3 BELGRADE EFIS SYMPOSIUM ON IMMUNOREGULATION
Immunity, Infection, Autoimmunity and Aging

Hotel Izvor, Arandjelovac Spa (Belgrade)
24-27 May 2015

IMMUNOLOGICALSOCIETY OF SERBIA

Organizers: Miodrag L. Lukic, Janko Nikolich-Zugich, Stipan Jonjic
UNIVERSITY OF KRAGUJEVACFACULTY OF MEDICAL SCIENCES

Welcome
It is our pleasure to invite you to welcome you in Arandjelovac at theoccasion of the 3rd Belgrade EFIS Symposium on Immunoregulation.
As the previous two, the meeting is organized under the auspices andwith support of European Federation of Immunological Societies.
The meeting will offer the lectures and discussions related to several
major topics of contemporary immunology. The list of speakers
includes outstanding scientists from Europe, Asia and North America
and oral presentations from the participants. There will be also time
for poster presentations and discussions with invited speakers.
Arandjelovac Izvor Spa is an ideal place for relaxed and productive
scientific meeting and offers pleasant environment for informal
discussions.
We have also organized sightseeing of Belgrade.
Belgrade is a hospitable city at the confluence of the two major
European rivers (Danube and Sava). It is one of the oldest cities in
Europe with tumultuous history and vibrant cultural life andentertainment at present.
Once again, we wholeheartedly welcome you.
Miodrag L. Lukic
Janko Nikolich-Zugich,
Stipan Jonjic

ORGANIZERS:
Miodrag L. Lukic M.D PhD
Center for Molecular Medicine
Faculty of Medicine, University of Kragujevac, Serbia
e-mail: miodrag.lukic@medf.kg.ac.rs
Stipan Jonjic, MD, PhD,
Department for Histology and Embryology
Center for Proteomics
School of Medicine, University of Rijeka, Croatia
e-mail: stipan.jonjic@medri.uniri.hr
Janko Nikolich-Zugich MD, PhD
Department of Immunobiology, University of Arizona, University of Arizona, Tucson, AZ
e-mail: jnikolich@medadmin.arizona.edu
LOCAL ADVISORY BOARD (Serbian Society of Immunology)
Nebojsa Arsenijevic, Chair, Faculty of Medical Sciences, University of Kragujevac
Miodrg Colic, Faculty of Medicine, Military, University of Belgrade
Marija Mostarica-Stojkovic, Faculty of Medicine, University of Belgrade
Stanislava Stosic-Grujicic, Institute of Biological Research, Belgrade
Nada Pejnovic, Faculty of Medical Sciences, University of Kragujevac
Gordana Leposavic, Immunology Research Center "Branislav Jankovic" Belgrade
INTERNATIONAL ADVISORY BOARD
Foo Y Liew (Glasgow) University of Glasgow
Hartmut Wekerle (Munich) Max Plank Institute for Neurobiology, Munich
Hannes Stockinger (Vienna) Medical University of Vienna
LOGISTICAL ORGANIZATION AND INQUIRIES:
Ana Miloradovic
e-mail: ana@medf.kg.ac.rs
Aleksandar Arsenijevic - participantse-mail: aleksandar@medf.kg.ac.rs
Dragana Nikolich-Zugich - non-European Invited Speakers
e-mail: draganaz@email.arizona.edu
THIS SYMPOSIUM IS SPONSORED BY:
EFIS European Federation of Immunology Societies
Center for Molecular Medicine, Faculty of Medical Sciences,
University of Kragujevac, Serbia
џ Serbian Society of Immunologyџџ                           ! !" ##PROGRAMDAY 1: SUNDAY

17.00-17.30 Welcome (M. L. Lukic, J. Nikolich-Zugich, S. Jonjic)Opening session
Chairs: A. Erdei - J. Nikolich - Zugich
17.30-18.00 Lorenzo Moretta (Institute Giannina Gasilini, Genova, Italy)- Natural killer cells: new approaches in the therapy of high risk leukemias

18.00-18.30 Rene Van Lier (Sanquin Blood Supply Foundation, Amsterdam, The Netherlands)- Properties of tissue-resident CD8Ά memory Tcells
18.30-19.00 Foo Y. Liew (University of Glasgow, Glasgow, UK)
19.30-- The role of cytokines in infections and inflammation
Conference dinner (by invitation)

DAY 2: MONDAY

07.30-08.30 Breakfast
Innate immune response in health and disease
Chairs: R. Van Lier - S. Jonjic
08.30-09.00 Marco Colonna (Washington University, St. Louis, MO, USA) - Innate lymphoid cells in immunity
09.00-09.30 Adrian Hayday (Kings College, London, UK) - Epithelial regulation of intraepithelial T cell
repertoires and response

09.30-10.00 Andrew N. McKenzie (University of Cambridge, Cambridge, UK) - Type-2 innate lymphoid cells
in immunity and disease

10.00-10.30 Andreas Diefenbach (Spemann Graduate School of Biology and Medicine, Institute of Medical
Microbiology & Hygiene, Germany) - Transcriptional control of innate lymphoid cell fatedecisions10.30-11.00 Coffee break
Lymphocyte repertoire and immune response
Chairs: B. Kyewsky - G. Leposavic
11.30-12.00 Kristin A. Hogquist (University of Minnesota, Minneapolis, MN, USA) - TCR reactivity in thymic
selection

12.00-12.30 Stephen Jameson (University of Minnesota, Minneapolis, MN, USA) - The transcription factor
KLF2 regulates T cell trafficking and differentiation                           ! !" ##12.30-13.00 Yousuke Takahama (Tokushima University, Tokushima, Japan) - Thymic microenvironments thatform T cell repertoire

13.00-13.30 Juan Carlos Zuniga-Pflucker (University of Toronto, Toronto, Canada) - Initiation of the T-lineage
development program in response to Notch signals requires GATA-3 for survival and fullcommitment of early progenitors

13.30-15.30 Break and poster viewing
15.30-17.30 Immunometabolism and glycoimmunology
Chairs: S. Gay - S. Stosic Grujicic
15.30-16.00 Marc Donath (University of Basel, Basel, Switzerland) - Targeting inflammation in treatment ofType 2 diabetes: time to start

16.00-16.30 Yvette Van Kooyk (University of Amsterdam, Amsterdam, The Netherlands) - Nanotechnology
aimed to design DC targeting vaccines for the induction of tumor-immunity

16.30-17.00 Nada Pejnovic (University of Kragujevac, Kragujevac, Serbia) - Galectin-3 in type 2 diabetes andimmunometabolism

17.00-17.30 Bojan Polic (University of Rijeka, Rijeka, Croatia) - NK cells link obesity-induced adipose stress to
inflammation and insulin resistance

17.30-19.00 Free time
19.00-20.00 Dinner
20.00-22.00 Poster discussion with refreshment

DAY 3: TUESDAY

07.30-08.30 Breakfast
08.30-12.30 Mechanism of tolerance, autoimmunity and immunopathology
Chairs: Y. Van Kooyk - M. L. Lukic
08.30-09.00 Bruno Kyewski (German Center for Cancer Research, Heidelberg, Germany) - Self-antigen
diversity in the thymus: what thymocytes see and don`t see

09.00-09.30 Steffen Gay (University of Zurich, Zurich, Switzerland) - Epigenetic regulation of inflammation in
autoimmune diseases

09.30-10.00 Sergei Nedospasov (Russian Academy of Science, Moscow, Russia) - Dissecting pathogenic
sources of TNF in disease and cell type-restricted cytokine targeting

10.00-10.30 Anna Erdei (Eotvos Lorand University, Budapest, Hungary) - Crosstalk between complement andToll-like receptors; regulation of human B cell responses under physiological and autoimmune
conditions

10.30-11.00 Coffee break
11.00-11.30 Burkhard Becher (University of Zurich, Zurich, Switzerland) - Cytokine networks in autoimmunity:
How helper T cells instruct macrophages                           ! !" ##11.30-12.00 Branka Horvat (International Center for Infectiology Research-CIRI, Lyon, France) - Emerging
contagion: immunopathogenesis of henipavirus infection

12.00-13.30 Short presentations
Chairs: Dj. Miljkovic – M. Colic
Megan Smithey (University of Arizona, College of Medicine, Tucson, USA) - Aging with MCMV maintains TCR
repertoire diversity in late life
Branka Popovic (Faculty of Medicine, University of Rijeka, Rijeka, Croatia) - IL-33-dependent immunosuppressive
Treg responses to liver damage during MCMV infection
Vivian Turner (University of Edimburgh, Edimburgh, UK) - Dysregulated function of marginal zone B cells inaged mice
Marija Milovanovic (Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia) - CMV infection
in neonatal and adult mice induces susceptibility to EAE in resistant BALB/c mice
Kamar Sulu Atretkhany (Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow,
Russia) - Systemic TNF ablation results in delayed tumor growth and reduced MDSC accumulation intransplantable tumor model
Bojan Jevtic (Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia) MicroRNA-155 Contributes to Re-activation of Encephalitogenic T Cells

13.30-14.30 Break and poster reviewing
14.30-17.00 Persistent and long term infections and their impact on aging
Chairs: M. Colonna – B. Polic
14.30-15.00 Beatrix Grubeck-Loebenstein (University of Innsbruck, Innsbruck, Austria) - Aging and adaptive
immunity in the human bone marrow

15.00-15.30 Arne Akbar (University College London, London, UK) - The regulation of T cell senescence andmetabolism by p38 MAPkinease signaling

15.30-16.00 Annette Oxenius (Swiss Federal Institute, Zurich, Switzerland) - Regulation of T cell Immunity
during viral infections

16.00-16.30 Stipan Jonjic (University of Rijeka, Rijeka, Croatia) - Vaccines and innate immunity: lessons fromcytomegalovirus immunoevasion

16.30-17.00 Janko Nikolich-Zugich (University of Arizona, Tucson, AZ, USA) - TGF-β impairs CD4 andantibody responses in old mice to increase Chikungunya virus disease severity and viral persistence

17.30-24.00 Excursion and conference dinner                           ! !" ##DAY 4: WEDNESDAY

07.30-08.30 Breakfast
Short presentation
Chairs: M. Smithey – V. Volarevic
Miljana Momcilovic (Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade,
Serbia) - In Vitro Effects of Binuclear (η6-p-cymene) Ruthenium (II) Complex Containing Bridging Bis (nicotinate)Polyethylene Glycol Ester Ligand on Differentiation Pathways of Murine Th Lymphocytes
Lovro Lamot (University of Zagreb School of Medicine, Zagreb, Croatia) -TRP channels overexpression
contributes to inflammasome activation in clavicular cortical hyperostosis
Gergely Toldi (Semmelweis University, Budapest, Hungary) -Impact of aging on calcium influx and potassiumchannel characteristics of T lymphocytes
Vladislav Volarevic (Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia) - Mesenchymal
stem cells attenuate acute liver injury mediated by NKT cells

Microbiota, Autoimmunity Inflammation
Chairs: S. Nedospasov – N. Pejnovic
Leo Joosten (University of Nijmegen, Nijmegen, The Netherlands - Trained Immunity:

09.30-10.00 consequences for inflammatory diseases?

Hannes Stockinger (Medical University of Vienna, Vienna, Austria) - Novel macrophage subsets

10.00-10.30 with potential implication in inflammatory diseases

Hartmut Wekerle- (Max Planck Institute for Neurobiology, Munich, Germany) - Ignitions of brain

10.30-11.00 autoimmune diseases in the gut

Ofer Mandelboim (Immunology and Cancer Research Hebrew University, Faculty of Medicine,

11.00-11.30 Jerusalem, Israel) - Recognition of bacteria by NK cells

Vishwa Deep Dixit (Yale University, New Haven, CT, USA) - Immunometabolic control of age-

11.30-12.00 related inflammation

Chairs: V. Pravica-M. Kataranovski
12.00-12.30 N. Avrion Mitchison (University College London, London, UK)
Clossing comments

NATURAL KILLER CELLS: NEW APPROACHES IN THE THERAPY OF HIGHRISK LEUKEMIAS
Lorenzo Moretta
Istituto Giannina Gaslini, Genova

Recently, haplo-HSCT has been further developed with the direct infusion,
together with CD34+ HSC, of donor-derived mature alloreactive NK cells
and TCRg/d+ T cells (obtained by depletion of TCRa/b+ T cells and CD19+
B cells). Both these cell types contribute to a rapid anti-leukemia effect
together with an efﬁcient defense against pathogens during the 6-8 week
interval required for the generation of alloreactive NK cells from HSC. The
results of this novel approach are particularly promising and further support
the usefulness of NK cell-based immunotherapy of otherwise fatal
leukemias.

5

SPEAKERS ABSTRACTS

NK cells are important effectors of the innate immunity and play a relevantrole in tumor surveillance and in defenses against viruses. Human NK cells
recognize HLA-class I molecules through inhibitory receptors (KIR andNKG2A) that block NK cell function upon interaction with their HLA
ligands. As a consequence, NK cells kill target cells that have lost (orunderexpress) HLA-class I molecules as tumors or virus-infected cells. NK
cell triggering is mediated by an array of activating receptors andcoreceptors that recognize ligands expressed primarily on tumors or virusinfected cells. NK cells have proven particularly useful in the therapy ofacute leukemias. Donor-derived “alloreactive” NK cells (i.e. that do not
express KIR speciﬁc for the HLA-class I alleles of the patient) play a majorrole in the cure of both adult and pediatrics high risk leukemias. In thesepatients, donor alloreactive NK cells kill leukemia blasts, thus preventing
relapses, and patient's DC, thus preventing graft-versus-host responses.
FACS analysis of KIRs expressed by NK cells allows to deﬁne the presence
and the size of the alloreactive NK subset in potential haploidentical donors(i.e. parents and/or siblings) and to select the best donor. We have recently
shown that the expression of activating KIRs, in particular the (HLA-C2speciﬁc) KIR2DS1, may also contribute to donor NK alloreactivity inpatients expressing C2 alleles. Importantly, a clear correlation wasestablished between the size of the alloreactive NK cell population and theclinical outcome. In this context, we have also shown that alloreactive NK
cells are generated from donor's HSC and persist in patients for long timeintervals.

Day 1
17⁰⁰

Opening session

18⁰⁰

Rene Van LierPROPERTIES OF TISSUE-RESIDENT CD8+ MEMORY T-CELLS
René A. van Lier1, Pleun Hombrink1 and Klaas van Gisbergen1,2
1

Adaptive Immunity Laboratory, Sanquin Research, Amsterdam, Netherlands;
Div of Molecular Immunology, WEHI, Parkville, Australia

SPEAKERS ABSTRACTS

2

Day 1
Lorenzo Moretta

The epithelial cells that line our body are constantly exposed to the external
environment. Within the airways, respiratory viruses speciﬁcally target
epithelial cells as initial site of entry and replication. After infection, a
specialized population of tissue-resident memory CD8+ T-cells (TRM) resides
in the epithelium to maintain constant immune surveillance and protection
against recurring respiratory infections1,2. In contrast to other recirculating
memory T-cells, Trm in solid tissues do not express CCR7 nor S1P1 thatmediate tissue egress allowing Trm retention at peripheral sites. The
transcriptional regulation of Trm differentiation is incompletely
understood.
We determined the transcriptional proﬁle of TRM from human lung resection
samples. A comprehensive set of transcription factors was identiﬁed thatcharacterizes lung resident TRM. To this set belongs Hobit (ZNF683), atranscription factor highly homologous to Blimp-13. Also in mice, Hobit isspeciﬁcally expressed in CD8+ Trm obtained from e.g. gut and skin. As
Blimp-1 is co-expressed with Hobit in Trm, we addressed the combined roleof Hobit and Blimp-1 in the differentiation of Trm cells using double
deﬁcient mice. Hobit and Blimp-1 were essential for the development ofskin-resident Trm after infection with HSV-1. In contrast, Hobit and Blimp1 did not regulate the formation of effector CD8 T-cells within the skin or theformation of memory CD8 T-cells within the spleen. Hobit and Blimp-1
suppressed the expression of CCR7 and S1P1, suggesting that these factors
directly regulated tissue retention of Trm. Similar to skin-resident Trm,
Hobit was expressed in other tissue-resident lymphocytes including NKT
cells, ILC1 and gut Trm after LCMV infection. The maintenance of all ofthese tissue-resident lymphocytes at peripheral sites required Hobit andBlimp-1. Thus, Hobit is speciﬁcally upregulated in tissue-resident
lymphocytes in bth humans and mice and collaborates with Blimp-1 tomaintain these cells within the tissues.
1

18⁰⁰
Foo Y. Liew

19⁰⁰

Blood and beyond: Properties of circulating and tissue-resident humanvirus-speciﬁc αβ CD8+ T cells. van Aalderen MC, Remmerswaal EB, Ten
Berge IJ, van Lier RA. Eur J Immunol. 2014 , [Epub ahead of print]
2
CD8+ T cells with an intraepithelial phenotype upregulate cytotoxic
function upon inﬂuenza infection in human lung. Piet B, de Bree GJ, SmidsDierdorp BS, van der Loos CM, Remmerswaal EB, von der Thüsen JH, vanHaarst JM, Eerenberg JP, ten Brinke A, van der Bij W, Timens W, van Lier
RA, Jonkers RE. J Clin Invest. 2011 Jun;121(6):2254-63
3
Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that
regulates NKT cell effector differentiation. van Gisbergen KP, Kragten NA,
Hertoghs KM, Wensveen FM, Jonjic S, Hamann J, Nolte MA, van Lier RA.
Nat Immunol. 2012 Sep;13(9):864-71.

6

THE ROLE OF CYTOKINES IN INFECTION AND INFLAMMATION
Eddy F Y Liew
Institute of Immunology, Infection and Inﬂammation, University of Glasgow, Glasgow G12 8AT, UK

SPEAKERS ABSTRACTS

Cytokines are hormones of the immune system. Cytokine-targeting
represents a major triumph in immunology scientiﬁcally, clinically andcommercially. There is therefore considerable interest in discovering novel
cytokines. I will illustrate the pleiotropic role of some of the novel
cytokines by focusing on interleukin (IL)-33. IL-33 is the latest member of
the IL-1 family. It is the ligand of ST2, which is expressed mainly on
activated Th2 cells, epithelial cells, neuronal cells and mast cells. IL-33 canskew a predominantly Th1 cell population to Th2 cells phenotype in vivo.
Furthermore IL-33 potently induces alternatively activated macrophages(M2). IL-33 signals via MyD88, the canonical pathway shared by all
members of the IL-1 family. Additionally, IL-33 activates type II cytokines
via mTOR (mammalian target of rapamycin). IL-33 mRNA is expressed
early during infection of the intestinal-dwelling nematode Trichuris murisin mice and IL-33 treatment enhances resistance to Trichuris infection by
inducing Th2. Furthermore, IL-33 reduces the pathology and mortality ofexperimental cerebral Malaria infection in mice by activating M2.
Importantly, IL-33 also effectively attenuates sepsis by mobilising the
innate cells neutrophils, to the site of infection and helps to clear thepathogens. Thus IL-33 is evolutionally preserved for the host defence
against infections. However, IL-33 can also induce Type 2 innate lymphoidcells (ILC2), which mediate airway inﬂammation. Furthermore, IL-33
contributes to the severity of mucocytis accompanying chemotherapy(Irinatecan). Blocking IL-33 prolongs the treatment and effectiveness of
chemotherapy in colorectal cancer. In contrast, IL-33 appears to restore thelong-term potentiation in a murine model of Alzheimer's disease. Therefore,
IL-33 is a double-edged sword and judicious regulation of IL-33 could haveconsiderable potential therapeutic effect.

Day 1

19⁰⁰
Conference dinner(by invitation)

7

INNATE LYMPHOID CELLS IN IMMUNITY
Marco Colonna, M.D.

SPEAKERS ABSTRACTS

Washington University School of Medicine
St. Louis, Missouri, USA

Day 2

Innate lymphoid cells (ILCs) are a heterogeneous population of
immune cells with two deﬁning characteristics: 1) they have lymphoidorigin, differentiating from the common lymphoid progenitor (CLP)and dependent on IL-2Rgc signaling. 2) They lack antigen-speciﬁc
receptors and therefore do not require the RAG proteins fordevelopment. Present throughout the body and enriched at mucosal
surfaces in both human and mouse, ILCs promptly produce effector
cytokines in response to stimulation with STAT-activating cytokines
and alarmins of the IL-1 family. Based upon similarities in effector
cytokine secretion and developmental requirements, ILCs can bedivided into three populations: group 1 ILCs, group 2 ILCs, and group
3 ILCs. IL-12, IL-21 IL-15 and IL-18 activate group 1 ILCs to produce
IFN-g; this group includes subsets such as T-bet+Eomes+ NK cells andT-bet+Eomes- ILC1. IL-25, thymic stromal lymphopoietin, and IL-33
trigger GATA-3+ ILC2 to produce type 2 cytokines such as IL-5 andIL-13. IL-23 and IL-1 prompt Rorgt+ ILC3 to produce IL-22 and/or IL17. As potent innate cytokine producers that respond to changes in thecytokine microenvironment, ILCs have demonstrated roles in early
infection control, adaptive immune regulation, lymphoid tissue
development, and in tissue homeostasis and repair. I will illustrate thecomplexity of ILC subsets, discuss novel functions, focusing on
emerging ILCs crosstalk with other immune cells and the microbiota.
Furthermore, I will highlight recent insights into the development ofILCs, the common pathways they share as well as points of divergence
between ILC groups and subsets.

8⁰⁰

9⁰⁰

Adrian Hayday

8

EPITHELIAL REGULATION OF INTRAEPITHELIAL T CELLREPERTOIRES AND RESPONSE
Adrian Hayday
The Francis Crick Institute, CRUK & King's College London.

