammary
glands isolated at different phases of carcinogenesis progression indicated that tumor lesions developed in the
lack of a functional IFN-I system showed an increased vascularization and the enlargement of blood vessels
when compared with immunocompetent counterpart. However, the analysis of the immune cells infiltrating the
mammary tumors as well as the immunophenotype of blood and lymphoid organs showed no major difference
between IFNARI+/+ vs IFNARI-/- mice.
Interestingly, microarray analysis of transcription profiles revealed that tumor-bearing mammary glands
isolated from both IFNARI-/- and IFNARI+/+ shared many of the most significantly up-regulated transcriptional
patterns already reported to be linked to HER-2/neu-carcinogenesis, including genes related to cell cycle and
associated with the inflammatory response. In the light of these results, we believe that the pathways of
inflammation activated in HER-2/neu tumors during carcinogenesis may overcome the lack of a functional IFNI system, as documented by the similar tumor transcriptional profiles, thus leading to similar downstream
immune-related phenomena.
In spite of the immune-related similarities, tumors arising in HER-2/neu+/- IFNARI-/- mice show the
overexpression of molecules involved in tumor invasiveness and metastasis, which was coupled with the
higher incidence of lung metastasis as compared to the wt counterpart.
Moreover, HER-2/neu+/- IFNARI-/- mammary tumors show increased tumorigenic capacity when implanted in
immunodeficient NOD SCID γ mice, thus suggesting a possible role of IFN-I in controlling cancer initiating
cells, which is currently under further investigation.
Poster 18
www.iss.it/ifnf/