SPEAKERS ABSTRACTS

The thesis of conventional immunology is one of centralised control,
whereby the response to infection within tissues is decided within lymph
nodes, from which effector T lymphocytes are despatched to quell regional
disturbances. But this cannot satisfactorily explain the interaction of theimmune system with tissues, since many tissues at steady-state are rich in T
cells. Moreover, discrete repertoires of T cells are associated with discrete
tissues. These observations raise many questions. For example, what is the
biological signiﬁcance of particular T cells being associated with particular
tissues, and how are these mutually compatible immune-stromalcompartments determined? To begin to answer these questions, we haveadopted a molecular genetic approach, identifying key receptor-ligand axes
by which epithelial tissues communicate with their local T cellcompartments from their development through to their function at rest andupon tissue dysregulation. These molecular axes offer new insight into
immunobiology within tissues in health and disease, with some moleculessuggesting new clinical avenues for precisely regulating immune cells in an
organ-speciﬁc fashion

Day 2
Marco Colonna

9⁰⁰
Andrew N. McKenzie

10⁰⁰

9

TYPE-2 INNATE LYMPHOID CELLS IN IMMUNITY AND DISEASE
Andrew N.J. McKenzie
MRC Laboratory of Molecular Biology, Cambridge UK

SPEAKERS ABSTRACTS

The innate lymphoid cell (ILC) family consists of interferon-γ (IFN-γ secreting group 1 ILCs (ILC1), type-2 cytokine-producing ILC2s andIL-22 and/or IL-17-positive ILC3s. ILC1 and ILC3 play critical roles
in protective immunity against bacteria, intracellular protozoan
parasites (ILC1), and fungi (ILC3), and in autoimmune disorders. By
contrast ILC2 associate with immune responses to parasitic worms
and allergy.
These functionally diverse cytokine-producing cells arise from acommon lymphoid progenitor (CLP) under the control of speciﬁctranscriptional regulators. These include upstream factors such as
inhibitor of DNA binding 2 (Id2), GATA binding protein 3 (GATA3),
nuclear factor interleukin-3 (Nﬁl3), and promyelocytic leukaemia zincﬁnger (PLZF) that restrict the differentiation of a common helper ILC
progenitor (CHILP) from the CLP. Downstream, lineage-speciﬁcfactors induce lineage commitment: ILC1s require the T-box
transcription factor T-bet (Tbx21); ILC2s require GATA3 andtranscription factor retinoic acid receptor-related orphan nuclear
receptor alpha (RORα); and ILC3s require ROR gamma t (RORγt).

Day 2
9⁰⁰

10⁰⁰

Adrian Hayday

Andreas Diefenbach

Although they comprise only a small proportion of the haematopoietic
compartment, ILC2s expand rapidly in response to the epitheliumderived type-2 initiator cytokines IL-25, IL-33 and TSLP, and are a
critical innate cellular source of type-2 cytokines. Their secretion of
IL-5 and IL-13, but also IL-9, IL-6, IL-4, GM-CSF and amphiregulin
induces eosinophilia, mucus production, muscle contractility, andtissue repair responses that are protective against parasitic helminth
infection, but also underlie inappropriate allergic inﬂammation.
Recent studies have shown that ILCs also play roles beyond
homeostasis and the acute response to infection or tissue damage, andact additionally to modulate adaptive T cell responses through theirexpression of MHCII and the presence/absence of additional costimulators. Indeed, as new reagents such novel mouse models aredeveloped to study ILCs in vivo previously unappreciated roles for
ILCs continue to be unearthed. Importantly, the presence of ILCs inhuman disease offers the possibility of targeting these cells
therapeutically.

10

TRANSCRIPTIONAL CONTROL OF ILC FATE DECISIONS

Andreas Diefenbach

Innate lymphoid cells (ILCs) are a recently discovered family of innate
lymphocytes that are substantially represented at mucosal surfaces andhave been implicated in the protection of epithelial barriers. Various
types of ILCs can be discriminated based on the expression of distinct
transcription factors controlling the expression of a distinct set ofcytokine genes endowing the various ILC subsets with a speciﬁc rangeof effector functions. Currently, three groups of ILCs are being
recognized. Group 1 ILCs (ILC1s) are a diverse group of ILCs
comprised of natural killer (NK) cells and other, poorly deﬁned subsets
of ILCs. It is believed that the ILC1 fate decision is controlled by the Tbox transcription factor T-bet endowing ILC1s with the capability to
produce large amounts of IFN- . ILC2s express high levels of GATA-3,
produce IL-5 and IL-13 and have been involved in immunity to helminth
infections and in the pathogenesis of allergic diseases. Group 3 ILCs
developmentally depend on the transcription factor ROR t andproduce the cytokines IL-22, IL-17A and IL-17F. ILC3s are believed to
be involved in the protection against intestinal bacterial infections and,
if inappropriately stimulated, can be important drivers of inﬂammatory
disorders. The transcriptional programs and effector cytokines of thevarious ILC subsets strikingly resemble those of the various T helper
cell effector fates suggesting that such transcriptional circuitry alreadyformed in the evolutionary older innate immune system. The various
ILC subsets are developmentally related as all ILC lineages depend on
the transcriptional regulator Id2 (inhibitor of DNA binding 2) thatinterfers with E2 protein-controlled gene expression. This raises theimportant issue if ILCs may derive from a common ILC progenitor(CILP). Identiﬁcation of such a progenitor would allow to identify themolecular signals required for the speciﬁcation of the various ILC
lineages. I will discuss progress towards our understanding of themolecular programs regulating ILC fate decisions and our current 10⁰⁰
models of transcriptional stability and plasticity of ILC fates. Finally, I
will discuss an unprecedented role of ILC3s in the protection against
mucosal virus infections.
Research in my lab is supported by grants from the European ResearchCouncil (ERC) and Deutsche Forschungsgemeinschaft.
11⁰⁰

11

SPEAKERS ABSTRACTS

Research Centre of Immunology and Institute of Medical Microbiology and Hygiene, University of Mainz
Medical Centre, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany

Day 2
Andrew N. McKenzie

Coffee breakT CELL REACTIVITY IN THYMIC SELECTION
Kristin A. Hogquist

SPEAKERS ABSTRACTS

Center for Immunology, University of Minnesota, Minneapolis MN

Day 2
11⁰⁰

The healthy adaptive immune system is responsive to foreign antigens,and tolerant to self. The reactivity of a T cell's receptor for self-peptide
MHC plays a determining role in T cell development. T cell signals
are integrated with other thymic environmental signals to allow
maturation and survival and to facilitate the development of diverse
types of T cells, including naïve CD4 and CD8 T cells, nTreg cells,
iNKT cells, and nIELs. Cells that receive a low afﬁnity signal during
development are positively selected, and migrate to the medulla where
they continue to undergo negative selection, functional maturation,and emigration to peripheral lymphoid organs. We sought to gain
further insight into the functional maturation process that occurs after
positive selection. By comprehensive ﬂow cytometric analysis of
RAG2GFP mice, we deﬁne the precise stage at which medullary
thymocytes acquire the competence to proliferate when stimulated
through the TCR. We also show that they become resistant to TNF as
they mature. Using microarray analysis we identiﬁed gene changes
unique to the later stages of thymic maturation. Gene set enrichment
analysis suggested these genes are regulated by NF-kB and IRF
transcription factors. We examined a number of mutant mice thatexhibit peripheral T cell lymphopenia. Of these, mice deﬁcient in
TAK1, a key signaling component of NF-kB and MAPK signaling
pathways, show normal positive selection, but a profound and speciﬁc
block in functional maturation. Restoration of the NF-kB signalingarm using a constitutively active IKKB mutant restored thecompetence to proliferate and emigrate, but did not allow peripheral T
cell survival. Alternatively, IFN receptor deﬁciency resulted inphenotypic abnormality of mature thymocytes, but competence toproliferate was normal. In summary, both NF-kB dependent andindependent signals downstream of TAK1 are required in medullary
thymocytes for the ﬁnal maturation of positively selected thymocytes
into naïve T cells that can proliferate and survive outside the thymus.
Funding source: RO1 AI088209

12⁰⁰

Andreas Diefenbach

12

THE TRANSCRIPTION FACTOR KLF2 REGULATES T CELL
TRAFFICKING AND DIFFERENTIATION
Stephen C. Jameson
Center for Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota
Medical School, Minneapolis MN 55414.

SPEAKERS ABSTRACTS

Previous studies showed that the transcription factor Kruppel-like factor 2(KLF2) is essential for egress of mature T cells from the thymus and for
peripheral recirculation of naïve T cells through lymphoid tissues.
However, less is known about the function of KLF2 in regulating antigenspeciﬁc responses of T cells and the establishment of T cell memory. In
studies on CD8+ T cells, we found that KLF2 is important for the generationof prototypical “resident memory” T cells (TRM) – a population that ismaintained in non-lymphoid tissues and has been shown to play the majorrole in protective immunity against infections at barrier tissue sites. Forced
expression of KLF2 opposes the maintenance of TRM, and these effects canbe mimicked by induced expression of a key transcriptional target of KLF2,
S1PR1. In addition, we found that differentiation of CD4+ T cells toward
the follicular helper T cell (Tfh) fate is opposed by KLF2. As with TRM
generation, S1PR1 expression contributes to inhibition of Tfh generation:
however, a more important pathway is KLF2 induction of the Blimp-1
transcription factor (which, through inhibition of Bcl-6 expression, blocks
Tfh differentiation). In addition, KLF2 can promote expression of T-bet and
GATA3 (involved in differentiation of Th1 and Th2 helper T cell subsets,
respectively). Our studies indicate that KLF2 expression is inﬂuenced bymultiple factors, including cytokine and costimulatory receptors, although
regulation of the PI3K pathway is emerging as a core mechanism for control
of KLF2 expression.

Day 2

Hence, for both CD4+ and CD8+ T cells, expression of KLF2 is pivotal forcontrol of distinct but complementary aspects of T cell differentiation, via
regulation of T cell localization and expression of lineage deﬁning
transcription factors. These data suggest KFL2 serves to direct the fate ofpost-activated T cells, shaping the distribution and function of effector andmemory T cell populations.
This work was supported by NIH award R37 AI-38903 (SCJ)

Kristin A. Hogquist

12⁰⁰
Yousuke Takahama

13⁰⁰

13

THYMIC MICROENVIRONMENTS THAT FORM T CELL REPERTOIRE
Yousuke Takahama

SPEAKERS ABSTRACTS

University of Tokushima, Tokushima, Japan

Day 2
12⁰⁰

Stephen Jameson

13⁰⁰

Juan Carlos Zuniga-Pﬂucker

During development in the thymus, newly generated repertoire ofdiverse TCR-ab recognition speciﬁcities in immature T cells is
selected to form the functionally competent and self-tolerant
repertoire of mature T cells. Positive selection supports the survival ofpotentially useful self-MHC-restricted thymocytes upon low-afﬁnity
TCR engagement, whereas negative selection deletes potentiallyharmful self-reactive thymocytes upon high-afﬁnity TCR
engagement. Recent advances in the biology of thymic stromal cells
have indicated that T cell formation in the thymus requires positive
selection by thymic cortical epithelial cells that express unique protein
degradation machineries, including the b5t-containing
thymoproteasome. It has also been revealed that the proximal interplay
among developing T cells, dendritic cells, and medullary epithelial
cells that promiscuously express tissue-speciﬁc self-antigens isessential for the establishment of self-tolerant TCR repertoire and thegeneration of regulatory T cells. These results suggest the vital roles
played by self-peptides speciﬁcally expressed by multiple thymic
microenvironments in the development of functionally competent andself-tolerant T cells.

14

THE TRANSCRIPTION FACTOR KLF2 REGULATES T CELL TRAFFICKINGAND DIFFERENTIATION
Juan Carlos Zúñiga-Pﬂücker
Department of Immunology, University of Toronto, and Sunnybrook Research Institute, Toronto, Ontario, Canada

SPEAKERS ABSTRACTS

The zinc-ﬁnger transcription factor, GATA-3, plays a crucial role during
early T-cell development and also dictates later T-cell differentiation
outcomes. However, its role and collaboration with the Notch signaling
pathway in the induction of T-lineage speciﬁcation and commitment havenot been fully elucidated. We show that GATA-3 deﬁciency inhematopoietic progenitors results in an early block in T-cell development
despite the presence of Notch signals, with a failure to up-regulate Bcl11b
expression, leading to a diversion along a myeloid, but not a B-cell, lineage
fate. GATA-3 deﬁciency results in dysregulated Cdkn2b expression,leading to apoptosis of early T-lineage cells due to inhibition of CDK4/6
function. We also show that GATA-3 induces Bcl11b, and together with
Bcl11b represses Cdkn2b expression. Our ﬁndings provide a signaling andtranscriptional network by which the T-lineage program in response to
Notch signals is realized.

Day 2
Yousuke Takahama

13⁰⁰

14⁰⁰

15

Break andposter viewing

TARGETING INFLAMMATION IN TREATMENT OF TYPE 2 DIABETES:TIME TO START
Marc Y Donath

SPEAKERS ABSTRACTS

University Hospital Basel, Switzerland

Day 2

Onset of Type 2 diabetes occurs when the pancreatic beta-cell fails toadapt to the increased insulin demand caused by insulin resistance.
Morphological and therapeutic intervention studies have uncovered an
inﬂammatory process in islets of patients with Type 2 diabetes
characterized by the presence of cytokines, immune cells, beta-cell
apoptosis, amyloid deposits, and ﬁbrosis. This insulitis is due to apathological activation of the innate immune system by metabolic
stress and governed by IL-1 signaling. We propose that this insulitis
contributes to the decrease in beta-cell mass and the impaired insulin
secretion observed in patients with Type 2 diabetes. Initially, theinﬂammatory response is probably deployed to promote beta-cell
repair and regeneration. Yet, as it becomes chronic activation of autoinﬂammatory processes may then become deleterious. Furthermore,
IL-6 emerges as an additional regulator of islet secretory function.
Indeed, IL-6 mediates a cross talk between fat, muscles and pancreatic
islets to adapt to changes in insulin demand via secretion of the incretin
glucagon-like peptide-1. Thereby, IL-6 reprograms the alpha cells to
produce glucagon-like peptide-1. This implicates IL-1 beta and IL-6 inthe regulation of beta cell insulin secretion in both health and disease.
It follows that modulation of inﬂammatory mediators, may present as apossible causal therapy with disease-modifying potential.

15⁰⁰

16⁰⁰

Yvette Van Kooyk

16

NANOTECHNOLOGY AIMED TO DESIGN DC TARGETING VACCINES FOR
THE INDUCTION OF TUMOR-IMMUNITY
Yvette van Kooyk
Department of Molecular Cell Biology and Immunology, VU University medical Centre, v.d. Boechorststraat 4,1081 BT Amsterdam, the Netherlands

SPEAKERS ABSTRACTS

Dendritic cells (DC) are specialized in the recognition of antigen and play apivotal role in the control of immunity and tolerance. For antigen
recognition DC express several C-type lectins, that function as innate
receptors that facilitate antigen uptake and cross-presentation. Many ofthese receptors also modify responses through signalling interference with
TLR. The natural ligands of C-type lectins are speciﬁc glycan structures thatcan be found on pathogens but also on tumors. These glycan structures caneither contribute to the tumor suppressive programming of the immune
system, or oppositely activate the immune system.
Moreover synthetic coupling of glycans to antigens can favour directionaltargeting of tumor antigens to human DC and Langerhans Cells (LC) in situ,
in the skin, facilitating the induction of tumor speciﬁc CD4+ and CD8+ T
cell responses and Th1 differentiation. Therapeutic vaccination of micewith glycan modiﬁed antigen show long term anti-tumor immunity whentumor induced T regulatory cells are temporarily reduced.
We identiﬁed that the glycosylation state of tumors may directly regulates
immune escape. In particular high sialylation of tumors results in increase of
FoxP3 CD4+ T cells (Treg) and lower frequencies of effector T cells.
Lowering the sialylation of tumors converts the frequencies Treg/Teff tofavourable anti-tumour immunity.
Our work sheds new light on the contribution of glycans to control tumour
immunity by either contributing to the induction of anti-cancer immunity or
tumour induced immune suppression.

Day 2
Marc Donath

16⁰⁰
Nada Pejnovic

17⁰⁰

17

GALECTIN-3 IN TYPE 2 DIABETES AND IMMUNOMETABOLISM
Nada Pejnovic

SPEAKERS ABSTRACTS

Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University ofKragujevac, Serbia

Day 2
16⁰⁰

Yvette Van Kooyk

17⁰⁰

Bojan Polic

Obesity, via gain of ectopic fat and by inducing metaﬂammation,
promotes insulin resistance, hepatic steatosis, and β cell failure thus
representing the major risk factor for type 2 diabetes and nonalcoholic
fatty liver disease (NAFLD). Galectin-3 (Gal-3), a β-galactosidebinding lectin, modulates immune/inﬂammatory responses andparticipates in a spectrum of diseases with sometimes opposite
functions. The evidence so far including our own data shows aprotective role for Gal-3 in obesity and diabetes. Galectin-3 ablation in
a setting of chronic overnutrition induces an increased visceral adipose
tissue (VAT) and pancreatic inﬂammatory response, hyperglycemia
and insulin resistance. Enlarged VAT in Gal-3 deﬁcient mice fed highfat diet contains more numerous proinﬂammatory myeloid cells, Type1 T and NKT cells and increased phosphorilated-NF-κB p65 protein
expression. Macrophage inﬁltration of islets, accumulation of AGE
and increased expression of RAGE, NLRP3 inﬂammasome, IL-1β andphosphorilated-NF-κB p65 were present in the absence of Gal-3.
Thus, Gal-3 controls excessive activation of NFκB and NLRP3
inﬂammasome in islets and VAT and downstream inﬂammationinduced by metabolic stimuli. Gal-3 deﬁcient mice developed more
pronounced high-fat diet induced liver steatosis and increased prosteatotic PPAR-γ, Cd36, Abca-1 and FAS in livers. However,hepatocellular damage, inﬂammation and ﬁbrosis were lower in Gal-3
deﬁcient mice which had less numerous proinﬂammatory myeloid
cells in livers, peripheral blood and bone marrow, and decreased
expression of genes related to inﬂammation, oxidative stress andﬁbrogenesis. Hepatic IL-33, ST2 and IL-13 mRNA levels were lower
in the Gal-3 absence. Exogenous IL-33 failed to induce ST2
upregulation and IL-13 production by CD11b+ myeloid cells in vitro
and in vivo. In vivo administered IL-33 enhanced liver ﬁbrosis in highfat diet-fed mice in both genotypes, albeit to a signiﬁcantly lower
extent in Gal-3 deﬁcient mice. Thus, Gal-3 attenuates steatosis, but
promotes liver injury, inﬂammation and IL-33 dependent liverﬁbrosis. Gal-3 is an important regulator of obesity-associatedimmunometabolic disorders.

18

NK CELLS LINK OBESITY-INDUCED ADIPOSE STRESS TO INFLAMMATION
AND INSULIN RESISTANCE
Felix M Wensveen1, Vedrana Jelenčić1, Sonja Valentić1, Marko Šestan1, Tamara Turk Wensveen2, Sebastian Theurich3,
Ariella Glasner4, Davor Mendrila5, Davor Štimac2, F. Thomas Wunderlich3, Jens C Brüning3, Ofer Mandelboim4,
Bojan Polić1
1

Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia;
Department of Internal Medicine, University Hospital Rijeka, Rijeka, Croatia;
Max Planck Institute for Metabolism Research Cologne, Cologne, Germany;
4
The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem, Israel;
5
Department of Surgery, University Hospital Rijeka, Rijeka, Croatia
2

Obesity is an increasingly common health issue that predisposes people tometabolic disorders such as insulin resistance (IR), which can progress todiabetes mellitus type 2 (DM2). An important underlying cause of obesityinduced IR is chronic systemic inﬂammation derived from accumulating
pro-inﬂammatory macrophages in visceral adipose tissue (VAT). Currently,
it is unknown which signal initiates adipose tissue macrophage (ATM)
activation in VAT. We ﬁnd that a phenotypically distinct VAT-resident NK
cells provide a crucial link between obesity-induced adipose stress and
ATM activation in VAT. Ligands for the NK cell- activating receptor
NKp46/Ncr1 are expressed in human and mouse VAT. Feeding with highfat diet causes up regulation of Ncr1-ligands on adipocytes, leading tolocalized activation and cellular increase of NK cells. IFNγ produced bythese cells drives early pro-inﬂammatory macrophage differentiation andpromotes obesity-induced insulin resistance. Lack of NK cells, Ncr1 or
IFNγ prevents macrophage activation in VAT and greatly ameliorates
glucose tolerance and insulin sensitivity. Therapeutic blocking of Ncr1signaling forestalls ATM activation. Our study identiﬁes NK cells as key
regulators of macrophage polarization and insulin resistance in response toobesity-induced adipose stress. The NK-ATM axis therefore provides anattractive new target for early treatment of patients with metabolic
syndrome to prevent progression to DM2.

Day 2
Nada Pejnovic

17⁰⁰
Free time

18⁰⁰

19

SPEAKERS ABSTRACTS

3

SELF-ANTIGEN DIVERSITY IN THE THYMUS: WHAT THYMOCYTES SEEAND DON'T SEE
Bruno Kyewski

SPEAKERS ABSTRACTS

Division of Developmental Immunology, Tumor Immunology Program, German Cancer ResearchCenter, D 69120 Heidelberg, Germany

In the course of central self-tolerance induction a highly diverse repertoire of T cell receptors(TCRs) is probed against a matching array of their ligands, namely self-peptide/MHC
complexes. While much has been learnt about the molecular mechanisms underlying thegeneration of TCR diversity, the cellular and molecular strategies responsible for intra-thymic
expression and presentation of self-antigen repertoires are less well understood. The diversity ofself-peptide display is, on the one hand, afforded by the remarkable heterogeneity of thymic
antigen presenting cells (APCs) and, on the other hand, by their unconventional molecularpathways of antigen expression, processing and presentation (1). Here I will elaborate on one
mechanism, namely promiscuous gene expression (pGE). PGE denotes the property of a speciﬁc
subset of thymic stromal cells - medullary thymic epithelial cells (mTECs) – to express a highly
diverse set of tissue-restricted antigens (TRAs) representing essentially all tissues of the body.
This allows self-antigens, which otherwise are expressed in a spatially or temporally restricted
manner to become continuously accessible to developing T cells. Failure of pGE at thequantitative or qualitative level will result in holes central tolerance and heightenedsusceptibility for organ-speciﬁc autoimmunity, e.g. type 1 diabetes mellitus, rheumatoid
arthritis or autoimmune myocarditis.
1. Klein, L, B. Kyewski, P.M. Allen and K.A. Hogquist. 2014. Positive and negative selection of
the T cell repertoire: what thymocytes see (and don't see). Nature Rev Immunology 14, 377-391.
Recent studies show that each APC subset appears to carry its speciﬁc antigen cargo as a result ofcell-type speciﬁc features: ﬁrstly, transcriptional control (i.e. promiscuous gene expression in
medullary thymic epithelial cells); secondly, antigen processing (i.e. proteasome compositionand protease sets); thirdly, intracellular antigen sampling (i.e. autophagy in thymic epithelial
cells) and fourthly, extracellular antigen sampling (i.e. immigrating dendritic cells sampling
extrathymic milieus). The combinatorial expression patterns of these attributes in distinct APC
subsets result in a self-peptide display partly unique to the cortex mediating positive selection
and to the medulla mediating tolerance induction.
The scope of central tolerance is to a large measure dictated by the pool of promiscuously
expressed genes.
Thus even lack of a single TRA can result in spontaneous organ-speciﬁc autoimmunity.
Promiscuously expressed gene, which have no structural or functional commonality, display
two prominent features, they are highly clustered in the genome and show a preference for
TRAs. In order to better understand these features, we set out to more precisely deﬁne thegenomic organization of this gene pool. In particular, we probed to which extent and according
to which rules predeﬁned genomic clusters of TRAs are transcribed in mTECs. Our analysis
proceeded from the bio-informatic deﬁnition of TRA clusters via gene expression analysis in
mTECs using whole genome arrays to the in depth analysis of selected TRA clusters by RT- PCR
at the population and single cell level. Patterns emerging from these studies will hopefully yieldinsight into molecular and evolutionary mechanisms responsible for selecting this gene pool.
Conceivably, positional cues in the genome and/or particular properties of self-antigens (e.g.
immunogenicity) could have been driving forces during the co-evolution of pGE and adaptive
immunity.

20

EPIGENETIC REGULATION OF INFLAMMATION IN AUTOIMMUNEDISEASES
Steffen Gay

Epigenetics has emerged to be a key regulator of gene expression in health and disease.
Most interesting has been the question how epigenetic information might be maintained throughDNA replication. In this regard it has been discussed that histone-modifying enzymes mightremain associated with the DNA through replication to translate the epigenetic information to
the newly-assembled chromatin (1).
Our laboratory has been studying the epigenetic modiﬁcations over the past 15 years ininﬂammation in general and in particular in rheumatoid arthritis (RA) (2,3), progressive
systemic sclerosis (scleroderma), pulmonary hypertension and ankylosing spondylitis. This
work was supported by EC grants, including FP6 Autocure, FP7 Masterswitch, Osteoimmune,
TEAM and the IMI 2 project BeTheCure (BTCure)
The complexity of epigenetics is illustrated by the involvement of multiple regulatory
biochemical and biological processes, such as acetylation, methylation, phosphorylation,sumoylation and non-coding RNAs, including microRNAs (miR), and long non-coding RNAs
(lncRNA).
The latest results from our laboratory are documented by the ﬁndings that bromodomains 2, 3
and 4 are expressed in the RA synovium and can be targeted with selective BRD inhibitors (4) toreduce the production of proinﬂammatory cytokines and matrix-metalloproteinases (MMP).
The earliest studies on hypomethylation of synovial ﬁbroblasts (RASF) (1) have been followedup (2) and most recently resulted in a novel therapeutic concept to silence the aggressive
phenotype of RASF (5).
One of our greatest surprises in our current research has been the observation that SF differ intheir phenotype depending from the localization in the body through the differential expressionof miRs and lncRNAs (6).
Based on the fact that we are not able to cure RA, despite all the impressive results withbiologicals and small molecules targeting inﬂammatory cytokines and signaling, it needs to be
stressed that the epigenome of the RASF is an important target to possibly cure RA (7).
1) Budhavarapu VN, Chavez M, Tyler JK. How is epigenetic information maintained through DNA
replication ? Epigenetics & Chromatin 6.32, 2013
2) Neidhart M et al. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue Association with genomic DNA hypomethylation and inﬂuence on gene expression. Arthritis Rheum 43:
2634-2647, 2000
3) Klein K, Gay S. Epigenetics in rheumatoid arthritis. Curr Opin Rheumatol 27:76-82, 20154) Klein K et al. The bromodomain protein inhibitor I-BET151 suppresses expression of inﬂammatory
genes and matrix degrading enzymes in rheumatoid arthritis synovial ﬁbroblasts. Ann Rheum Dis. 2014
[Epub ahead of print]5) Neidhart M et al. Inhibition of spermidine/spermine N1-acetyltransferase (SSAT1) activity - a new
therapeutical concept in rheumatoid arthritis. Arthritis Rheum 66:1723-1733, 2014
6) Ospelt C et al. Joint speciﬁc positional differences in coding and noncoding transcriptome of synovial
ﬁbroblasts as a determinant of the susceptibility of synovial joints to rheumatoid arthritis. Arthritis Rheum
66:S1216, 2014
7) Frank-Bertoncelj M, Gay S. The epigenome of synovial ﬁbroblasts: an underestimated therapeutic target
in rheumatoid arthritis. Arthritis Res Ther 16:117, 201421

SPEAKERS ABSTRACTS

Department of Rheumatology, University Hospital, CH-8091 Zurich, Switzerland

DISSECTING PATHOGENIC SOURCES OF TNF IN DISEASE AND CELL
TYPE-RESTRICTED CYTOKINE TARGETING
Nedospasov S.A.1,2,3
1Engelhardt Institute of Molecular Biology, Moscow, Russia
Lomonosov Moscow State University, Moscow, Russia
Lobachevsky University, Nizhni Novgorod, Russia

2

SPEAKERS ABSTRACTS

3

Day 3
9⁰⁰

Steffen Gay

10⁰⁰

Anna Erdei

Proinﬂammatory cytokines TNF, IL-6 and IL-1 may contribute topathogenesis of autoimmunity and therefore systemic inhibition ofthese cytokines became an essential part of the therapy for systemic
autoimmune diseases. Our recent studies in experimental arthritis
suggest that TNF from particular cellular sources may play an antiinﬂammatory role. If so, systemic cytokine inhibition may be like adouble-edged sword disrupting both pathogenic and protective
signaling.
Based on these ﬁndings we are developing an approach to cell typerestricted cytokine neutralization by utilizing bispeciﬁc antibody
constructs that would attach to the cell surface of a particular type of
immune cells, and capture the cytokine released by these cells. Ourconstructs are based on single domain antibodies (VHH) speciﬁc forhuman TNF and for cell type-speciﬁc markers, in particular, for F4/80
surface molecule expressed on macrophages. We ﬁnd that suchantibodies can effectively attach to the cell surface, capture and retain
released cytokines preventing their dissemination. Using mice
humanized for the TNF system and with macrophages isolated fromsuch mice we assessed activity of these constructs in vitro and in vivo.
Our ﬁndings may serve as a basis for bioengineering of new type ofcytokine inhibitors.
Supported by Russian Scientiﬁc Fund grant 14-25-00160 and Russian
Ministry of Science and Education grant 14.Z50.31.0008

22

CROSSTALK BETWEEN COMPLEMENT AND TOLL-LIKE RECEPTORS;REGULATION OF HUMAN B CELL RESPONSES UNDER PHYSIOLOGICALAND AUTOIMMUNE CONDITIONS
Anna Erdei

SPEAKERS ABSTRACTS

Immunology Department, Eötvös University, Budapest, HUNGARY

The complement system and TLRs are two major components of innate
immunity which provide a fast ﬁrst-line defence against harmful agents.
Since many pathogen associated molecular patterns are able to activate botharms of the innate immune system, their effect should be investigatedsimultaneously under physiological as well as pathological conditions. We
have shown earlier that complement receptor 1 (CR1, CD35) is a potent
inhibitor of the B cell receptor (BCR)-induced functions of human B cells(Jozsi et al, JI, 2002, Kremlitzka et al, Int.Immunol. 2013). Now we foundthat ligation of CR1 on B cells treated with the TLR9-ligand CpG
oligonucleotide strongly reduced the BCR-induced activation, proliferation
and Ig-production of the cells. Although in SLE and RA patients the numberof CR1 molecules is markedly diminished on B cells, clustering of thiscomplement receptor resulted in efﬁcient inhibition of the major B cellfunctions similarly to healthy controls.
Our results reveal a novel link between complement and Toll-like receptors
in the regulation of humoral immunity and pave the way for developing a
new tool for the inhibition of autoreactive B cells.

Day 3
Sergei Nedospasov

10⁰⁰
Coffee break

11⁰⁰

23

Burkhard Becher

CYTOKINE NETWORKS IN AUTOIMMUNITY: HOW HELPER T CELLSINSTRUCT MACROPHAGES
Burkhard Becher
University of Zurich, Switzerland

SPEAKERS ABSTRACTS

There is a long-standing debate as to the role and function of T cell
polarization in autoimmune disease. We call pathogenic T cells THpath
cells based on their ability to cause tissue inﬂammation. It is becoming
increasingly clear that none of the previously coined TH cell
polarization patterns (TH1, TH2 or TH17) fulﬁl the criteria of deﬁned
THpath cells.
In other words, how a pathogenic molecular signature is translated into
the ability to trigger autoimmune tissue inﬂammation is a subject ofgreat debate. I will discuss a few of the features of THpath cells, the roleof cytokines in autoimmune disease and propose that TH path cells
themselves must recruit myeloid cells into the inﬂamed tissue tomediate tissue damage. But exactly how TH path cells achieve this featis the subject of my seminar in which I will discuss a number of recent
important observations made by immunologists.

Day 3
Anna Erdei

11⁰⁰
Branka Horvat

12⁰⁰

24

EMERGING CONTAGION: IMMUNOPATHOGENESIS OF NIPAH VIRUS
INFECTION
Branka Horvat

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus ofHenipavirus genus that causes human outbreaks annually in South-East
Asia. Similarly to previous Ebola occurrences, the Henipavirus outbreaks
remain sporadic until now and seem to affect only small areas; however,NiV may have a global pandemic potential and is an agent of particularconcern in the ﬁeld of human and agricultural biodefense.
Immunopathogenesis of this recently emerged virus is still poorly
understood. Although lymphocytes are not susceptible to Henipavirus
infection, they bind efﬁciently the virus and transinfect endothelial and Verocells even several days after binding. This transinfection is mediated byheparan sulfate and could be inhibited with heparin, opening thus novel
perspectives for the development of heparan sulfate–targeting therapeutic
approaches against these emerging infection. We have further analyzed thesusceptibility of different type of mice, bearing defects in either innate oradaptive immune system, to NiV infection. In contrast to wild-type, micedeﬁcient for type-I interferon (IFN-I) receptor were shown to be highly
susceptible to NiV. Although viral sensing through either TLR or RLR alone
was not critical in anti-viral defense, mice devoid in both TLR and RLR
signaling succumbed to the infection, with similar survival rate as IFN-I
deﬁcient mice. Utilization of mice with tissue-speciﬁc deletion of IFN-I
receptors suggested that IFN-I signaling in any of single cell population,
including macrophages, dendritic cells (DC), natural killer cells andneurons, neither in plasmacytoid DC - specialized in IFN-I production, is
not crucial for the protection from lethal NiV infection. Interestingly,
presence of T-cell but not B-cell compartment was critical in allowing
resistance to the infection, and this effect was independent on perforin
production. Finally, depletion of macrophages allowed rapid systemic
propagation of NiV infection and high lethality in mice, suggesting theirimportant role at the crossroads between innate and adaptive immunity. 11⁰⁰
Altogether, these results revealed some novel aspects of immuno-regulationof NiV infection, which could help in development of new strategies tocontrol this highly lethal infectious disease.

12⁰⁰

25

SPEAKERS ABSTRACTSInternational Center for Infectiology Research (CIRI), Inserm U1111, CNRS, UMR5308, ENS-Lyon, University Lyon 1, Lyon,
FranceDay 3
Burkhard Becher

Short presentations

AGING AND ADAPTIVE IMMUNITY IN THE HUMAN BONE MARROW
Beatrix Grubeck-Loebenstein
Institute for Biomedical Aging Research, University of Innsbruck, Innsbruck, Austria

SPEAKERS ABSTRACTS

With aging the immune system undergoes signiﬁcant age-relatedchanges. These age-dependent changes are referred to as immunosenescence and are partially responsible for the poor immune response
to infections and the low efﬁcacy of vaccination in elderly persons.
Immunosenescence is characterized by a decrease in innate andadaptive cell-mediated immune function in the peripheral blood and thebone marrow.
The aging of bone marrow cells and in particular, of adaptive immune
cells in the bone marrow has been addressed relatively rarely. Agerelated changes of human bone marrow, T, B and plasma cells will be
discussed in this talk.

Day 3
14⁰⁰

15⁰⁰

Arne Akbar

26

THE REGULATION OF T CELL SENESCENCE AND METABOLISM BY P38
MAPKINASE SIGNALLING
Lanna A, Henson SM and Akbar AN
Division of Infection and Immunity, University College London, U.K.

SPEAKERS ABSTRACTS

Persistent viral infections, ageing and inﬂammatory syndromes induce theaccumulation of senescent human T cells. However the mechanism that
regulates the function of these end-stage cells is unclear. Human CD8+
effector memory T cells that re-express CD45RA (CD27-CD45RA+;
EMRA) express surface KLRG-1 and CD57, exhibit reduced replicative
capacity and telomerase activation, decreased survival and high expressionof nuclear gH2AX after T cell receptor (TCR) activation. We investigated
the involvement of p38 MAP kinase signalling in these senescencecharacteristics of these cells. The expression of both total andphosphorylated p38 was highest in the EMRA compared to other CD8+ T
cell subsets. Furthermore, the inhibition of p38 signalling, especially inCD4+ EMRA T cells, signiﬁcantly enhanced their telomerase activity andsurvival after TCR activation. EMRA T cells preferentially utilize
glycolysis to fuel their effector functions. The inhibition of p38 enhances
mitochondrial function, reduces reactive oxygen species production andincreases proliferation and telomerase activity, however, these cells still
utilize glycolysis instead of oxidative phosphorylation for energy. The
precursors for glycolysis are generated by autophagy, that is increased after
p38 blockade. Thus activation of the p38 MAPK pathway is directly
involved in the senescence characteristics of highly-differentiated CD4+ T
cells and this pathway also regulates the metabolic function in thispopulation.

Day 3

Henson SM et al J. Clin Invest. 2014.

Beatrix Grubeck-Loebenstein

15⁰⁰
Annette Oxenius

16⁰⁰

27

T CELL CONTROL OF CYTOMEGALOVIRUS INFECTION IN GLANDULAR
TISSUES
Jenny Thom and Annette Oxenius
Institute of Microbiology, ETH Zurich, Zurich, Switzerland

SPEAKERS ABSTRACTS

Infecting 60-90% of the world population, human cytomegalovirus(HCMV) is a pathogen of high epidemiological relevance andrepresents a major cause of morbidity and mortality in immunocompromised individuals. CMV maintains its existence in the
population by prolonged replication in and secretion from mucosal
tissues, including the salivary glands (SG) which supports viral
replication for months after virus is controlled in all other organs.
Experimental infection of mice with murine CMV (MCMV) has serves
to explore tissue-speciﬁc control of CMV infection and reveals thatvirally mediated MHC class I down-regulation in SG-resident acinar
glandular epithelial cells renders this tissue uniquely resistant to CD8 T
cell mediated virus control. Instead, CD4 T cells are strictly required tocease virus replication during primary CMV infection. Both CMVspeciﬁc CD4 and CD8 T cells gradually acquire a tissue resident
memory T (TRM) cell phenotype in the SG, with the inductive
requirements differing between the two subsets.
Upon intraglandular reinfection the CMV-speciﬁc CD8 TRM cells - andnot CD4 TRM cells - confer immediate protection. The reasons for thisapparently disparate requirement of CD4 and CD8 T cells conferring
protection in SG tissue during primary and secondary CMV infection
will be discussed.

Day 3
15⁰⁰

Arne Akbar

16⁰⁰

Stipan Jonjic

Grant funding: This work was supported by ETH Zurich, the Swiss
National Science Foundation and the ETH Foundation.

28

VACCINES AND INNATE IMMUNITY: LESSONS FROM CYTOMEGALOVIRUS
IMMUNOEVASION
Stipan Jonjić
Stipan Jonjić, Faculty of Medicine University of Rijeka, Rijeka, Croatia

SPEAKERS ABSTRACTS

Cytomegalovirus (CMV) establishes life-long infection of its host, ensuringcontinuous supply of effector memory CD8+ T cells. CMVs possess
numerous immunoevasion genes able to modulate basically any part ofimmune response, including NK cell and CD8+ T cell response. It is wellestablished that deletion of these viral inhibitors leads to virus attenuationin vivo.These features make CMV a very attractive CD8+ T cell vaccinevector candidate. Control of CMV infection is in great part dependent on
NKG2D, an activating receptor when expressed on NK cells and costimulatory one when expressed on CD8+ T cells. We have constructed
highly attenuated mouse CMV (MCMV) expressing NKG2D ligand RAE1γ inserted in place of its viral inhibitor (Slavuljica et al, 2010) and foreign
CD8+ T cell epitope as well (Trsan et al, 2013). Such a recombinant vaccinevector provided outstanding and long-lasting CD8+ T cell-mediatedprotection against challenge infections. Moreover, RAE-1γMCMV-vector
circumvented MCMV interference of antigen presentation, improved
antigen presentation to CD8+ T cells and potentiated memory CD8+ T cell
response. Surprisingly, these immuno enhancing properties of RAE-1γ
expressing MCMV vector were retained even in NKG2D deﬁcient mice,pointing to additional NKG2D-independent immune function of RAE-1γ.
In my talk, I will discuss the capacity of MCMV expressing RAE-1γ as a
vaccine vector against other pathogens, as well as tumors.

Day 3
Annette Oxenius

16⁰⁰
Janko Nikolich-Zugich17⁰⁰

29

TGF-β IMPAIRS CD4 AND ANTIBODY RESPONSES IN OLD MICE TO INCREASECHIKUNGUNYA VIRUS DISEASE SEVERITY AND VIRAL PERSISTENCE
Jennifer L. Uhrlaub1, Vesna Pulko1, Victor R. DeFilippis2, Gary D. Coleman3, and Janko Nikolich-Zugich1
1

Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, AZ, USA
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, USA
WIL Research, Ashland, OH USА

2
3

SPEAKERS ABSTRACTS

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus endemic
to Africa and Asia, which causes sudden onset of fever, rash anddebilitating poly-arthralgia in peripheral joints that can persist for years,
particularly in older individuals. In 2013, CHIKV spread to the western
hemisphere resulting in more than one million cases in the Carribean.
Autochthonous transmission in the United States was conﬁrmed in
2014. In response to the increased geographic distribution of CHIKV
and the likelihood that elderly immune-naïve populations may
experience severe and life-threatening disease, we have developed amouse model of age-related vulnerability to CHIKV infection. We
demonstrate reduced ability of old mice to mount effective immune
responses to CHIKV and control viremia, leading to increased disease
severity and viral persistence in the joints. Ineffective immune
responses were due, in part, to dyscoordinated cytokine production.
Speciﬁcally, CXCL9 and TGFβ, were identiﬁed as key contributors to
impaired CD4 and antibody responses against viral epitopes in old
mice. Moreover, neutralization of TGFβ reduced acute joint swelling,
restored Ab responses, and virtually eliminated chronic joint pathology.
These results provide a valuable tool for further mechanistic dissectionof age-related vulnerability to CHIKV and point to possible targets in
CHIKV disease treatment.

Day 3
16⁰⁰

ABBREVIATIONS: A - adult mice, 3-5 mo old; CHIKV - chikungunya
virus; O - old mice, 18-20 months old

Stipan Jonjic

17⁰⁰
Excursion andconference dinner

30

TRAINED IMMUNITY: CONSEQUENCES FOR INFLAMMATORY DISEASES?
Leo A.B. Joosten
Radboudumc Nijmegen The Netherlands

SPEAKERS ABSTRACTS

The inability of innate immunity to build an immunological memory,considered one of the main characteristic differentiating it form adapative
immunity, has been recently challenged by studies in plants, invertebrates,
and mammals. The concept of trained immunity has been described and theimplications of this phenomenon on inﬂammatory diseases is not known
yet. Monocyte reprogramming is the basis of trained immunity which leads
to a state of activation of the monocytes. The innate memory of monocytes,
macrophages or NK-cells will have also an impact on adaptive immune
responses.
Here we discuss innate memory of monocytes and the mechanisms of that
leads to reprogramming of the innate immune cells. Besides the well-known
protective effects of trained immunity on infectious diseases, the role ofmonocyte reprogramming on chronic inﬂammation is not elucidated yet.
The consequences for inﬂammatory diseases as atherosclerosis, rheumatoid
arthritis or gout will be discussed.

Day 4

10⁰⁰

31

Hannes Stockinger

NOVEL MACROPHAGE SUBSETS WITH POTENTIAL IMPLICATION IN
INFLAMMATORY DISEASES
Hannes Stockinger and Anna Ohradanova-Repic
Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and
Immunology, Medical University of Vienna, Vienna, Austria

SPEAKERS ABSTRACTS

Macrophages are strategically located within the tissues where theyplay a crucial role in immune surveillance. In response to tissue damage
or invading pathogens, macrophages effectively ingest and eliminate
the source of danger and by concomitant release of proinﬂammatory
cytokines they attract and activate other immune cells including
macrophage precursors and T cells. However, exacerbated macrophage
responses underlie the pathology of several inﬂammatory diseases
including rheumatoid arthritis. A subset of macrophages found within
the affected joints was characterised by expression of folate receptor β(FRβ). In order to study the function of FRβ-positive macrophages
within the diseased tissue in more detail, we prepared in vitrodifferentiated macrophages from peripheral blood monocytes. Our
analyses, comprising a comprehensive gene expression proﬁling,
revealed that FRβ is strongly upregulated in several subsets ofmacrophages that can be distinguished by other markers as well as byfunctional properties. While FRβ-positive subsets share highphagocytic potential, they display marked differences in their capacity
to stimulate T cells. Our results revealed an important aspect ofmacrophage plasticity and potentiating a transition to an
immunosuppresive phenotype in vivo could lead to a novel way oftreatment for inﬂammatory diseases.

Day 4
Leo Joosten

10⁰⁰
Hartmut Wekerle

11⁰⁰

32

IGNITION OF BRAIN AUTOIMMUNE DISEASES IN THE GUT
Hartmut Wekerle
Max Planck Institute for Neurobiology, Munich, Germany

SPEAKERS ABSTRACTS

Over millions of years of evolution, the human organism has adopted
microbial societies that colonize speciﬁc outer inner surfaces. Rather thanmenacing our body, these microbiota crucially contribute to our wellbeing.
Thus, our microbial gut ﬂora, one of the densest bacterial habitats known,
contributes to the essential digestion of diets, produces essential nutrient
factors, protects against invading pathogens and warrants proper immune
function within the gut associated lymphatic tissues. Beyond this local
functions, intestinal microbiota act systemically, on the immune, endocrineand nervous systems.
Unfortunately, the beneﬁcial interactions of body and microbiota can go
awry. We found that under particular conditions, gut microbes activate
autoimmune T lymphocytes to attack the body's own nervous tissues.
Studying an animal model of human multiple sclerosis (MS), we detailinnate and adaptive immune mechanisms that underlie these triggering
events. In addition, we have clinical trials to learn, whether similar
activation leads to the development of human MS. First emerging results
will be presented.
Supported by Hertie-Foundation, Max-Planck Society, DFG (KoselleckAward, SFB218), KKNMS, and SyNergy.

Day 4

33

10⁰⁰

Hannes Stockinger

11⁰⁰

Ofer Mandelboim

RECOGNITION OF BACTERIA BY NK CELLS
Chamutal Gur1,2, Gilad Bachrach3 and Ofer Mandelboim1*
1

The Lautenberg Center of General and Tumor Immunology, The Hebrew University Hadassah Medical School, IMRIC
Jerusalem, Israel.
2
The Rheumatology Research Center, Hadassah-Hebrew University, Jerusalem, Israel.
3
The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

SPEAKERS ABSTRACTS

Bacteria, such as Fusobacterium nucleatum, are present in the tumor
microenvironment. However, the immunological consequences of
intra-tumoral bacteria remain unclear. Here, we have shown that naturalkiller (NK) cell killing of various tumors is inhibited in the presence ofvarious F. nucleatum strains. Our data support that this F. nucleatummediated inhibition is mediated by human, but not by mouse TIGIT, an
inhibitory receptor present on all human NK cells and on various T cells.
Using a library of F. nucleatum mutants, we found that the Fap2 proteinof F. nucleatum directly interacted with TIGIT, leading to the inhibitionof NK cell cytotoxicity.
We have further demonstrated that tumor-inﬁltrating lymphocytes
expressed TIGIT and that T cell activities were also inhibited by F.
nucleatum via Fap2. Our results identify a bacterium-dependent, tumor
immune evasion mechanism in which tumors exploit the Fap2 proteinof F. nucleatum to inhibit immune cell activity via TIGIT.

Day 4
Hartmut Wekerle

11⁰⁰
Vishwa Deep Dixit

12⁰⁰

34

IMMUNOLOGICAL REGULATORS OF HEALTH-SPAN
Vishwa Deep Dixit,

Age is the greatest risk factor for chronic diseases. Data from several
clinical studies suggest an association between increase in proinﬂammatory markers and emergence of age-related degenerative changes.
However, it remains unclear whether downregulating inﬂammation, a
critical protective response for host-defense and tissue remodeling,
represents a viable therapeutic approach during aging process. Here we
detail a mechanism by which the Nlrp3 inﬂammasome controls systemic
low-grade age related ''sterile'' inﬂammation in both periphery and brain
independently of the noncanonical caspase-11 inﬂammasome. The Nodlike receptor (NLR) family of innate immune cell sensors, such as thenucleotide-binding domain, leucine-rich-containing family, pyrin domaincontaining-3 (NLRP3) inﬂammasome controls the caspase-1 activation in
myeloid-lineage cells in several organs during aging. The NLRP3
inﬂammasome is especially relevant to aging as it can get activated in
response to structurally diverse damage-associated molecular patterns(DAMPs) such as extracellular ATP, excess glucose, ceramides, amyloids,
urate and cholesterol crystals, all of which increase with age. Ablation of
Nlrp3 inﬂammasome protected mice from age-related increases in the
innate immune activation, alterations in CNS transcriptome, andastrogliosis. Consistent with the hypothesis that systemic low-grade
inﬂammation promotes age-related degenerative changes, the deﬁcient
Nlrp3 inﬂammasome-mediated caspase-1activity improved glycemic
control and attenuated bone loss and thymic demise. Notably, IL-1 mediatedonly Nlrp3 inﬂammasome-dependent improvement in cognitive function
and motor performance in aged mice. In addition, ablation of Nlrp3
inﬂammasome activation within thymic macrophages prevented agerelated thymic atrophy and resulted in preservation of T cell receptor
repertoire diversity in aged mice. These studies reveal Nlrp3 inﬂammasome
as an upstream target that controls age-related inﬂammation and offer an 11⁰⁰
innovative therapeutic strategy to lower Nlrp3 activity to delay multiple
age-related chronic diseases and enhance healthspan.
Acknowledgements: The research in Dixit lab is supported in part by US
National Institutes of Health grants AI105097, AG043608, DK090556 and 12⁰⁰
AG045712

35

SPEAKERS ABSTRACTS

Department of Immunobiology and Comparative Medicine, Yale School of Medicine, New Haven CT

Day 4Ofer Mandelboim

N. Avrion Mitchison

36IL-33-dependent immunosuppressive Treg responses to liver damage during
MCMV infection
Branka Popović1, Mijo Golemac1, Lidija Bilić-Zulle2, Miodrag Lukić3, Astrid Krmpotić1, Stipan Jonjić1
1

Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Croatia
Department of Medical Informatics,Faculty of Medicine, University of Rijeka, Croatia
Department of Microbiology and Immunology, Centre for Molecular Medicine and Stem Cell Research, Faculty of Medicine,
University of Kragujevac, Serbia

2

Regulatory T cells (Treg) are critical for preventing autoimmunity mediatedby self-reactive T cells, but also in modulating immune responses toinfections and therefore controlling balance between activation andtolerance. Murine cytomegalovirus (MCMV) is a herpesvirus withpathogenic potential that can induce high levels of viral replication andmodulate antiviral responses. Thus, early immune mechanisms are essential
in controlling virus replication and protecting the host from virus-induced
pathology. Studies on Treg cells reveal their role in suppressing early NK
and T cell responses activated by MCMV in infection. However, absence ofTreg cells does not inﬂuence viral clearance. Here we show that Treg cells
are indispensable for preventing liver pathology induced by MCMV
infection. In addition, their suppressive capacity is dependent on tissue
alarmin, IL-33, which promotes Treg recruitment and function in liver of
MCMV infected mice. Moreover, mice lacking IL-33 receptor showed
higher death rate mediated by stronger liver pathology after infection withhigh viral dose. This is a consequence of decreased inﬁltration of Treg cells
and lower expression of anti-inﬂammatory IL-10 and TGF-β, compared tocontrol mice. In the light of recent discovery of IL-33's importance foraccumulation and maintenance of Treg cells in acute colitis model, ourresults suggest protective and homeostatic role of IL-33 signaling ininﬂammation induced by MCMV infection.

PRESENTATION

3

1

37

Diagnostic utility of soluble interleukin-2 receptor in children with suspected
hemophagocytic lymphohistiocytosis
Srdja Janković1, Lidija Dokmanović1,2, Nada Krstovski1,2, Jelena Lazić1,2, Predrag Rodić1,2, Goran
Milošević1, Dragana Janić1,2
lUniversity Children's Hospital, Tiršova 10, 11000 Belgrade, Serbia
University of Belgrade Faculty of Medicine, Doktora Subotića 8, 11000 Belgrade, Serbia

PRESENTATION

2

The levels of soluble interleukin-2 receptor (sIL-2R) in the plasma reﬂect
the activation of the adaptive immune system and are extremely elevated inhemophagocytic lymphohistiocytosis (HLH). Cutoff value of sIL-2R
according to the HLH-2004 criteria, devised by The Histiocyte Society, is2400 U/ml. Other criteria include fever, hepatosplenomegaly, bi- or
pancytopoenia, hypoferritinemia, hypertriglyceridemia and/or
hypoﬁbrinogenemia, reduced NK cell activity and hemophagocytosis. Fiveof the eight criteria are necessary for the diagnosis of HLH. Measurement ofsIL-2R levels by enzyme-linked immunosorbent assay (ELISA) is routinely
performed in the Laboratory for Immunology of the University Children's
Hospital in Belgrade since August 2008. Up to March 2015, we have
performed this test for 41 child suspected to suffer from HLH. Twenty-six
children (63,4%) were found to have HLH based on the HLH-2004 criteria,
while in 15 (36,6%) HLH was not conﬁrmed. Of 26 diagnosed children, sIL2R was above the cutoff value in 21, yielding sensitivity of 80,8%. Only twoof the 15 children who did not turn out to have HLH had sIL-2R above thecutoff. The speciﬁcity of sIL-2R measurement in diagnosing HLH wastherefore 13/15 (86,7%). Given that different HLH-2004 criteria are often
not fulﬁlled at the same time and that ELISA test can be performed rapidly
and efﬁciently, the presented results warrant consideration of routine use ofsIL-2R measurements in laboratories of tertiary care medical institutions,particularly if combined with meticulous patient selection, in order toreduce time to diagnosis and thus enable timely treatment of HLH.

2

38

Deletion of CMV inhibitor of PVR (CD155) results in enhanced virus susceptibility
to innate immune control and dramatic attenuation in vivo
Tihana Lenac Rovis1, Noa Stanietsky2, Vanda Juranic Lisnic3, Stefan Jordan4, Paola Kucan1, Adriana
Tomic1, Marina Babic3, Pinchas Tsukerman2, Ofer Mandelboim2, Astrid Krmpotic3, Stipan Jonjic1,3
1

Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
The Lautenberg Center for General and Tumor Immunology, The Hebrew University, The BioMedical Research Institute,
Hadassah Medical School, Jerusalem, Israel
3
Department for Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
4
Max von Pettenkofer-Institute, Ludwig Maximilians-University, Munich, Germany

Poliovirus receptor (PVR), or CD155, is highly conserved and ubiquitously
expressed molecule which plays a role in immune response to tumors andviruses. It is recognized by activating (DNAM-1 and Tactile) and inhibitory(TIGIT) receptors expressed on variety of immune cells. Humancytomegalovirus (HCMV) protein UL141 downmodulates the expressionof PVR, suggesting its role in immunosurveillance of CMV infection(Tomasec et al., Nat Immunol. 2005). Here we show that murine CMV(MCMV) also downregulates the surface expression of PVR and further, wecharacterized the viral protein involved. This protein is encoded by m20
gene region which displays a highly complex transcriptional proﬁle: ﬁve 3'co-terminal highly-overlapping transcripts are transcribed from this generegion and each has the potential to encode different proteins. Indeed, we
have detected two different proteins with different temporal kinetics andlikely different functions. Furthermore, we demonstrated the biologicalsigniﬁcance of PVR regulation on viral pathogenesis and virus
susceptibility to various immune response mechanisms. Our results indicatethat MCMV affects the maturation of PVR, rather than directly removing itfrom the surface of infected cells. Since PVR can ligate both inhibitory andactivating immune receptors, the consequence of its viral downregulation in
immune response was hardly predictable. Yet, deletion of MCMV inhibitorof PVR resulted in dramatic virus attenuation which could only partially bereversed by depletion of NK cells, suggesting the impact of other immune
control mechanisms, in addition to NK cells.

PRESENTATION

2

3

39

The Hepatitis C Virus Contributes to the Aggravation of the Immunosuppressive
Environment by Increasing the Suppressive Activity of Natural Regulatory T cells andInducing the Emergence of Type 1 Regulatory T Cells
Ouaguia Laurissa1, Moralès Olivier 1, Wychowski Czeslaw 2, Carpentier Arnaud 3, Aoudjehane Lynda 4,
Dubuisson Jean2, Calmus Yvon 4,5,6, de Launoit Yvan 1, Conti Filomena 4,5,6, Delhem Nadira 1
1

CNRS- UMR 8161 - Institut de Biologie de Lille, Lille, France
Inserm U1019-CNRS UMR8204- Centre d'Infection et d'immunité de Lille, France
National Institute of Health (NIH), Bethesda, USA
4
UPMC Université Paris 06 & INSERM, UMR_S 938, CdR Saint-Antoine, F-75012, Paris, France
5
AP-HP, Hôpital Saint-Antoine, Pôle Digestif, Centre de Transplantation Hépatique, F-75012, Paris, France
6
Faculté de Médecine René Descartes Université Paris 05, F-75014, Paris, France
2

PRESENTATION

3

4

Background & Aims: HCV infection is characterized by a high risk ofchronicity. We have shown in a previous work that natural and induced
human regulatory T cells (nTreg and Tr1) play an important role in the
progression of hepatitis C to hepatocellular carcinoma and are associatedwith the severity of viral recurrence after liver transplantation. However,nothing is known concerning the speciﬁc impact of HCV on these tworegulatory T cell sub-populations. Our hypothesis is that HCV may promote
the recruitment of regulatory T cells in the infected liver, and alter theirphenotype and suppressive activity for the progression of liver
pathogenesis.
Methods: Treg cells were isolated from the blood of healthy donors andinfected in vitro with HCV. The impact of HCV infection on nTreg and Tr1
phenotype and function has been assessed.
Results: We showed that Treg infection with HCV signiﬁcantly increases
the expression of viral receptors and that the viral proteins are internalized
into Treg. We also showed that viral infection raises the Treg anergy andpromotes the recruitment of infected Treg cells by HCV-infected
hepatocytes and primary intrahepatic ﬁbroblasts. In addition, HCV
infection induced a signiﬁcant increase in the expression of markers
associated with Treg cells, thus potentiating their "suppressive phenotype."
These results are correlated with the functional analysis of infected Treg
cells, showing (i) a signiﬁcant increase in the expression of markers
associated with their suppressive activity, (ii) a signiﬁcant increase in thesecretion of immunosuppressive cytokines and (iii) an increase inimmunosuppressive function. Finally, we have shown that HCV promotes
conversion of conventional T lymphocytes into induced T regulatory type 1
cells.
Conclusion: This study shows for the ﬁrst time that HCV can be able to beinternalized into human Treg cells and could promote Treg recruitment into
the infected liver. This may contribute to explain the mechanisms by which
HCV escapes the immune system and promotes the progression of hepatitis
C to cirrhosis and HCC.

40

Ocular conjunctiva as a mucosal immunization route: humoral and cellular immune
responses against Escherichia Coli Nissle 1917 bacterial ghosts
Aleksandra Inic-Kanada1, Sandra Belij1, Marijana Stojanovic2, Emilija Marinkovic2, Ivana Lukic2, Jacqueline
Montanaro1, Nadine Schürer1, Elisabeth Stein1, Nora Bintner1, Ulrike Beate Mayr3, Werner Lubitz3, Talin
Barisani-Asenbauer1

Mucosal delivery of vaccines has the potential to eliminate the requirement
for needles and induce strong immunity against pathogens. However, mostsubunit protein antigens are poor immunogens and there is an urgent needfor the development of new and improved routes of immunization, as well
as safe and efﬁcacious delivery systems and adjuvants. In this study wecharacterized humoral and cellular immune responses after conjunctival
immunization with corpuscular Escherichia coli Bacterial Ghosts (EcN
BGs) in BALB/c mice and evaluated tolerability at the ocular surface after
conjunctival application. BALB/c mice were immunized via theconjunctiva or subcutaneously with 12,5 µg EcN BGs per mouse. Three
immunizations were performed at 2-week intervals and immune responses
were evaluated 2 weeks after the last immunization. There was no pathology
at the ocular surface of mice immunized with EcN BGs via the conjunctiva.
EcN BGs speciﬁc secretory IgA levels in tears was signiﬁcantly higher inthe group immunized conjunctively compared to subcutaneously
immunized mice. Furthermore, immunization with EcN BGs via theconjunctiva promoted the establishment of a EcN BGs-speciﬁc Th1/Th17
immune response. Increased levels of IgA and Th1-biased immune
response without any resultant pathology indicate that EcN BGs applied via
the conjunctiva merit further investigations as adjuvantic carriers inconjunctival vaccine development.

PRESENTATION

1
Medical University Vienna, Laura Bassi Centres of Expertise, OCUVAC - Centre of Ocular Inﬂammation and Infection, Institute ofSpeciﬁc Prophylaxis and Tropical Medicine, Centre for Pathophysiology, Infectiology and Immunology, Vienna, Austria
2Institute of Virology, Vaccines and Sera – TORLAK, Belgrade, Serbia
3
BIRD-c GmbH&CoKG, Vienna, Austria

541

The Protective Potential of an individual Anti-Tetanus Monoclonal Antibody isimproved if used in a combination with another MAb able to bind Tetanus Toxin
simultaneously
Ana Filipovic

PRESENTATION

Institute of Virology, Vaccine and Sera "Torlak" Belgrade

Monoclonal antibodies (MAbs) are considered for a long time as a reagentof choice for prevention of tetanus as they can be produced in large scale invitro and their quality is consistent. In this work we explored whether the
MAbs mixture would confer better protectiveness than each MAb alone.
The protective potential of anti-TTn MAbs, MAb 51 and MAb71, wasevaluated in murine model system. MAbs were applied separately (10 µg of
MAb in PBS, i.p) or as equimolar mixture (5 µg of each MAb in PBS, i.p),
simultaneously or 2h, 6h and 10h upon i.p. challenge with lethal dose ofTTn. Mice were monitored on a daily basis for two weeks for symptoms oftetanus disease (max. pathology score 5). MAb51 and MAb71 can bind TTn
simultaneously without any steric hindrance (index of aditivity 89 ± 7).
When applied separately, simultaneously with TTn challenge, MAb51
confer full protection (survival rate 100%, mean daily pathology score2.10.18, no signs of pathology 7 days upon treatment) while survival ratewith MAb71-treated mice was 60% (mean daily pathology score 3.80.18,
30% of alive mice had slight symptoms of tetanus two weeks upon achallenge). Clinical picture of mice treated in the same way with
MAb51/MAb71 mixture was signiﬁcantly better than with MAb51-treatedmice (no pathology with 80% mice, pathology score 1). Separately or in
equimolar mixture, MAbs also provided protection when they were applied
early after TTn challenge (up to 10h, pathology score 1). In all cases thetiming of MAb(s) administration did not signiﬁcantly inﬂuence survival
rate but the intensity of pathology and recovery time (positive correlation).
Obtained results show that pooling of MAbs that are able to bind TTn
simultaneously can improve their individual protective potentials.

642Deletion of Galectin 3 Enhances Primary Biliary Cirrhosis in Mice by Enhanced
Apoptosis of Biliary Epithelial Cells and Release of Autoantigens
Aleksandar Arsenijevic1, Marija Milovanovic1, Jelena Milovanovic1, Bojana Stojanovic1, Natasa Zdravkovic2,
Patrick Leung3, Fu-Tong Liu3, Erick Gershwin3, Miodrag L. Lukic1
1Center for Molecular Medicine and Stem Cell Research, Faculty of Mecial Sciences, University of Kragujevac, Serbia
Department of Internal Medicine, Faculty of Mecial Sciences, University of Kragujevac, Serbia
University of California, Davis, CA, USA

2

Galectin-3 (Gal-3) is expressed in various cell types and is involved in thepathogenesis of many chronic inﬂammatory diseases. Increased expressionof Gal-3 in epithelial cells protects them from apoptosis. Apoptotic death ofbiliary epithelial cells (BECs) has important role in the pathogenesis ofprimary biliary cirrhosis (PBC). PBC is the liver-speciﬁc autoimmune
disease characterized by a multilineage response against PDC-E2. When
BECs undergo apoptosis the major mitochondrial autoantigen, PDC-E2,
remains immunologically intact and is expressed at the apical surface of thesmall bile duct cells, perpetuating autoimmune response. The aim of thisstudy was to analyze the role of Gal-3 in PBC pathogenesis. Female
C57BL/6 wild type and Gal-3 KO mice were immunized with 2OA-BSA
and disease was evaluated by histological examinations and measurementof the serum levels of PDC-E2 speciﬁc antibodies. Immunophenotyping oflymphocytes and dendritic cells was done by ﬂow cytometry. Expression of
Gal-3 and cytokeratin in BECs was detected by immunohistochemistry
while apoptosis of BECs was analyzed by TUNEL assay and Annexin V
staining. We report here that deletion of Gal-3 molecule ampliﬁes PBC
induced by 2OA-BSA mice in C57BL/6 mice on histopathological ﬁndings
and serum analysis. Periportal inﬁltrations, bile duct damage, and ﬁbrosis inlivers of Gal-3 KO mice were more pronounced with granuloma formation
observed only in Gal-3 KO mice. Four week after immunization, serumanti- PDC-E2 IgA antibody was signiﬁcantly increased in Gal-3 KO micecompared to WT mice. Liver inﬁltrates of Gal-3 KO mice contained higher
number of IFN-γ positive CD8+ lymphocytes. Enhanced PBC in Gal-3 KOmice is accompanied with reduced apoptosis of BECs. Besides BECs of
Gal-3 KO mice had inherently higher response to apoptotic stimuli. There
were more proinﬂammatory dendritic cells in the livers of Gal-3 KO 2OABSA immunized mice. Accelerated 2OA-BSA induced PBC in Gal-3 KOmice appears to be the result of enhanced release of autoantigen andconsecutive stimulation of inﬂammatory antigen presenting and effector
cells.
Key words: PBC, 2OA-BSA, Gal-3, BECs, apoptosis
This work was supported by the Ministry of Science, Republic of Serbia,
grants ON175069, ON175071 and ON175103Faculty of Medical Sciences,
University of Kragujevac, Serbia grants MP 01/14 and 02/14.

43

PRESENTATION

3

7

Genetic deletion of Galectin-3 attenuate dextran sodium sulphate colitis in mice
Bojana Simovic Markovic, Marina Gazdic, Aleksandar Nikolic, Irena Djordjevic, Nebojsa Arsenijevic,
Miodrag Stojkovic, Miodrag Lukic and Vladislav Volarevic

PRESENTATION

Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Serbia

8Introduction. Galectin-3 (Gal-3) is an endogenous lectin with a broad
spectrum of immunoregulatory effects: it plays an important role inautoimmune/inﬂammatory and malignant diseases, but the precise role ofGal-3 in pathogenesis of ulcerative colitis is still unknown.
Methods. We used a model of dextran sulphate sodium (DSS)-induced
colitis, that has a high degree of uniformity and reproducibility to humancolitis, to test susceptibility of wild-type C57BL/6 and Gal-3-deﬁcient mice(Gal-3-/-) to this disease. DSS (3%, molecular weight 40kDa) was dissolvedin water and given to C57Bl/6 and Gal-3-/- mice in place of normal drinking
water (ad libitum) for 7 days. Disease Activity Index (DAI: weight loss,
stool consistency, visible blood in feces), was used to assess the clinicalsigns of colitis. The cellular make up of colon and phenotype of coloninﬁltrated immune cells were determined by ﬂow cytometry.
Results. Genetic deletion of Gal-3 signiﬁcantly reduce the damage of colon
tissue of DSS-treated mice. Level of pro-inﬂammatory cytokines (IL-1β,
TNF-α и IL-6) were signiﬁcantly lower in sera and colons of DSS-treatedGal-3-/- mice when compared to WT DSS-treated mice. The total number ofCD11c+ inﬂammatory dendritic cells (DC) which expressed CD80 and I-A
and produce pro-inﬂammatory cytokines (TNF-α and IL-6) as well as TNFα and IL-1β producing CD45+CD11c-Ly6G+ neutrophils weresigniﬁcantly lower in colons of Gal-3-/- DSS-treated mice. In addition, thetotal number of inﬂammatory colonic (F4/80+CD11b+SiglecF-,
F4/80+CD11b+I-A+, IL-1, IL-6 and IL-12 producing) macrophages weresigniﬁcantly lower in Gal-3-/- mice compared with WT DSS-treated mice.
On contrary, there was signiﬁcantly higher number of IL-10 producing
regulatory DC and alternatively activated M2 macrophages in colon tissueof Gal3-/- DSS-treated mice. In vitro lipopolysaccharide (LPS) and DSSstimulated peritoneal macrophages isolated from untreated Gal-3-/- mice
produce lower amounts of TNF-α and IL-1β when compared to WT cells.
Adoptive transfer of WT macrophages managed to signiﬁcantly enhance
the severity of DSS-induced colitis of Gal-3-/- mice. Antibiotic treatment
did not affect differences between DSS-treated WT and Gal-3-/- mice.
Conclusion. Gal-3 expression promotes acute DSS-induced colitis. Thiseffect is due to its pro-inﬂammatory role in particular on peritoneal
macrophages rather than its role as a receptor for pathogens.
Keywords. DSS, colitis, Gal-3
This study was ﬁnanced by Grant of European Crohn's and Colitis
Organisation (ECCO) and by Serbian Ministry of Science and Education(projects no.175069 and 175103)

44

Mesenchymal stem cells attenuate acute liver injury mediated by NKT cells
Vladislav Volarevic, Marina Gazdic, Bojana Simovic Markovic, Aleksandar Nikolic, Irena Djordjevic,
Nebojsa Arsenijevic, Miodrag Stojkovic and Miodrag Lukic

The effects of mesenchymal stem cells (MSC) on phenotype and function ofnatural killer T (NKT) cells, major effector cells in acute liver injury, is not
fully understood. We used two well-established experimental models of
NKT-cell mediated acute liver pathology: Concanavalin A (Con A)-induced
hepatitis (Con A; 12 mg/kg i.v) and α-galactosylceramide (α-GalCer; 50μg/kg i.v)-induced liver injury to evaluate effects of MSC on liver damage
and NKT cell functions.
MSC (single iv. injection, 500 000/mouse, given immediately after Con A or
α-GalCer) signiﬁcantly attenuate Con A and α-GalCer mediated liver
damage, as demonstrated by histopathological analysis and liver enzyme
tests. MSC-treatment attenuates inﬂux of inﬂammatory NKT cells in theliver (TNF-α-, IFN-γ-, T-bet+ CD4+ and CD1d tetramer+ as well as Gata3+, IL-4-producing NKT cells) and down-regulates inﬂammatory cytokines(TNF-α, IFN-γ, and IL-4) in the sera of Con A and α-GalCer-treated
C57Bl/6 mice. Serum levels of anti-inﬂammatory IL-10 and percentage of
IL-10-producing NKT cells in the liver were signiﬁcantly higher in MSCtreated mice. MSC did not signiﬁcantly affect phenotype of macrophages
and dendritic cells, suggesting that MSC modulate production of cytokines
directly in liver NKT cells. Signiﬁcantly lower amounts of inﬂammatory
cytokines (TNF-α, IFN-γ, IL-4), and higher amounts ofimmunosuppressive IL-10 were noticed in supernatants of in vitro α-GalCer(100 ng/mL)-stimulated liver NKT cells cultured with MSC in transwellsystems when compared to α-GalCer-stimulated liver NKT cells whichwere cultured alone. MSC treatment attenuate expression of FASL, CD107
and TRAIL, receptors known to be responsible for NKT cell mediatedapoptosis and cytotoxicity. Accordingly, MSC treatment signiﬁcantly
reduced cytotoxic potential of liver NKT cells. The results obtained by
xCELLigence system for monitoring real-time cytotoxicity showed thatNKT cells isolated from MSCs+α-GalCer-treated mice were signiﬁcantly
less cytotoxic against HEPG2 hepatocyte cells then NKT cells isolated frommice treated with α-GalCer-only. Human MSC managed to signiﬁcantly
attenuate production of inﬂammatory cytokines in α-GalCer-stimulated
peripheral blood mononuclear cells and attenuate their cytotoxicity against
HEPG2 cells. In conclusion, MSC protect from acute liver injury byattenuating cytotoxicity and capacity of liver NKT cells to produce
inﬂammatory cytokines.
This study was ﬁnanced by Serbian Ministry of Science and Education(projects no.175069 and 175103) and Faculty of Medical Sciences
University of Kragujevac (MP01/14)

45

PRESENTATION

Centre for Molecular Medicine and Stem Cell research, Faculty of Medical Sciences University of Kragujevac Serbia

9Ethyl Acetate Extract of Origanum Vulgare Exerts Immunomodulatory Effect In Vitro
and Reduces Hyperglycemia in Mice
Milica Vujičić1, Ivana Nikolić1, Tamara Saksida1, Bobana Milenković1, Dušan Radojević1, Andreas Tzakos2,
Stanislava Stošić-Grujičić1 and Ivana Stojanović1
1

Institute for Biological Research “Siniša Stankovic”, Department of Immunology, University of Belgrade, Belgrade, Serbia
University of Ioannina, Department of Chemistry, Ioannina, Greece

PRESENTATION

210Type 1 diabetes (T1D) is autoimmune inﬂammatory disease with hallmark
of pancreatic beta cell destruction via various proinﬂammatory mediators.
Focus of T1D treatment is shifting towards establishing novel therapeutic
approaches which could be more efﬁcient than standard insulin
replacement. Origanum vulgare, besides being a spice, is also known as asource of natural antioxidants and exerts anti-fungal, anti-bacterial and antiinﬂammatory effects. The main aim of this study was to determine effects ofethyl acetate extract of Origanum vulgare (EAO) on immunomodulationand cellular viability, as well as its effect on diabetes development. EAO
extract was prepared from Oregano leaves by polar extraction method. In
vitro effects of EAO were determined by measuring proliferation (MTT
test) and cytokine production (ELISA) of murine lymph node and peritoneal
cells, and nitric oxide (NO) production of peritoneal cells (Griess reaction).
To evaluate the effect of EAO on diabetes development, the extract wasadministered intraperitoneally to C57BL/6 mice that were subjected to T1D
induction by multiple low doses of streptozotocin (MLDS). Our results
indicate that 48h treatment with EAO did not alter basic or concanavalin Ainduced proliferation of lymph node cells, as well as IL-4 secretion, while
reducing IFN-γ and IL-17 secretion. EAO reduced IL-1β secretion of
peritoneal cells, while secretion of TNF remained unchanged. Also, EAO
did not affect NO production or viability of peritoneal cells. Finally, 10-day
of EAO treatment signiﬁcantly reduced the diabetes incidence and the levelof hyperglycemia in MLDS-treated mice. In conclusion, EAO alters
secretory patterns of immune cells in vitro by reducing proinﬂammatory
Th1 and Th17 response, and protects mice from developing hyperglycemia
in vivo. Observed immunomodulatory and anti-diabetogenic effects of
EAO are being further analyzed for potential treatment of diabetes.
This work was supported by the Ministry of Education, Science andTechnological Development, Republic of Serbia (grant no. 173013).

46

Carbon Monoxide-Releasing Molecule (CORM)-A 1 Potentiates Regulatory Arm of
the Immune Response and Exerts Protective/Regenerative Effects on Beta Cells
Ivana Nikolić, Tamara Saksida, Milica Vujičić, Ivana Stojanović and Stanislava Stošić-GrujičićType 1 diabetes (T1D) is an autoimmune disease characterized by insulitis
and islet beta cell loss. Thus, an effective therapy requires beta cellpreservation and immune suppression. Using mouse model of T1D inducedby multiple low doses of streptozotocin (MLDS), we have recently reported
that carbon monoxide-releasing molecule (CORM)-A1 can achieve bothgoals efﬁciently by acting on both: the islet beta cells and immune system.
CORM-A1 therapy exerted an anti-inﬂammatory effect and produced amore favorable cytokine proﬁle by shifting the M1/Th1/Th17 balance
towards a M2/Th2 response, while directly interfered with beta cell
apoptosis through the reduction of cytochrome c and caspase 3 levels. In thepresent work we tested the hypothesis that CORM-A1 therapy might
inﬂuence regulatory arm of the immune response, as well as beta cellregeneration. We found that in CORM-A1-treated MLDS-induced mice theimprovement of hyperglycemia was lost after depletion of
cyclophosphamide-sensitive FoxP3+ Treg cells. Of note, the improvement
was accompanied by decreased levels of IL-12, IL-2 and early activation
marker CD25 in the spleen and pancreatic lymph nodes and increased TGFβ, resulting in reduced lymphocyte proliferation in both organs. In parallel,
decreased transcript levels of IL-2, but increased mRNA expression of
TGF-β was observed within pancreas, with concomitant local up-regulationof Ki-67 protein expression, suggesting proliferation of surviving beta cells.
Taken together, our results indicate that CORM-A1 may attenuate
autoimmune diabetes not only through immune cell resetting, but also by
improving the survival/growth of insulin-producing pancreatic beta cells.
These data shed new light on the mechanisms that underlie CORMA1–mediated immunomodulation and provide insights into the role of
CORM-A1 in regulating islet cell function and glucose homeostasis, which
may ﬁnd clinical application.
Supported by Ministry of Education, Science and Technological
Development, Republic of Serbia (grant no. 173013).

47

PRESENTATION

Department of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia

11Subchronic oral intake of low cadmium doses affects intestinal immune
responses in rats
Ninkov Marina1, Popov Aleksandrov Aleksandra1, Demensku Jelena1, Mirkov Ivana1, Mileusnic Dina1,
Grigorov Ilijana2, Petrovic Anja2, Zolotarevski Lidija3, Nikolic Milica4, Kataranovski Milena1,5
1

PRESENTATION

Immunotoxicology group, Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University ofBelgrade, Belgrade, Serbia
2
Department of Molecular Biology,Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade,
Serbia3
Institute of Pathology, Military Medical Academy, Belgrade, Serbia
4
Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade,
Belgrade, Serbia
5Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia

12

Gastrointestinal (GI) tract is one of the main targets of cadmium (Cd),important food and drinking water contaminant. In this study, the effect ofsubchronic (30 days) oral (in drinking water) intake of environmentally
relevant doses of cadmium (5µg/ml and 50µg/ml) on intestinal [(tissue of
duodenum and mesenteric lymph node (mLN) cells)] was examined in
Dark Agouti (DA) rats. Atomic absorption spectrophotometry (AAS)
analysis revealed signiﬁcant cadmium load in duodenum,which was
associated with changes of both intestinal bacterial load and composition(Denaturing Gradient Gel Electrophoresis/DGGE).Shortening of villi,
damage to epithelium, increases in goblet-like vacuoles and mononuclear
cell inﬁltration in lamina propria were histologically evident at both
cadmium doses, with massive necrosis at higher Cd dose (judging by High
Mobility Group Box-1/HMGB-1 Western blot analysis).Increased levels of
proinﬂammatory cytokines (IL-1β, IFN-γ, IL-17) were observed at both Cd
doses (TNFα at higher dose solely). Cadmium administration affecteddraining lymph nodes as well, judging by signs of stress response (drop of
cellular reduced glutathione/GSH at higher dose, rise of
metallothionein/MT mRNA at both doses).Increased cellularity of mLN
was observed at higher Cd dose, but with no changes in proliferative
responses. Intake of both Cd doses resulted in higher (compared to controls)
levels of IFN-γ and IL-17 mRNA as well as increased mLN cell
responsiveness to ConA stimulation.Signiﬁcant increases in numbers of
CD68+ cells and stimulation of innate immune activities (numbers of
dihydrorhodamine/DHR+ cells and intracellular myeloperoxidase/MPO+
cells, LPS-stimulated nitric oxide/NO production and ex vivo IL-1β
expression) were observed at higher dose of cadmium.Proinﬂammatory
effects of cadmium might have resulted from changes in gut microbiota andtissue damage.Interactions of this metal with intestinal immune systemshould be taken into account when assessing dietary cadmium as health risk
factor.
Funded by Ministry of Education, Science and Technological Developmentof Serbia (#173039).

48

Characterization of single sorted ANTI-ADAMTS13 speciﬁc B cells from the spleenof acquired thrombotic thrombocytopenic purpura (ATTP) patients
Magdalena Skowronska
Clinic of Hemat Central Hematology Laboratory and Department of Clinical Research, University Hospital Bern, Inselspital, Bern,
Switzerland

Methods: Splenic mononuclear cells from 7 aTTP patients (2 treatedwith
Rituximab) were analysed by ﬂow cytometry. We calculated the frequenciesof highly positive anti-ADAMTS13B-cellsamong naïve, un-, switchedmemory B-cells and plasma cells. B-cells bearing anti-ADAMTS13 IgG
were individually sorted based on binding to ﬂuorescently labelled
rADAMTS13. Gene transcripts of single cells were reverse-transcribedfollowed by nested PCR of IgG heavy/light chains. Gene analysis
identifying the Variable domains (V) and V-(D)-J junctions was performed
using IMIGT/V-QUEST tool.
Results: Anti-ADAMTS13 cells were detected in the spleen of all patients(average 0.057% among B-cells populations) with highest prevalence
among plasma cells. The two Rituximab treated patients had decreased levelof CD19/CD20 positive cells (1% and 20% of lymphocytes after Tcell/monocyte exclusion), compare to untreated patients (~63%). Splenicanti-ADAMTS13 speciﬁc B-cells of 4 aTTP patients revealed 149
antibodies sequences from whichwe analysed the CDR3diversity. Most
frequently used V-genes were of IGHV1-69, IGHV3-30, IGHV4-31 (20%,
12%, 7%).
Conclusion: Speciﬁc anti-ADAMTS13 B-cells were found in the spleen of
all aTTP patients, in a range similar to ITP patients, including plasma cells
known to be unaffected by Rituximab. Currently we clone selected single
sorted monoclonal antibodiesfor functional tests to select the inhibitory Abs
as tool to develop antigen-speciﬁc therapies for aTTP.

49

PRESENTATION

Introduction: Autoantibodies(Abs) that neutralize and/or accelerate theclearance of ADAMTS13 cause acquired Thrombotic Thrombocytopenic
Purpura (aTTP). Since increasing evidence points at the spleen as a major
reservoir for antigen speciﬁc memory B-cells, we investigated the splenic
B-cell population and the frequency of ADAMTS13-speciﬁc B-cells as aprerequisite for understanding the humoral autoimmune response and fordeveloping an optimized and specialised treatment.

13

Level of regulatory cytokines correlate with the outcome of the inﬂammation in theprocess of PEX production
Tatjana Sarenac Vulovic1, Sladjana Pavlovic2, Nemanja Zdravkovic2
1

Clinic for Ophthalmology, Clinical Centre Kragujevac, Kragujevac, Serbia
Department of Microbiology and Immunology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia

PRESENTATION

2

14

Objective: The purpose of this study is to reveal the participation ofdifferent regulatory cytokines within the process of pseudoexfoliation(PEX).
Methods: Our study included 120 patients, referred to cataract surgery withearly and late stage of pseudoexfoliation syndrome (XFS) or
pseudoexfoliation glaucoma (XFG). Humour and serum levels ofcytokines: TNF-α, IL-17, IL-6, TGF-β, PDGF, EGF, IGF IL-8 and ITAC
were measured in a sample with high sensitivity enzyme-linked
immunoabsorbent assay (ELISA) kit.
Results: Aqueous humour levels of proinﬂammatory cytokines TNF-α andIL-17 are increased in patients with the early and late stage of XFS and XFG,
while IL-6 levels are increased in the early stage of XFS and XFG. Serumlevel of IL-6 showed a signiﬁcantly increased level of IL-6 in patients withthe early and late stage of XFS. Aqueous humour level of regulatory
cytokine TGF-β is increased in the early stage of XFS. Early and late stageof XFS show increased level of PDGF. XFG patients have an increased levelof EGF, IL-8, and ITAC; but IGF levels were detected only in XFG. Locally,
proﬁbrotic action was sustained by elevated TGF-β, followed by PDGF
action in the late stage of XFS, and with the ﬁnal action of EGF, IGF, IL-8
and ITAC in glaucoma. This process is supported by increased TGF-β in theserum within early stage of XFS, and enhanced PDGF, EGF and IL-8 withinlate stage, and IGF and ITAC in the glaucoma. Our results indicated that in
the early and late stage of XFS and in XFG IL-17 and TNF-α play the mainrole in inﬂammation activation in the tissue; overlooked by IL-6 action in
the early stage and in the phase of glaucoma. The ﬁnal result of ﬁbrous tissue
deposition is the development of glaucoma without vasculogenesis (de novo
blood vessel formation) and angiogenesis (new capillary branches fromexisting blood vessels) in the eye.
Conclusions: The purpose of this study is to indicate the signiﬁcance ofdifferent cytokine actions in stress conditions, as well as to reveal their roles
in different stadiums of PEX production and accumulation process. The
mechanism of the development of XFS syndrome via cytokine formation
will provide us new therapeutic insights for the treatment.
Key words: pseudoexfoliation, cytokines, ﬁbrosis.

50

Standardized bovine colostrum attenuates development of experimental autoimmune
diabetes
Ivana Nikolić , Milica Vujičić, Ivana Stojanović, Stanislava Stošić-Grujičić, Tamara Saksida

Bovine colostrum is a rich source of immune components that play a role inconveying passive immunity to the offspring, protection and maturation of
newborn's gastrointestinal tract and protective host immunity of the
mammary gland itself. Colostrum has exerted positive effects in diseases
affecting gastrointestinal tract, as well as type 2 diabetes. The aim of thisstudy was to investigate therapeutic value of standardized bovine colostrum(SBC) in the model of type 1 diabetes (T1D). Autoimmune T1D was
induced in C57BL/6 mice with multiple low doses of streptozotocin(MLDS, 5 x 40 mg/kg b.w./day). SBC was administered per os (8 g/kg
b.w./day) for 10 consecutive days starting from the day of MLDS induction.
The disease severity was evaluated by weekly measurement of blood
glucose level and by histological analyses of the pancreas at the end ofexperiment (35th day). Ex vivo analysis of cytokine expression andproduction was measured on the 10th day in the spleen and pancreatic (PLN)and mesenteric lymph nodes (MLN). SBC administration to MLDS induced
mice prevents diabetes development, indicated by euglicaemia and by theabsence of insuilitis. In the MLN cells SBC disrupted harmful Th17
response, as judged by reduced expression and secretion of IL-17 compared
to diabetic mice. Also, colostrum down-regulated two Th17 associatedcytokines, IL-6 and IL-23. IFN-γ expression was down-regulated in MLN
cells of SBG treated mice, while IL-4 secretion was up-regulated incomparison to MLDS diabetic group. Modulation of the immune response
seen in MLN protrudes to the spleen and PLN, giving overall less inﬁltrationof immune cells to the pancreas. SBC acts on immune cells and halts(auto)aggression towards pancreatic beta cells. Hence, this derivative could
be tested in diabetes and other diseases with aberrant immune response.
(Supported by Ministry of Education, Science and Technological
Development, Republic of Serbia #173013).

51

PRESENTATION

Department of Immunology, Institute for Biological Research “Siniša Stanković”, Belgrade, Serbia

15

The Role of Trichinella spiralis Muscle Larvae Secretory Antigens in Shaping of
Immune Response
Jelena Cvetković, Alisa Gruden-Movsesijan, Nataša Ilić, Ljiljana Sofronić-Milosavljević

PRESENTATION

Institute for the Application of Nuclear Energy – INEP, University of Belgrade, Serbia

16

The helminth Trichinella spiralis communicates with the host immune
system through excretory-secretory products (ES L1). ES L1 antigens drive
immune response towards Th2 and regulatory type. However, there is no
information about mechanisms by which ES L1 inﬂuences suchpolarization. Here, we investigated the impact of ES L1 and its components
on immune response, Pattern Recognition Receptors (PRRs) involved in theinteraction with these antigens and subsequently provoked signaling eventsin dendritic cells (DCs). ES L1 antigens signiﬁcantly increased the
production of IL-4 and IL-10, while they did not affect the levels of TGF-β
and IFN-γ in vivo. Changes in the structure of ES L1 glycans greatly
reduced the production of IL-4 indicating their importance for Th2 immune
response. In vitro experiments with ES L1 and ES L1 components:
7C2C5Ag (containing 45, 49, and 53-kDa glycoproteins) and Tsp53(recombinant p53) resulted in the same semimature DC phenotype
accompanied by reduced production of IL-12p70 and elevated productionof IL-10 and the capacity to promote Th2 and regulatory responses.
Modiﬁcation of ES L1 glycans (periodate treated ES L1 - pES L1) did notchange DC phenotype but had an impact on cytokine production of DCs(reduced IL-12p70 and IL-10). DCs treated with pES L1 provoked low
production of IL-10 and TGF-β in T cells, indicating the importance of
intact glycans for Th2 and regulatory response induction. Considering
investigation of PRRs involved in the interaction with ES L1 antigens, wehave found that TLR2 is engaged by ES L1 and 7C2C5Ag, while TLR4
interacts only with ES L1. These interactions are impaired with changes incarbohydrate structure of ES L1. ES L1, 7C2C5Ag and Tsp53 transiently
activated ERK MAP kinase, weakly activated p38 kinase and these
activations were glycan dependent. This study indicates for the ﬁrst timethat some components of ES L1 (7C2C5Ag and Tsp53) possess
immunomodulatory properties and that glycans on ES L1 are important for
immune response polarization.
(This work was supported by Ministry of Education, Science andTechnological Development, R. Serbia, Grant No. 173047).

52

Immunometabolic Differences in Prototypical Th1- And Th2-Type Mouse Strains in
High-Fat Diet Induced Obesity
Nemanja Jovicic1,2, Nada Pejnovic1,3, Ilija Jeftic1,3, Ivan Jovanovic1, Gordana Radosavljevic1,
Nebojsa Arsenijevic1, Miodrag L. Lukic1
1Center for Molecular Medicine and Stem Cell ResearchInstitute of Histology
Institute of Pathophysiology, Faculty of Medical Sciences, University of Kragujevac, Serbia

2Introduction & Aims: The inherent susceptibility to obesity-related
metabolic disorders and type of immune/inﬂammatory response inmetabolic tissues in mouse strains are markedly dependent on the genetic
background which may affect the translation of experimental data to humanpathology. Therefore, we have investigated the strain-dependent
differences of visceral adipose tissue (VAT) and liver immunophenotype,
liver steatosis, inﬂammation and ﬁbrosis in experimental model of high-fat
diet induced obesity in C57BL/6 and BALB/c, the prototypical Th1 and Th2mouse strains.
Methods: Male 8-week old C57BL/6 and BALB/c mice were placed on
HFD (60% kcal fat) or standard chow diet (10% kcal fat) for 24 weeks. We
performed histological and liver and adipose tissue immunophenotypic
analyses as well as expression of hepatic proﬁbrogenic and lipid
metabolism-related genes.
Results: After 24 weeks of dieting BALB/c mice exhibited higher weightgain on standard diet, while C57Bl/6 mice exhibited higher weight gain on
HFD. In comparison to BALB/c mice, the amount of visceral fat and fastingblood glucose levels was higher in C57Bl/6 mice on both, chow and HFD. In
contrast to BALB/c mice, HFD induced a signiﬁcant increase of the amountof VAT and number of VAT associated CD3+CXCR3+ Th1 cells, dendritic+
cells (DCs) and F4/80 macrophages in C57Bl/6 mice. In livers, more
numerous CD3+ and CD8+ T lymphocytes, myeloid DCs, proinﬂammatory
macrophages (F4/80+CD11b+CD11+ andF4/80+IL-1β+) and CD11b+Ly6C+high
monocytes and higher levels of IL-6, TNF-α and IFN-γ were detected in
HFD-fed C57Bl/6 mice than in diet-matched BALB/c mice. HFD-fed
C57Bl/6 mice had scarce liver steatosis in contrast to BALB/c mice which
had marked hepatic steatosis and increased expression of genes related tolipid metabolism with higher serum levels of cholesterol and triglycerides
and lower glycogen deposition in the liver. HFD induced prominent liverﬁbrosis in C57Bl/6 mice while BALB/c mice developed scarce livercollagen deposition. The expression of mRNA for procollagen,proﬁbrogenic IL-13 and TGF-β in liver and the levels of IL-33, IL-13 andTGF-β in sera and liver homogenates were higher in HFD-fed C57Bl/6 micecompared to diet-matched BALB/c mice.
Conclusion: The obtained results indicate that Th1-type mice aresusceptible to obesity, liver inﬂammation and ﬁbrosis while Th2-type mice
to liver steatosis in response to obesogenic HFD which is associated withdifferential phenotypes of immune cells in metabolic tissues.
Immunometabolic differences in relation to Th1 and Th2 dominance may be
relevant for studies of obesity-associated metabolic diseases in humans.

53

PRESENTATION

317

Galectin-3 Promotes Hepatic Inﬂammation and Fibrosis in Obesogenic Mouse Modelof Nonalcoholic Steatohepatitis
Ilija Jeftic1,3 ,Nemanja Jovicic1,2, Jelena Pantic1, Nebojsa Arsenijevic1, Miodrag L. Lukic1, Nada Pejnovic1,3
1

Center for Molecular Medicine and Stem Cell ResearchInstitute of Histology
Institute of Pathophysiology, Faculty of Medical Sciences, University of Kragujevac, Serbia

2

PRESENTATION

318

Introduction & Aims: Galectin-3 (Gal-3), a β-galactoside-binding lectin,
is involved in the regulation of obesity, metaﬂammation, and type 2
diabetes, but its role in the pathogenesis of obesity-associated non-alcoholicsteatohepatitis (NASH) is incompletely deﬁned. In this study, we aimed todissect the role of Gal-3 in liver inﬂammatory response and ﬁbrosis, key
parameters in the pathogenesis and progression of NASH, induced byobesogenic high-fat diet (HFD).
Methods: Gal-3-deﬁcient (LGALS3 -/-) and wild-type (LGALS3 +/+)C57Bl/6 mice received HFD (60% kcal fat) or standard chow diet (10% kcalfat) for 24 weeks and metabolic parameters, gene expression andimmunophenotypic analyses were performed.
Results: In comparison to WT mice, HFD-fed LGALS3-/- mice developed
increased obesity, type 2 diabetes and more pronounced liver steatosis
which was accompanied by upregulation of hepatic FAS, PPAR-γ andCd36 expression. However, ALT and AST levels, liver injury, inﬂammationand ﬁbrosis scores, and hepatic procollagen and α-SMA mRNA expression
were signiﬁcantly increased in HFD-fed WT mice compared to dietmatched LGALS3-/- mice. The more pronounced hepatic ﬁbro-inﬂammatory
response induced by obesogenic diet in WT mice was associated withincreased myeloid DCs and proinﬂammatory monocytes/macrophages
inﬁltrated into the livers, and higher hepatic CCL2, NLRP3 inﬂammasome
and IL-1β mRNA expression. Furthermore, the levels of proﬁbrogenic IL33 and IL-13 in liver homogenates and IL-33, ST2 and IL-13 mRNA
expression in liver were markedly higher in WT than in LGALS3-/- mice on
HFD, while hepatic expression of TGF-β were similar. Moreover, incontrast to WT macrophages, in vitro stimulated LGALS3-/- peritoneal
macrophages with recombinant mouse IL-33 failed to upregulate ST2
expression and IL-13 production.
Conclusion: Gal-3 attenuates steatosis, but promotes liver injury,
inﬂammation and ﬁbrosis, thus participates in the progression of NASH
induced by obesogenic diet in mice. Further, we show for the ﬁrst time thatGal-3 plays an important regulatory role in the newly described proﬁbrotic
IL-33/ST2/IL-13 pathway.

54

Molecular genetic analysis of metastasis associated colon cancer 1 (macc1) and c-met
expression in chronic myeloid leukemia
Stevic I1, Kapur-PojskićL2, Lojo-Kadric N2, Ler D1,3
1

Genetics and Bioengineering, International University of Sarajevo (IUS), Sarajevo, BiH
Laboratory for Human Genetics, Institute for GeneticEngineering and Biotechnology (INGEB),Sarajevo,BiH
Laboratory for MolecularDiagnostics, 'MojaKlinika', Sarajevo, BiH

3Chronic myeloid leukemia (CML) is a hematopoietic neoplasm
characterized by the Philadelphia chromosome and the related BCR-ABL1
oncoprotein. Several proangiogenic molecules have been implicated indisease acceleration, including the hepatocyte growth factor (HGF) and cMet. Both c-Met and HGF have been shown to be deregulated and socorrelated with poor prognosis in a number of major human cancers. The
gene encoding the HGF receptor, c-Met, is a transcriptional target of
MACC1.The newly identiﬁed proto-oncogene MACC1 (MetastasisAssociated Colon Cancer 1) gene stimulates proliferation, motility andinvasion in colon cancer cells through up-regulation of c-Met. Once
activated, c-Met can result in activation of several downstream signalingcascades (MAPK and PI3K/Akt pathways). The MACC1 was ﬁrstly
identiﬁed to be over-expressed in primary and metastatic tumor tissue ofcolon cancer compared to healthy tissue. This study was directed to reveal
the potential role of MACC1 in CML. Aim of this study was to investigate
the level of mRNA expression of MACC1 and c -Metin patient with CML
who was on treatment with imatinibmesilate (Gleevec). For this purpose,
Real-Time PCR methodology was optimized and validated. Results areshowing positive correlation between BCR-ABL and MACC1 expression,and between MACC1 and c-Met expression on transcript level.
Additionally, results showed higher sensitivity of MACC1 in comparison to
the BCR-ABL indicating so the potential role of MACC1 as novel
biomarker in CML. To conclude, this pilot project could have an important
impact on further studies and new perspectives about diagnostics as well as
therapy for CML patients that could improve their life quality and survival.
Keywords: MACC1, c-Met, biomarkers, CML

PRESENTATION

2

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55

Cytomegalovirus infection enhances development of diabetes mellitus type 2 in obesity
Marko Šestan1, Felix M Wensveen1, Sonja Valentić1, Bojan Polić1
1

PRESENTATION

Department of Histology and Embryology, University of Rijeka School of Medicine, Rijeka, Croatia

20

Diabetes mellitus type 2 (DM2), main clinical complication of obesity, ischaracterized by chronic high blood glucose levels and insulin resistance,
but also with chronic low-grade systemic inﬂammation that originates in thevisceral adipose tissue (VAT) of obese individuals. As body weightincreases, pro-inﬂammatory cells (macrophages, neutrophils, activated T
cells, etc.) accumulate in VAT. These cells secrete pro-inﬂammatory
cytokines (TNFα IL-1β), which play a key role in the development ofobesity-associated insulin resistance. Very little is known whether and how
viral infections contribute to chronic VAT-inﬂammation and induction of
DM2. Cytomegaloviruses (CMVs) are species-speciﬁc beta-herpesviruses
which can infect a variety of cell types like ﬁbroblasts,monocytes/macrophages, endothelial cells, dendritic cells, etc. Majority ofhuman population (40 – 90%) is infected with CMV already duringadolescence, and, after usually clinically inapparent acute infection in
immunocompetent host life long latency is established. Infection of micewith mouse CMV (MCMV) represents a well accepted model for studying
similar biological features of human CMV infection.
In this work we investigated how MCMV infection inﬂuences thedevelopment of a chronic systemic inﬂammation and insulin resistanceunder condition of high fat diet (HFD) – induced obesity. We observed that
VAT can be efﬁciently infected with MCMV and the infection shows
relatively fast clearance kinetics similar to the one observed in the liver.
Infection of VAT is associated with a rapid accumulation of various immune
cells. NK cells are activated on day 3 and their numbers rapidly increase
until day 6 after infection. The augmentation of NK cell population isfollowed by a dramatic increase of CD4 and CD8 T cells. Very early, fourweeks after induction of HFD and MCMV infection mice already develop
insulin resistance and glucose intolerance. Moreover, upon establishment of
MCMV latency (16 weeks p. i.), only in the group of MCMV and HFD
treated mice we observed signs of glomerulopathy in juxtaglomerularcortex of the kidneys: enlarged glomeruli, mesangiosis, increased thicknessof the ﬁltration membrane and impaired podocytes.
Altogether, our results show that MCMV infection can cause signiﬁcantenhancement of the VAT associated inﬂammatory process, insulin
resistance and associated complications in obesity. However, theimmunological mechanisms how MCMV infection inﬂuences theseprocesses in VAT still need to be determined.

56

Impact of aging on calcium inﬂux and potassium channel characteristics ofT lymphocytes
Gergely Toldi, Szonja Kollár, László Berta, Zsóﬁa Eszter Vásárhelyi, Attila Balog, Barna Vásárhelyi, János
Rigó Jr

Adaptive immunity and T cell function are affected by aging. Calcium
inﬂux patterns, regulated by Kv1.3 and IKCa1 potassium channels,
inﬂuence T cell activation. We aimed to compare calcium inﬂux kinetics in
CD8, Th1 and Th2 cells in human peripheral blood samples obtained fromﬁve different age groups (cord blood, 10-15 ys, 25-40 ys, 45-55 ys, 60-75
ys).
We measured calcium inﬂux using ﬂow cytometry in samples treated withor without speciﬁc inhibitors of Kv1.3 and IKCa1 channels (MGTX and
TRAM, respectively).
Calcium inﬂux was higher in Th1 cells of adults, however, its extent
decreased again with aging. Importantly, these changes were not detected inTh2 cells, where the pattern of calcium inﬂux kinetics is similar throughout
all investigated age groups. MGTX had a more pronounced inhibitory effecton calcium inﬂux in Th2 cells, while in Th1 cells the same was true for
TRAM in the 25-40 ys and 45-55 ys groups. Calcium inﬂux of CD8 cells
were inhibited to a similar extent by both applied inhibitors in these groups,
and had no effect in the elderly.
Altered lymphocyte potassium channel inhibitory patterns, regulators ofcalcium inﬂux kinetics, might contribute to the development of age-relatedchanges of T cell function.

PRESENTATIONFirst Dept of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary

21

57

Aging affects rat inﬂammatory peritoneal exudate composition and macrophage
inﬂammatory mediator production in a strain-dependent manner
Stanislava Stanojević1, Ivana Ćuruvija1, Veljko Blagojević1, Ivana Vujnović1, Raisa Petrović1, Mirjana
Dimitrijević1, Gordana Leposavić2
1

Immunology Research Center “Branislav Janković”, Institute of Virology, Vaccines and Sera “Torlak”, Belgrade, Serbia
Department of Physiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

PRESENTATION

2

22

The present study was designed to examine inﬂuence of aging on
macrophage proinﬂammatory/anti-inﬂammatory capacity in rat model ofthioglycollate-induced peritonitis. Peritoneal macrophages were isolated
from young (3-months-old) and aged (18-months-old) Dark Agouti (DA)and Albino Oxford (AO) rats seven days post-injection of thioglycollate
medium. Freshly isolated peritoneal exudate cells were examined for the
expression of CD163, CCR7, CD14 and TLR4, whereas cytokine
production (TNF-α, IL-6 and IL-10) and arginine metabolism end-products(NO and urea) were assayed in vitro under basal conditions and following
stimulation with LPS. In DA rat inﬂammatory peritoneal exudate, aging
diminished the frequency of cells with a “resolving macrophage”
CD14+CD163+ phenotype. However, in AO rats, which exhibited stablefrequency of CD14+CD163+ cells in inﬂammatory peritoneal exudate withaging, the proportion of CCR7-bearing peritoneal cells, presumably
immigrating inﬂammatory monocytes, was diminished in aged animals.
Under basal culture conditions, macrophages from aged rats of both strains
released less amount of TNF-α, IL-6 and IL-10, but produced more urea
than cells from young strain-matched rats. However, these changes weremore pronounced in peritoneal macrophages from AO rats. Additionally,
age-related decrease in the frequency of TLR4-expressing cells wasobserved among fresh peritoneal exudate cells from AO rats. Upon LPS
stimulation, the production of prototypic inﬂammatory cytokines (TNF-α
and IL-6) was diminished in macrophages from aged AO rats, whereas
aging had the opposite effect on their production in DA rat macrophages.
Moreover, aging increased NO production in LPS-stimulated macrophages
from DA rats, whereas urea production was enhanced in macrophages fromboth strains, but this increase was strikingly more pronounced inmacrophages from AO rats. Collectively, results suggest that aging affects
inﬂammatory peritoneal exudate cellular composition and macrophage
proinﬂamatory/immunomodulatory capacity in a strain- speciﬁc manner.
(Supported by the grant 175050 from Ministry of Education, Science andTechnology of the Republic Serbia).

58

Aging with MCMV maintains TCR repertoire diversity in late life
Megan J. Smithey1, Vanessa Venturi2, Miles P. Davenport2 and Janko Nikolich-Zugich1
1

Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, AZ 85724
Computational Biology Group and ‡Complex Systems in Biology Group, Centre for Vascular Research, University of New South
Wales, Kensington, New South Wales 2052, Australia

Cytomegalovirus (CMV) is a ubiquitous persistent herpesvirus (worldwide
infection rate 45-100%). CMV has coevolved with mammalian hosts formillennia. It has been postulated that lifelong persistent immune
interactions between host and CMV may contribute to age-related immune
senescence. We previously reported that mice aged with persistent murine
CMV (MCMV) show impaired CD8 T cell function upon challenge withseveral microorganisms, including Listeria monocytogenes expressing themodel OVA antigen (Lm-OVA) in late life. To further explore how lifelong
persistent MCMV infection may inﬂuence aged immune responses, wechallenged control adult and old mice, as well as lifelong MCMV-infected
old mice (hereafter referred to as MCMV+) with Lm-OVA, and assessed theOVA-speciﬁc TCRb repertoire by single-cell sorting and TCRb CDR3
sequence analysis. We found that compared to old MCMV-negative
animals, OVA-speciﬁc CD8 effector T cells generated in old MCMV+ micecontained diverse clonotypes exhibiting (i) non-canonical TCR genes; (ii) aloss of a conserved amino acid motif; (iii) reduced avidity for cognate
antigen; and (iv) broader recognition of altered peptide ligands. Thus,
lifelong MCMV infection appeared to maintain/broaden diversity within
the CD8 T cell response to a new infection in late life, by recruitment ofclonotypes with broad cross-reactive antigen recognition capacity. These
results have profound implications for our understanding of naïve T cellmaintenance over a lifespan, and suggest that our coevolution with CMV
may include surprising, potentially positive impacts on adaptive immunity
in late life.

PRESENTATION

2

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59

Age-related effects on invariant NKT cells
Mari Pattison, Neil Mabbott, Barry McColl

PRESENTATIONThe Roslin Institute and Royal School of Veterinary Studies, University in Edinbourgh

24

The immune system protects us against bacteria, viruses and otherpathogens, and if compromised can result in susceptibility to infection.
Immunosenescence refers to age-related immune impairments in immune
function that may contribute to increased prevalence and severity of
infectious disease in the elderly. Despite increasing understanding ofmolecular and cellular age-related immune alterations, knowledge isincomplete, particularly on dynamic and functional cellular changes.
Initial studies, using immunohistochemistry and ﬂow cytometry,havedeﬁned key structural and cellular changes occurring at 3, 12 and 22 monthsof age and identiﬁed invariant NKT (iNKT) cells as a cell population ofinterest. iNKT cells are a subpopulation of T cell with an invariant TCR
which recognises self and foreign lipids,for instance the glycolipid alphagalactosylceramide (α-GalCer), presented on the CD1d molecule. iNKT
cells play a role in the defence against a range of pathogens, including thosethat lack glycolipid, for example Escherichia coliand
Schistosomamansoni[1], showing that iNKT cells can be activated in theabsence of strong TCR signals. They have also proved to be protective or
harmful in a range of autoimmune diseases and cancer.
In our aged model we observed a signiﬁcant increase in the population sizeof iNKT cells, however no alterations in their distributions in spleen or
inguinal lymph nodes. Using the surface markers CD44 and NK1.1, we canfollow iNKTcell development in the thymus through stage 1 (CD44lo NK1.1hi
hi+), stage 2 (CD44 NK1.1 ) and stage 3 (CD44 NK1.1 ). Our data show a
reduction in mature stage 3 iNKT cells corresponding with an increase instage 2 in the thymus, spleen and liver. Current experiments will determine
if age affects iNKT cell expression of activation markers and release ofcytokine in response to in vitro stimulation
An improved understanding of how ageing affects functional activity in the
immune system may reveal novel targets for intervention to alleviate agerelated immune dysfunction and possibly lighten the medical burden ofageing.
1. Tupin, E., et al The unique role of natural killer T cells in the response to
microorganisms. Nature Reviews Microbiology 5, 405-417.

60

Impaired CD4 and antibody responses in old mice increase Chikungunya virus disease
severity and viral persistence
J. Uhrlaub, V. Pulko, J. Nikolich-Zugich: Department of Immunobiology and the Arizona Center on Aging,
University of Arizona College of Medicine, Tucson, AZ, 85724 USA

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus endemic to
Africa and Asia, which causes sudden onset of fever, rash, and debilitating
poly-arthralgia in peripheral joints, which can persist for years, particularly
in older individuals. The virus has been spreading around the world with
>800,000 cases of autochthonous CHIKV infection in the Americas since its
arrival in the western hemisphere in 2013 including the ﬁrst conﬁrmed
transmissions of CHIKV in the U.S. In response to the increased geographic
distribution of CHIKV and the likelihood that elderly immune-naïve
populations may experience severe and life-threatening disease, we havedeveloped a mouse model of age-related vulnerability to CHIKV infection.
We demonstrate reduced ability of old mice to mount effective immune
responses to CHIKV and control viremia, leading to increased disease
severity and viral persistence in the joints. Ineffective immune responses
were due, in part, to uncoordinated cytokine production. Speciﬁcally,CXCL9 and TGFβ, were identiﬁed as key contributors to impaired CD4 andantibody responses against viral epitopes in old mice. These results provide
a valuable tool for further mechanistic dissection of age-related
vulnerability to CHIKV and point to possible targets in CHIKV disease
treatment.

PRESENTATION

V. DeFilippis: Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, 97005 USA

25

61

Dysregulated function of marginal zone B cells in aged mice
Turner, V.M.1 and Mabbott, N.1
1

PRESENTATION

Neurobiology Division, The Roslin Institute and the R(D)SVS, University of Edinburgh, UK

As the average age of our population continues to climb the burden on thehealth system increases. This is due in part to the decreased ability of theaged immune system to deal with infection. Aging results in increased
immunosenescence and along with this an increased susceptibility in aged
individuals towards opportunistic pathogens and inﬂammatory conditions.
In aged mice a disruption of the localization of immune cells within the
spleen has been shown. Our laboratory is speciﬁcally interested in the
disruption of the localisation of B cells and macrophages within themarginal zone (MZ) of the spleen as this has been demonstrated to besigniﬁcantly altered in aged mice. First,an investigation intowhen the
disruption of the splenic structure begins was performed. Spleens of micefrom two months up to thirty months were analyzed via FACS and IHC. This
analysis demonstrated that the number of B cells within the spleen increased
with age. However, by 30 months there was a signiﬁcant depletion in thenumber of B cells, predominately affecting the follicular B cell population
as the representation of MZ B cells remained relatively intact.IHC analysis
demonstrated an increasingly signiﬁcant disturbance to the splenic structurewith age. This disruption primarily resulted in an increased thickness of themarginal metallophilic macrophage and MZ B cell populations. Further
analysis to assess the functioning of the B cell populations were performed
via survival and chemotaxis assays. These assays unveiled an increased
survival of aged MZ B cells. There also appeared to be analtered sensitivityof the aged MZ B cells towards S1P, a important chemoattractant, theimplications of which will be discussed. These results demonstrate alteredfunctioning of the MZ B cells with age, potentially altering the ability ofcells to respond during an immune response.

26

62

Nitrosative events in asthma as a selective pulmonary disorder marker
O.O. Parilova, B.V. Pylypchak, T.T. Volodina

Allergic disorder pathogenesis is modulated by NO at the level of immune
system. In the current study, we provided evidence of iNOS overexpression
and NO overproduction under the chronic ovalbumin-induced asthma andconsidered the elaborated non-invasive approach to assess airway NO
generation in asthmatic subjects.
Guinea pigs were randomly assigned to: intact group (normal); ovalbumin(OVA) sensitized animals (OVA/0,9%NaCl); OVA challenged group (0,9%NaCl/OVA); OVA-induced asthma group (OVA/OVA). Since IL-4 evokes
switch for IgE, IgG1 and Th2 cell differentiation, sensitization to allergen(OVA/0,9%NaCl) was indicated with 4,5–fold growth of plasma IL-4
content compared with normal (p(U)<0,05). IL-13 values attained 1,5-foldincrease in OVA/OVA and 0,9%NaCl/OVA groups versus normal(p(U)<0,05). IL-13 lung level corellated with lung tissue histology, thatdeﬁned mucus hypersecretion by goblet cells and mucous glands, basement
membrane thickening in 0,9%NaCl/OVA, and acute obstructive
emphysema, sclerosis of interalveolar walls, ﬁbrosis in OVA/OVA. The
dynamics of NO metabolism under the chronic allergic asthma revealed,that iNOS lung content using antibodies increased markedly in 2,4- and2,65-fold among OVA/OVA and 0,9%NaCl/OVA groups respectively versus
normal (p(U)<0,05). OVA multiple challenge procedures resulted in 2,4fold and 2,3-fold rises of intracellular NO in the immune cells inﬁltrating
airway of OVA/OVA and 0,9% NaCl/OVA groups versus OVA/0,9%NaCl(p(U)<0,05).
Considering an important role of NO in the adaptive immune responses
associated with allergic diseases, the experimental model of the device
consisting of electrochemical gas sensor type NO2-B4 (Alphasense) andTriton 6000U data acquisition system with measurement accuracy ±10E-6
V was developed. Because NO has a short half-life in an unbound state due
to one unpaired electron, we assert that measuring NO2 reﬂects theendogenic NO concentration produced in the lungs. Baseline sensor output
signal and typical charts as a function of NO2 concentration wererecordered. The obtained data give an incentive for further studies.

63

PRESENTATION

Department of Metabolism Regulation, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine (NASU),
Kyiv, Kyiv region, Ukraine27

IFN-γ and IL-17A drive distinct airway changes in a murine model ofsteroid-unresponsive severe asthma
Mahesh Raundhal1,2, Christina Zinger-Morse1, Timothy BOriss1, Anupriya Khare1, Jay Kolls2, Sally Wenzel1,
Anuradha Ray1,2, Prabir Ray1,2

PRESENTATION

Departments of Immunology1 & Medicine2, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

28

Background: Asthma is an inﬂammatory disease of the airways
characterized by airway obstruction due to inappropriate immune
responses against some environmental moieties like house dust mite(HDM) and pollens. Depending on the frequency and intensity of
exacerbations experienced, asthma can be categorized as mild-moderateasthma (MMA) that is corticosteroid (CS)-responsive and severe asthma(SA) that is poorly managed by CS. The presence of neutrophils in asthma
has been correlated with refractoriness to CS, fatal exacerbations andseverity. To date, our understanding of the pathological mechanisms
central to neutrophil-prominent SA is minimal primarily because of thelack of a suitable animal model, limiting the development and testing of
new therapeutics.
Aim: The aim of this study was to elucidate the immune mechanisms
underlying the difference in steroid-responsiveness in MMA and SA.
Method: Human asthma samples and a novel mouse model of SA wereused to study cytokine responses, airway inﬂammation andhyperreactivity in MMA and SA.
Results: Unlike patients with milder disease, SA patients consistently
have a mixedTh1/Th2/Th17 immune phenotype in their airways despitehigh dose CS treatment. The combination of anallergen, HDM, withagents that mimic infection induces a SA phenotype in mice. As in humansevere asthmatics, the immune response in this mouse model elicits mixed
granulocytic airway inﬂammation and airway hyperresponsiveness thatare poorly responsive to CS. IFN-γ-deﬁcient and IL-17A-deﬁcient micesubjected to the SA model highlight the differential role played by eachcytokine in modulating hallmark features of asthma such as airway
inﬂammation, airway hyperreactivity and steroid responsiveness.
Proteases and lipid mediators were identiﬁed as potential biomarkers ofsevere asthma using biological network analysis of RNA-Seq expression
data.
Conclusion: The immune responses that contribute to the pathogenesis ofsevere and mild-moderate asthma are distinctly different.

64Ragweed Allergic Subjects Have Decreased Serum Levels of the Chemokines CCL2,
CCL3, CCL4 and CCL5 out of the Pollen Season Compared to the Healthy Individuals
Zhivka Kostova, Tsvetelina Batsalova, Dzhemal Moten, Ivanka Teneva, Balik Dzhambazov

CC-chemokines are important mediators of the allergic responses andregulate the cell trafﬁcking. The aim of this study was to examine the serum
levels of CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1 and CCL5/RANTES,
and to determine whether there are differences between ragweed allergic
subjects and healthy individuals out of the pollen season. Peripheral blood
samples were collected from 14 subjects allergic to ragweed pollen and 12
healthy controls. Serum concentrations of chemokines/cytokines were
measured by enzyme-linked immunosorbent assay. We observed
signiﬁcantly decreased concentrations of CCL2/MCP-1, CCL3/MIP-1,
CCL4/MIP-1 and CCL5/RANTES in the sera of ragweed-allergic patientscompared to the healthy individuals (32.2 vs 106.4 pg/ml, 89.5 vs 135.7
pg/ml, 63.4 vs 119.2 pg/ml and 11.2 vs 18.1 ng/ml, respectively, p<0.01). In
contrast to the CC-chemokines, the serum levels of IL-8/CXCL8 showed asigniﬁcant increase (p<0.05) in the allergic group compared to the nonallergic subjects. IL-4 levels were similar in both groups. In the sera ofallergic patients, we have also detected signiﬁcantly elevated levels of
ragweed-speciﬁc IgE and IgG. However, decreased serum concentrationsof the four CC-chemokines and elevated levels of IL-8 can be used as
biomarkers for more accurate evaluation of the allergic status of patientswith pollen allergy out of the season, to study the mechanisms for
activation/inhibition of the subclinical allergic responses and fordevelopment of therapeutic strategies.

PRESENTATION

Developmental biology, Plovdiv University, Plovdiv, Bulgaria

29

65

Adjuvanticity Of Lactobacilli In A Mouse Fes p1 Allergy Model
R Minić 1, M Gavrović-Jankulović2, G Mathiesen 3, I Živkovic 1, L Dimitrijević 1
1

Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia,
Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Serbia
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway

2

PRESENTATION

330

Allergies of the respiratory tract represent a major health hazard to
predisposed individuals. Species of the Lactobacillus genus have been
examined extensively and oral/intranasal delivery of the bacteria wasshown to be beneﬁcial in different allergy models. We have chosen group I
grass pollen allergen Fes p1 as an object of our study. Group I grass pollen
allergens from different grass species show high sequence similarity, andhigh cross reactivity and is therefore a good target in an allergy model.
The aim of this study was to make a Fes p1 allergy model in Balb/c mice andto examine whether oral application of different species of Lactobacillus
produces differences in antibody levels i.e. to test their adjuvanticity. Twochosen species were L. plantarum WCFS1, an extensively analyzed strain
and L. rhamnosus LB64, a newly analyzed strain in this context.
Fes p1 allergen was isolated from pollen of Festuca pratensis with ion
exchange chromatography, and 7 μg premixed with alum was injected i.p.
into Balb/c mice, on days 0, and 7. Lactobacilli were given by oral gavage,
and at the end of the experimental period antibody classes and subclasses
were analyzed with ELISA. Peripheral blood smears were used to counteosinophil content.
Untreated animals had high Fes p1 speciﬁc IgE, and IgG1 levels, andeosinophilia. Oral application of either of the strains reduced peripheral
blood eosinophilia. The administration of L. plantarum led to an increase in
Fes p1 speciﬁc IgG2a, and L. rhamnosus increased both IgG2a, IgA, andIgE levels in mice.
The application of either of the tested Lactobacillus species had a beneﬁcial
effect in a Fes p1 mouse allergy model, with L. rhamnosus LB64 being
superior because of its capacity to induce high serum IgA levels, which
positively correlates to protective mucosal IgA.

66

Cell death of ED1+ cells in the central nervous system of Dark Agouti rats at the peakof experimental autoimmune encephalitis
Neda Djedović1, Irena Lavrnja2, Katrin Wendrich3, Miljana Momčilović1, François Paquet-Durand3, Dragana
Trifunović3, Djordje Miljković1
1

Department of Immunology, Institute for Biological Research "Siniša Stanković", University of Belgrade, Serbia
Department of Neurobiology, Institute for Biological Research "Siniša Stanković", University of Belgrade, Serbia
Institute for Ophthalmic Research, University Eye Clinic,University of Tubingen

3Inﬁltration of macrophages into the central nervous system (CNS), as wellas activation of microglia is a hallmark of multiple sclerosis and its animalmodel - experimental autoimmune encephalomyelitis (EAE). Cell death in
EAE has been demonstrated as an essential mechanism in the local
regulation of the inﬂammatory reaction, but also as one of the major factors
contributing to the destruction of the CNS tissue. Here, cell death of ED1+
cells (macrophages/microglia) in the spinal cord of EAE rats wasinvestigated. Cell death in general was assessed using the TUNEL assay,
while cleaved caspase-3 immunostaining was employed as the marker of“classical” apoptosis. Dark Agouti (DA) rats were immunized with spinal
cord homogenate emulsiﬁed in complete Freund's adjuvant. Inﬁltrates of
immune cells were detected both in white matter (WM) and grey matter(GM) of spinal cords in DA rats at the peak of EAE. ED1+, TUNEL+ andcaspase-3+ cells were detected within, but also outside the inﬁltrates. While
there were no differences in the proportion of TUNEL+ ED1+ cells between++
inﬁltrates and non-inﬁltrated areas in WM, there were more ED1 TUNEL
cells in GM in inﬁltrates than in non–inﬁltrated areas. A similar distribution
was observed for ED1+caspase-3+ cells. The observed discrepancy in
distribution of dead ED1+ cells in inﬁltrates and non-inﬁltrated areas in GM
and WM of spinal cord indicated that differential spatial regulation ofmacrophage/microglia cell death occurred in DA rats. These ﬁndings
contribute to the understanding of pathogenesis of EAE in DA rats. It also
opens new perspectives for a research aiming at more efﬁcient treatment ofmultiple sclerosis.

PRESENTATION

2

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67

Expression of miR146-a, an inﬂammation-associated microRNA, in Mesial Temporal
Lobe Epilepsy
B. Leal1, C. Carvalho1, J. Chaves2, A. Bettencourt1, J. Freitas2, J. Lopes2, J. Ramalheira2, A. Martins da Silva2, P.
P Costa1,3, B. Martins da Silva1
1

Univ Porto, Lab Imunogenetics & Autoimmu and NeuroScien, Unidade Multidisciplinar Invest Biomed, Inst Ciencias Biomed
Abel Salazar
Hospital Santo António Centro Hospitalar do Porto
3
Instituto Nacional Saúde Dr. Ricardo Jorge – Delegacção do Porto

PRESENTATION

2

32

Background: Neuroinﬂammation appears as an important epileptogenic
mechanism. MicroRNAs (miRNA) are small non-coding RNA moleculesthat function as post-transcriptional regulators of gene expression.
MicroRNas control different biological process including immune systemhomeostasis and function. Several evidences, both in patients and animalstudies, have demonstrated an abnormal brain expression of miR-146a in
Mesial Temporal Lobe Epilepsy. Knowing that miR expression is very
stable in biological ﬂuids such as plasma or serum our aim was tocharacterize miR146a expression in serum of MTLE patients.
Methods: Expression levels of miR146a and U6B small nuclear RNA gene(reference gene) were quantiﬁed by Real-Time PCR in serum of 14 MTLE
patients all with Hippocampal Sclerosis (6F, 8M, mean age= 44.1±11.7
years, age of onset= 13.5±10.6 years, 7 with Febrile Seizures antecedents).
A group of 10 healthy individuals was used as control. Relative expression
values were calculated using the 2-ΔΔCt method.
Results: We observed that expression of miR146a was 2 fold higher in
MTLE-HS patients than in controls.
Conclusion: The results obtained in serum are in accordance with theresults obtained from brain tissue of epileptic patients. This may conﬁrm
that miR-146a is a suitable biomarker of epileptogenesis. Additionally, it isthought that miR-146a has a role in ﬁne-tuning the response to cytokines
during epileptogenesis. Nevertheless its importance in epilepsy
development it is yet not fully understood. The comprehension of this role
may be relevant for the development of new therapeutic strategies.
Supported by a BICE Tecnifar Grant 2014

68

CMV infection in neonatal and adult mice induces susceptibility to EAE in resistant
BALB/c mice
Jelena Milovanovic1, Marija Milovanovic1, Aleksandar Arsenijevic1, Bojana Stojanovic1, Branka Popovic2,
Nebojsa Arsenijevic1, Stipan Jonjic2 Miodrag L. Lukic1
1Center for Molecular Medicine and Stem Cell Research, Faculty of Medicine University of Kragujevac, Serbia
Center for Proteomics, Department for Histology and Embryology, School of Medicine, University of Rijeka

In contrast to C57BL/6 mice BALB/c mice are relatively resistant to the
induction of autoimmune encephalomyelitis evaluated by inﬂammation anddemyelinination in CNS after challenge with MOG35-55 peptide as
encephalitogen. The aim of this study was to analyze the role of CMV
infection in EAE pathogenesis. Female BALB/c mice were infected withmurine cytomegalovirus before immunization with MOG35-55 and disease
was evaluated by clinical and histological examinations.
Immunophenotyping of CNS inﬁltrates was done by ﬂow cytometry. Here
we provide ﬁrst evidence that both neonatal and adult infection with murine
CMV (MCMV) abrogates this resistance. Infected BALB/c mice developed
clinical and histological signs indistinguishable from those seen insusceptible C57BL/6 mice. MCMV infected and MOG35-55 immunized
mice had massive inﬁltration in brain parenchyma, cerebellum and spinal
cord. Further, analysis of inﬁltrating cells in infected mice revealed almost
equal number of CD4+ and CD8+ inﬁltrating cells in CNS, similar withﬁndings in multiple sclerosis. This study clearly indicates the inﬂuence of
CMV in EAE pathogenesis and also reveals new experimental model moresimilar to MS. We also demonstrate that viral infection enhances the inﬂux
of MOG35-55 speciﬁc cells in CNS and favors pro-inﬂammatory type of
APC in draining lymph nodes.
Key words: EAE, MCMV, BALB/c mice, CD8 lymphocyte
This work was supported by the Ministry of Science, Republic of Serbia,
grants ON175069, ON175071 and ON175103Faculty of Medical Sciences,
University of Kragujevac, Serbia grants MP 01/14 and 02/14.

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69

New Strategies for Development of Antigen-speciﬁc T Regulatory Cells
Đorđe Miljković, Tamara Saksida, Maja Kojdić, Ivana Nikolić, Milica Vujičić, Stanislava Stošić-Grujičić andIvana Stojanović

PRESENTATION

Department of Immunology, Institute for Biological Research “Siniša Stanković“, University of Belgrade, Belgrade, Serbia

34

T regulatory cells (Treg)-based therapy of autoimmune diseases is rapidly
developing. However, the low numbers of endogenous Tregs that can beisolated from an individual or laboratory animal have limited their study andpotential therapeutic use. Therefore, various approaches are tested for invitro Treg expansion. Antigen-speciﬁc Treg compared to polyclonal exert
stronger suppressive effects in animal models of autoimmune diseases.
During autoimmune process, the number of autoantigen-speciﬁc effector T
cells is by far larger compared to autoantigen speciﬁc Tregs. Having in mind
the plasticity of T cells, we hypothesized that it would be feasible to convert
antigen-speciﬁc effector T cells isolated from animals with autoimmune+
disease to antigen-speciﬁc FoxP3 Tregs. To this end several manipulations,
including inhibition of Th17 cell differentiation, epigenetic modiﬁcations
or redox modulations have been performed. To obtain autoantigen-speciﬁc
T cells, NOD mice were immunized with Myelin Oligodendrocyte
Glycoprotein (MOG) peptide emulsiﬁed in Complete Freund Adjuvant.
Cells of draining lymph nodes and spleens were treated with MOG in vitro
to reactivate MOG-speciﬁc effector T cells. After 96h of culture CD4+ cells
were separated using magnetic beads (1,4% FoxP3+) and stimulated with
pro-Treg cocktail (anti-CD3+anti-CD28 antibody+TGF-b+IL-2). After
four days, the percentage of FoxP3+ cells reached 9,4% (as determined by
ﬂow cytometry). Simultaneous inhibition of Th17 cells with STAT3
inhibitor increased FoxP3+ population to 13,3%. On the other hand,promotion of histone acetylation by all trans retinoic acid and mytramicine(inhibitors of histone deacetylases) did not further increase the percentage
of FoxP3+. Although simultaneous treatment with hydrogen peroxide(H2O2) had no effect on FoxP3+ cells, the administration of H2O2 72h after
stimulation with pro-Treg cocktail promoted generation of Treg by 5-fold.
These results implicate that Treg population can be enriched from the poolof effector CD4+ T by various in vitro approaches.
Supported by the Ministry of Educations, Science and Technologicaldevelopment, Republic of Serbia (grants no. 173013 and 173035).

70

Anti-inﬂammatory effects of benfotiamine in LPS stimulated microglia: role of
NF-κB signaling
I. Božić1, D. Savić1, D. Laketa2, N. Nedeljković2, S. Peković1, I. Lavrnja1
Department of Neurobiology; Institute for Biological Research “Sinisa Stankovic”, University of Belgrade; Belgrade; Serbia
Institute of Physiology and Biochemistry; Faculty of Biology; University of Belgrade; Belgrade; Serbia

Microglial cells are immune cells of the central nervous system (CNS) thatplay a major role in its surveillance. Changes in CNS homeostasis, invading
pathogens or neuron impairment, lead to activation of microglial cells thatquickly proliferate, acquire amoeboid morphology and produce toxic
mediators such as nitric oxide (NO) and pro-inﬂammatory cytokines. Thesechanges are regulated by transcription factors, most importantly NF-κB.
Although microglial activation is important for maintaining tissue
homeostasis, excessive activation leads to chronic inﬂammation that candamage healthy neurons. Substances that suppress microglial activation arepotential therapeutics for neurodegenerative diseases. Benfotiamine (Sbenzoylthiamine-O-monophosphate) is a synthetic derivative of vitamin B1
that has anti-inﬂammatory properties. In this study we investigated antiinﬂammatory properties of benfotiamine on activated microglia in vitro.
BV-2 microglia were pre-treated with benfotiamine, stimulated with LPS
and their viability and morphology were evaluated. LPS induced prominent
alterations in cell morphology, enlargement of cell bodies and spreading ofmultiple processes. Pre-treatment with benfotiamine before LPS
stimulation suppressed these morphological changes. Additionally,
benfotiamine diminished LPS induced NO production, without altering cell
viability. Furthermore, benfotiamine decreased LPS induced production of
pro-inﬂammatory cytokines TNF- α and IL-6, while increasing productionof anti-inﬂammatory cytokine IL-10. Analysis of NF-κB activation
revealed that benfotiamine's effects were mediated by this transcription
factor.
In conclusion, benfotiamine exerts anti-inﬂammatory properties in LPS
activated microglia in vitro and should be further investigated as a potential
therapeutic for neurodegenerative diseases.

71

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1
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35

Cellular and molecular correlates of sexual dimorphism in the clinical presentationof experimental autoimmune encephalomyelitis in rats
Mirjana Nacka-Aleksić1, Jasmina Djikić1, Ivan Pilipović2, Zorica Stojić-Vukanić3, Duško Kosec2, Biljana
Bufan3, Nevena Arsenović-Ranin3, Mirjana Dimitrijević2, Gordana Leposavić1
1

Department of Physiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia
Immunology Research Centre “Branislav Janković”, Institute of Virology, Vaccines and Sera “Torlak”, Belgrade, Serbia
Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

2

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36

Male Dark Agouti rats immunized for experimental autoimmune
encephalomyelitis (EAE) exhibited more severe disease compared with
their female counterparts. To elucidate the cellular and molecularmechanisms underlying this phenomenon, CD4+ T lymphocytes andantigen presenting cells from spinal cord and/or draining lymph node
(DLN) were examined for activation molecule expression and cytokine
proﬁle. At the peak of EAE, consistent with the greater maximal
neurological score in male rats, the number of re-activated CD4+ T-cells andproportion of highly activated microglial cells/macrophages (CD45high)among CD11b+ cells (producing more TNF-α, IL-1b, IL-6 in vitro) in theirspinal cord were greater. Furthermore, IL-17+/IFN-y+ T-cell ratio in male
rat spinal cord was shifted towards the former on the account of the highly
pathogenic IL-17+IFN-γ+ cells. This was in accordance with upregulated
IL-23/p19, IL-1β and IL-6, but downregulated TGF-β expression in male rat
spinal cord mononuclear cells (MNC). In the inductive phase of disease,greater frequency of re-activated CD4+ and IL-17+ T-cells was also
observed in male rat spinal cord. Consistently, greater frequency of CD45highcells among male rat spinal cord CD11b+ cells and increased expression ofcytokines favouring Th17 polarization in their spinal cord MNC wereregistered. On the contrary, in DLN from rats of the same sex lower
frequency of activated cells within CD4+ T lymphocytes and cells
expressing MHCII and CD40 within the CD11b+ population was found.
Additionally, in male compared with female rats, the frequency of IL-17+
and IFN-γ+ T-cells was reduced in DNL and DNL T-cells produced less IL17 and IFN-γ upon MBP stimulation in vitro. Collectively, the results
suggest that: i) DLN and spinal cord microenvironments differ in CD4+ Tcell activation and polarization capacity, and ii) the target organ
microenvironment has important role in shaping a sexually dimorphic
clinical outcome of EAE.

72

Longitudinal plasma lipid proﬁle in multiple sclerosis patients during three years oftreatment with interferon-beta
Irena Dujmovic Basuroski1, Emina Milosevic2, Milos Markovic2, Sarlota Mesaros1, Radmila Obrenovic3,
Jelena Drulovic1, Tatjana Pekmezovic4, Marija Mostarica Stojkovic2, Dusan Popadic2
1

Clinic of Neurology, Clinical Centre of Serbia, University of Belgrade School of Medicine, Belgrade, Serbia
Institute of Microbiology and Immunology, University of Belgrade School of Medicine, Belgrade, Serbia
Institute for Medical Biochemistry, Clinical Centre of Serbia, Belgrade, Serbia
4Institute of Epidemiology, University of Belgrade School of Medicine, Belgrade, Serbia
2Background: It has been recently suggested that interferon (IFN)-β, theﬁrst-line disease-modifying treatment in relapsing remitting (RR) multiple
sclerosis (MS), might inﬂuence plasma lipid proﬁle in treated patients.
However, data on longitudinal plasma lipid proﬁle in MS patients treatedwith IFNβ are scarce and with conﬂicting results.
The aim of this study was to analyze longitudinal plasma levels of totalcholesterol (TC), low-density lipoprotein cholesterol (LDL-C), highdensity lipoprotein cholesterol (HDL-C) and total triglycerides (TG) in
RRMS patients treated with IFNβ-1a or IFNβ-1b (both subcutaneously)over 3 years and correlate those levels with clinical treatment response.
Materials and methods: We determined plasma TC, LDL-C, HDL-C andTG levels prior to IFNβ-treatment (baseline) and at treatment months 6, 12,
24 and 36 by commercial methods. Neurological disability was scored by
the Expanded Disability Status Scale (EDSS). Treatment response criteria
were: no EDSS score increase ≥1 points sustained over ≥6 months and no
relapse during 3 years of follow-up. Forty-ﬁve patients (n=45) wereconsidered responders and 25 were found to be non-responders according tothese clinical criteria.
Results: TC, LDL-C increased signiﬁcantly at 6 months, but returned tobaseline values at treatment month 12 and remained similar afterwards. By
contrast, HDL-C decreased at treatment month 6, but continued to growafterwards. TG levels peaked at month 6 and month 12 of treatment. At any
study time point, analyzed plasma lipid proﬁle parameters did not differ
signiﬁcantly between responders and non-responders and did not correlatewith the EDSS score.
Conclusion: Our results further support a notion that IFNβ treatment in MS
patients might originate changes in plasma lipid proﬁle. Further research isneeded to investigate whether those changes might reﬂect changes indisease activity in patients with MS treated with IFNβ.
Supported by Serbian Ministry of Education, Science and Technological
Development (Grants No. 175038, 175031 and 175087).

73

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337

MicroRNA-155 Contributes to Re-activation of Encephalitogenic T Cells
Bojan Jevtić1, Gordana Timotijević2, Suzana Stanisavljević1, Miljana Momčilović1, Marija Mostarica
Stojković3 and Đorđe Miljković1
1

Institute for Biological Research “Siniša Stanković“, University of Belgrade, Serbia
Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
Institute for Immunology and Microbiology, School of Medicine, University of Belgrade, Serbia.

2

PRESENTATION

338

MicroRNAs (miR) are small non-coding RNAs that have a profound role inregulating immune response. It has been suggested that miR-155
signiﬁcantly contributes to the pathogenesis of multiple sclerosis and its
animal model experimental autoimmune encephalomyelitis (EAE). Here,the role of miR-155 in re-activation of encephalitogenic CD4+ T cells thatare considered as the major pathogenic population in multiple sclerosis and
EAE was investigated. Dark Agouti (DA) rats were immunized with myelin
basic protein (MBP) emulsiﬁed in complete Freund's adjuvant. CD4+ T cells
were puriﬁed from draining lymph node cells (DLNC) isolated on day 6
after the immunization (inductive phase of EAE) and from spinal cord
immune cells (SCIC) isolated at the peak of EAE. CD4+ T cells obtained
from SCIC (i.e. in vivo re-activated cells) had markedly higher expressionof miR-155 in comparison to those puriﬁed from DLNC. Further, in vitro reactivation of CD4+ T cells obtained from DLNC with MBP also led toincrease in miR-155 expression. In order to examine if the observed
elevation of miR-155 expression during re-activation of encephalitogenic
CD4+ T cells had a functional signiﬁcance, DLNC, SCIC and CD4+ T cells
puriﬁed from them were transfected with an inhibitor of miR-155 (antimiR-155). As a consequence, expression of important CD4+ T cell effector
cytokines IFN-γ and IL-17 was reduced. These results imply that miR-155
contributes to encephalitogenicity of CD4+ T cells in autoimmune
neuroinﬂammation.
This work was supported by the Ministry of Education, Science andTechnological Development, Republic of Serbia (grant no. 173035).

74

IL-33/ST2 axis mediates resistance to EAE by promoting regulatory B and tolerogenic
dendritic cells
Bojana Stojanovic1 Marija Milovanovic1, Aleksandar Arsenijevic1, Jelena Milovanovic1, Branka Popovic2,
Stipan Jonjic2, Nebojsa Arsenijevic1, Miodrag L. Lukic1
1Center for Molecular Medicine and Stem Cell Research, Faculty of Medicine University of Kragujevac, Serbia
Center for Proteomics, Department for Histology and Embryology, School of Medicine, University of Rijeka

BALB/c mice are resistant to EAE induction with MOG35-55. ST2 molecule isexpressed on immune cells and its signaling is accompanied by accentuated
ST2 response. We recently published (Milovanovic et al. PlosOne 2012)that deletion of ST2 molecule abrogates resistance to EAE, as evaluated byclinical and histological ﬁndings. Adoptive transfer of ST2 KOlymphocytes restimulated with MOG35-55 induced clinical signs of thedisease in ST2 KO and BALB/c mice. MOG35-55 speciﬁc CD4+ lymphocytesof ST KO mice had higher expression of transcriptional factor T-bet,
inﬂammatory cytokines IFNγ, IL-17, GM-CSF, TNFα and chemokine
receptors important for migration to CNS (CCR6, CXCR5 and CXCR3).
ST2 KO mice had higher incidence of activated antigen presenting cells,
myeloid/regulatory dendritic cells (DC) ratio and percentages of myeloid
cells containing inﬂammatory cytokines, IL-1, IL-12 and IL-6. ST KO DC+
induced higher in vitro proliferation of CD4 cells and stimulated in vitro
with TLR1/2 agonist produced more cytokines involved in Th1 and Th17
differentiation compared with DC isolated from BALB/c mice.
Interestingly we also observed the increase of regulatory CD11b+CD5+ B
lymphocytes, IL-10 producing B1 lymphocytes and decrease of IL-6
producing B cells in BALB/c mice after immunization with MOG35-55
peptide. The opposite results were found in ST2 KO mice.
Results indicate that ST2 signaling inﬂuences the activity of regulatory B1cells and differentiation of proinﬂammatory antigen presenting cells,
consecutively affecting the differentiation of encephalytogenic T cells in the
periphery.
Key words: EAE, ST2 molecule, dendritic cells, regulatory B cells,
BALB/c miceThis work was supported by the Ministry of Science, Republic of Serbia,
grants ON175069, ON175071 and ON175103Faculty of Medical Sciences,
University of Kragujevac, Serbia grants MP 01/14 and 02/14.

75

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T helper cells 1/17-related genes in interferon-β-treated relapsing-remitting multiple
sclerosis patients – correlation to clinical response
Emina Milosevic1, Irena Dujmovic Basuroski2, Milos Markovic1, Sarlota Mesaros2, Jelena Drulovic2, Marija
Mostarica Stojkovic1, Dusan Popadic1
1

Institute of Microbiology and Immunology School of Medicine, University of Belgrade Belgrade, Serbia
Institute of Neurology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia

PRESENTATION

240Interferon (IFN)-β, ﬁrst-line treatment in relapsing remitting multiple
sclerosis (RRMS), alters the expression of more than a hundred genes, but
we still lack the explanation why many patients do not respond to therapy. In
multiple sclerosis (MS), T helper cells (Th) 1 and Th17 are considered theorchestrators of the myelin destruction, thus we assessed expression of
Th1/17-related genes in IFN-β-treated RRMS patients over 36 months.
RRMS patients (n=45) on IFN-β1b therapy were considered responders (R,n=20), if without relapses or progression on EDSS during 3 year follow-up
and the rest as non-responders (NR, n=25). Relative gene expression wasdetermined in peripheral blood mononuclear cells by qPCR for marker ofIFN-β biological response (MX1, OAS2), Th1/17 polarizing cytokines (IL12/23p40, IL-12p35, IL-23p19), respective receptors (IL-12Rβ1, IL12Rβ2, IL-23R), transcription factors (T-bet, RoRγt) and effector cytokines(IFN-γ, IL-17A, IL-17F, GM-CSF) before start and at 6, 12, 24 and 36
months of IFN-β therapy.
All mRNA levels changed statistically signiﬁcantly during the therapy,
with, on average, similar values in R and NR. MX1 and OAS2 mRNA levels
were up-regulated at every time point, and statistically highly correlated, inboth groups. IL-12/23p40, IL-12p35, IL-23p19 and GM-CSF mRNA
expression decreased, while IL-23R, RoRγt and IFN-γ statisticallysigniﬁcantly increased at 6 months. All mRNA levels reverted to baseline at
24 months. At 36 months, IL-12/23p40, IL-23p19 and T-bet mRNA levels
decreased, IFN-γ and IL-12Rβ1 levels were similar to 24 months ones, andthe rest increased statistically signiﬁcantly.
MX1 and OAS2 showed R and NR did not differ in IFN-β biologicalresponse. Although expression levels of Th1/17 axes' genes did not
distinguish R and NR, we observed large inter-individual variations in
temporal patterns of particular genes. Furthermore, overall changes ofmRNA levels due to IFN-β treatment warrant further investigation.

76

Cytokine proﬁles in ﬁrst episode psychosis and schizophrenia in relapse
Milica Borovcanin1, Ivan Jovanovic2, Slavica Djukic Dejanovic1, Gordana Radosavljevic2, Nebojsa
Arsenijevic2, Miodrag L. Lukic2
2

Department of Psychiatry, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia
Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia

The ﬁeld of “psychoneuroimmunology” has gained increasing interest andresearch work during the last 20 years. Several epidemiological studies
demonstrate a co-occurrence of autoimmune diseases, chronic
inﬂammatory conditions, and mental disorders. Since in many cases theelevated prevalence of autoimmune/chronic inﬂammatory diseases existed
before the onset of psychosis, the hypothesis was put forward that the onsetof psychosis could be induced by an inﬂammatory process elicited by the
autoimmune reaction. The aim of our study was to analyze the serum
concentrations of type-1, type-2, type-17 and regulatory cytokines in drugnaive patients with ﬁrst episode psychosis and schizophrenia in relapse. We
have demonstrated that type-1 and type-17 responses are blunted and type-2
overweight in schizophrenia. Our results also implicate anti-inﬂammatory
response through TGF-β production. Analysis showed that TGF-β and IL23 can be valuable markers for psychosis. The presence of enhanced antiinﬂammatory/immunosuppressive activity in schizophrenia may be an
attempt to counteract or limit ongoing pro-inﬂammatory processes anddownregulating chronic inﬂammation. A chronic pro-inﬂammatory stage
with a robust type-2 response, including high IL-6 levels, may also
predominate in later stages. Our results suggest that immune pattern inschizophrenia can be similar with those in atopic disorders and this cytokine
disbalance can be corrected with antipsychotics.
1. Borovcanin M, Jovanovic I, Radosavljevic G, Djukic Dejanovic S,
Bankovic D, Arsenijevic N, Lukic ML. Elevated serum level of type-2
cytokine and low IL-17 in ﬁrst episode psychosis and schizophrenia in
relapse. J Psychiatr Res. 2012; 46(11): 1421-6.
2. Borovcanin M, Jovanovic I, Radosavljevic G, Djukic Dejanovic S,
Stefanovic V, Arsenijevic N, Lukic ML. Antipsychotics can modulate thecytokine proﬁle in schizophrenia: attenuation of the type-2 inﬂammatory
response. Schizophr Res. 2013; 147(1): 103-9.
3. Borovcanin M, Jovanovic I, Radosavljevic G, Djukic Dejanovic S,
Stefanovic V, Arsenijevic N, Lukic ML. Increase systemic levels of IL-23 asa possible constitutive marker in schizophrenia.
Psychoneuroendocrinology. 2015; 56: 143-7.

77

PRESENTATION

1

41

Antigen-afﬁnity mediates B cell survival through differential BAFF receptor signaling
via the PI3K/Mcl-1 cascade
Felix M. Wensveen1, Rianne van der Windt2, Arnon Kater2, Robert Brink3, Eric Eldering2
1
2

PRESENTATION

3

Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia;
Academic Medical Center, University of Amsterdam, Amsterdam, The Nederlands
Garvan Institute of Medical Research, Sydney, Australia

Upon antigen encounter, the responsive B cell pool undergoes spectacularafﬁnity increase through a stringent selection mechanism that eliminates
cells with low B cell receptor (BCR) afﬁnity. B cell selection in theGerminal Center (GC) has been well described, but the crucial phase of
afﬁnity-dependent selection before GC entry is relatively obscure.
Previously, we showed that mice deﬁcient for the pro-apoptotic protein
Noxa have an antibody response of reduced afﬁnity, due to increased
survival of low-afﬁnity clones that enter the GC. In this study, weinvestigated the mechanism behind early afﬁnity-mediated B cell selection.
We ﬁnd that in the ﬁrst days after activation, B cells depend on BAFF forsurvival and induce expression of the BAFF receptor in a BCR-afﬁnity
dependent fashion. In response to BAFF, PI3K signal intensity is larger inhigh-afﬁnity compared to low-afﬁnity B cells, resulting in increased levelsof the pro-survival protein Mcl-1. Inhibition of PI3K following BAFF
stimulation in activated B cells reduced Mcl-1 protein levels and negatedsurvival differences between cells of high- and low afﬁnity. Deﬁciency for
the Mcl-1 antagonist Noxa reduced dependence on PI3K for survival inactivated B cells. In the presence of redundant BAFF, or in absence of Noxa,
immunization resulted in a relative increase of low-afﬁnity B cells and an
overall reduction of the total antigen-responsive B cell pool upon
immunization. Our ﬁndings elucidate an important mechanism of antigenafﬁnity dependent selection in the earliest phases of B cell activation.

42

78

Inﬂammatory Microglial Activity is Inhibited by Short Term Exposure to
Ethyl Pyruvate
Suzana Stanisavljević, Bojan Jevtić, Neda Đedović and Đorđe Miljković

Ethyl pyruvate (EP) is a potent redox and anti-inﬂammatory agent that has
recently been shown protective in experimental autoimmune
encephalomyelitis (EAE), an animal model of multiple sclerosis.
Microglial cells basically have protective role in the central nervous system,
but their inappropriate pro-inﬂammatory activity has been shown
detrimental in various neurodegenerative disorders, including multiple
sclerosis. It was previously shown that EP potently inhibits inﬂammatory
activity in microglia. The aim of this study was to determine whether
microglial inﬂammatory activity is sensitive to short term exposure to EP.
As the result, short term treatment of BV2 microglial cells with EP inhibited
production of pro-inﬂammatory cytokines (interleukin-6 and tumor
necrosis factor) and nitric oxide in the cells. At the same time, activation of
NFκB in BV2 cells was not affected by EP, implying that the observed
effects of the agent on pro-inﬂammatory molecules was achieved in NFκB
independent way. The observed immunomodulatory effects are importantfor understanding therapeutic potential of EP in treatment ofneuroinﬂammatory disorders.
This work was supported by the Ministry of Education, Science andTechnological Development, Republic of Serbia (grant no. 173035).

PRESENTATION

Institute for Biological Research "Siniša Stanković", Department of Immunology, University of Belgrade, Serbia

43

79

The Potential Immunomodulatory Role of Cortisol on NKG2D Activating NK Cells
Receptor in Ovarian Cancer
Ahmed Adel Seida1,2,3
1

Leibniz Research Institute for Environmental Medicine, Molecular Immunology Department, Düsseldorf, Germany
Interdisciplinary Center for Clinical Research, University of Würzburg, School of Medicine, Würzburg, Germany
Department of Obstetrics and Gynecology, University of Würzburg, School of Medicine, Germany

2

PRESENTATION

3

44

Background: Tumour-inﬁltrating myeloid-derived suppressor cells(MDSC) or tumour-associated macrophages (TAM) which are abundant inovarian cancer show a high expression of the enzyme 11BetaHydroxysteroid dehydrogenase I (11β-HSD1) which able to convert
inactive cortisone into active cortisol which has been detected in serum,
ascitic ﬂuid and tumour exudates from ovarian cancer patients. Consideringthat cortisol has a strong suppressive effect on immune cells, weinvestigated the effect of cortisol or TGF-β or both since they do not act
separately of each other but modulate each other's activities on theactivating NK cell receptor (NKG2D) expression. This shall now be
investigated by pharmacological inhibitors of cortisol (RU486) and TGF-β(SD208).
Material and methods: Using immunohistochemistry, real-time PCR,
luminescent immunoassays (LIA), Flow cytometry and immunoﬂuorescent
double staining.
Results: We found that 11b-HSD1 enzyme is highly expressed in humanand murine ovarian cancer tissue via real-time PCR and
immunohistochemistry. Luminescent immunoassays (LIA) showedelevated cortisol in serum, ascitic ﬂuid and tumour exudates from ovariancancer patients. Immunoﬂuorescent double staining revealed a colocalization of 11b-HSD1 with CD14, CD68, and CD85, but not with
EpCAM. Expression of 11β-HSD1 can thus be attributed to TAM or MDSC.
Expression of NKG2D on NK cells was analyzed by ﬂow cytometry. The
result indicated that NKG2D is downregulated by both cortisol and TGF-β1.
Cortisol and TGF-β1 were further found to act synergistically indownregulating NKG2D. However, RU486 and SD208 were able to restore
the activating NKG2D receptor expression on NK cells.
Key words: Ovarian Cancer, 11Beta-Hydroxysteroid dehydrogenase I,
Cortisol, TGF-β, NKG2D; NK Cells.

80IL-33 modulate innate immunity to tumours
Ivan P. Jovanovic, Nada N. Pejnovic, Gordana D. Radosavljevic, Jelena M. Pantic, Marija Z. Milovanovic,
Nebojsa N. Arsenijevic and Miodrag L. Lukic

The role of IL-33/ST2 pathway in antitumor immunity is not elucidated. We
have previously shown that ST2 deletion enhanced anti-tumor immune
response in a murine model of metastatic 4T1 breast carcinoma which was
associated with potentiated Th1/Th17 cell polarization and enhanced NK
cell cytotoxic activity (Jovanovic I, et al. Eur J Immunol.
2011;41:1902–12). In this study we demonstrate time dependent increase ofendogenous IL-33 at both the mRNA and protein levels in primary 4T1
tumors and metastatic lungs during breast cancer progression. Further,exogenous administration of recombinant murine IL-33 accelerated tumor
growth and development of lung and liver metastases, which was associatedwith increased intratumoral accumulation of CD11b+Gr-1+ TGF-b1+
myeloid-derived suppressor cells (MDSCs) that expressed IL-13α1R, IL13-producing Lin - Sca-1 + ST2 + innate lymphoid cells (ILCs) and++++CD4 Foxp3 ST2 IL-10 Tregs. Higher incidence of monocytic vs.
granulocytic MDSCs and plasmocytoid vs. conventional dendritic cells(DCs) was present in mammary tumors of IL-33-treated mice. Intratumoral
NKp46+NKG2D+ and NKp46+FasL+ cells were markedly reduced after IL33 treatment, while increased frequencies of these tumoricidal natural killer(NK) cells were found within primary tumors in ST2-deﬁcient micecompared to wild-type mice. Administered IL-33 promoted intratumoral
cell proliferation and neovascularization, which was attenuated in theabsence of ST2. Tumor-bearing mice given IL-33 had increased
percentages of splenic MDSCs, Lin-Sca-1+ ILCs, IL-10-expressing CD11c+
DCs and alternatively activated M2 macrophages and higher circulatinglevels of IL-10 and IL-13. A signiﬁcantly reduced NK cell, but not CD8+ Tcell cytotoxicity in IL-33-treated mice was observed and the mammary
tumor progression was not affected by in vivo depletion of CD8+ T cells. We
demonstrate a previously unrecognized role for IL-33 in promoting breast
cancer progression through increased intratumoral accumulation of
immunosuppressive cells and by diminishing innate antitumor immunity.
Therefore, IL-33 may be considered as an important mediator in theregulation of breast cancer progression.
This work was supported by grants from the Serbian Ministry of Scienceand Technological Development (175069, 175071 and175103), Serbia andfrom the Faculty of medical sciences Kragujevac (project MP 01/12),
Serbia.

81

PRESENTATION

Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia

45Antimelanoma and chemosensitizing properties of hop plant derived compoundisoxanthohumol
Tamara Krajnović1, Danijela Maksimović-Ivanić1, Goran N. Kaluđerović2, Ludger A. Wessjohann2 and Sanja
Mijatović1
1

Department of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany

PRESENTATION

2

46

It is well known that many synthesized and natural compounds from plants
apart from anti-inﬂammatory properties possess strong anticancer activity.
Since it has been recently demonstrated that beer and hop plant (Humulus
lupulus) derived compound, isoxanthohumol (IXN), down regulates
inﬂammation, the aim of this study was to investigate possible anticancer
potential of IXN in vitro and in vivo.
Our results showed remarkable viability decrease of mouse (B16)
melanoma cells in dose-dependent manner after the treatment with IXN.
Oppositely, the autophagic process was not responsible for the decrease ofcell viability. On the other hand, certain percentage of apoptotic andnecrotic cells followed diminished division rate of survived melanoma
cells. At the intracellular level, inhibited activity of well-known regulatorsof cell proliferation, p70-S6 Kinase and S6 ribosomal protein wasdetermined. Microscopic observation of the melanoma cells exposed to
IXN revealed remarkable morphological transformation synchronized byelevated tyrosinase activity, indicating enhancement in pigment synthesis
as biochemical marker of differentiation toward melanocytes. In addition toobserved direct tumoricidal effect, IXN potentiated the effectiveness ofpaclitaxel in vitro. Moreover, chemosensitization was conﬁrmed insyngeneic melanoma model where mice were exposed to subtoxic doses ofthis chemotherapeutic.
Taken together, these results point out multiple beneﬁts of IXN manifested
through direct anticancer potential as well as remarkable capacity tosensitize melanoma cells to conventional therapy with paclitaxel.
Supported by Ministry of Education, Science and Technological
Development, Republic of Serbia (Grant No. 173013).

82

Systemic TNF ablation results in delayed tumor growth and reduced MDSC
accumulation in transplantable tumor model
Atretkhany K-S1,2, Nosenko M.A.1,2, Zvartsev R.V.1, Kruglov A.A.1,3, Eﬁmov G.A.1,4, Qin Z.5, Nedospasov S.A.1,2,3,
Drutskaya M.S.1,*
1

Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Moscow, Russia
Immunology Department, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
4
National Research Center for Haematology, Moscow, Russia
5Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
2

Myeloid-derived suppressor cells (MDSC) represent a heterogeneous
population that consists of immature myeloid cells (IMC). Under normal
conditions, these cells differentiate into mature macrophages, granulocytes
and dendritic cells. However, under pathological conditions such as
inﬂammation, cancer or infection, there is an inhibitory effect on
differentiation of IMC that leads to their expansion. MDSC have immune
suppressive activity through upregulation of arginase 1 (Arg1) and
inducible nitric oxide synthase (iNOS). These suppressive factors canenhance tumor growth by repressing T cell–mediated antitumor responses.
Recently, it was shown that TNF-TNFRII axis sustains survival of MDSC
through upregulation of cellular FLICE-inhibitory protein (c-FLIP) andinhibition of caspase-8 activity [1].
In our work we established an experimental model to study the effect ofsystemic TNF ablation on the tumor-induced expansion of MDSC in vivo.
We used Etanercept, a soluble fusion protein of p75 TNF receptor and theconstant end of IgG1 antibody, as an inhibitor of murine TNF in C57Bl/6
mice transplanted with MCA205 ﬁbrosarcoma. Inﬂiximab, a chimericmonoclonal antibody against human TNF, was used as control since it does
not bind mTNF [2].
Mice were injected with 10µg/g of Etanercept or Inﬂiximab every threedays. Tumor cells were injected one week after the initiation of anti-TNF
therapy. Tumor growth was monitored every 2–5 days. We also assessed theaccumulation of MDSC in the peripheral blood by ﬂow cytometry using
epitope-speciﬁc antibodies against CD11b and Gr-1.
We show that systemic anti-TNF therapy results in delayed growth kineticsof MCA 205 ﬁbrosarcoma. Mice injected with Etanercept have signiﬁcantly
reduced tumor volume and accumulate less MDSC in the peripheral blood
three weeks after MCA tumor cell inoculation. Our data suggests that
Etanercept-based therapy reduces solid tumor growth and tumor-induced
accumulation of MDSC in MCA-205 ﬁbrosarcoma tumor model.
This work is supported by RFBR grant #14-04-91186 and RNF grant #1425-00160 (for IL-6 dependent MDSC differentiation).
1. Xueqiang Zhao., et al. 2012. J Clin Invest.; 122(11):4094–4104.
2. Knight D., et al. 1993. Mol. Immunol. 30 (16): 1443–53

83

PRESENTATION

347

Decreased level of various peripheral blood lymphocyte signaling molecules inmetastatic melanoma patients
Katarina Mirjačić Martinović1, Ana Vuletić1, Tatjana Srdić-Rajić1, Nada Babović2, Radan Džodić3, 4, Vladimir
Jurišić5, Gordana Konjević1
1

Department of Experimental Oncology , Institute of Oncology and Radiology of Serbia , Belgrade, Serbia
Department of Medical Oncology, Institute of Oncology and Radiology of Serbia , Belgrade, Serbia
Surgical Oncology Clinic, Institute of Oncology and Radiology of Serbia
4
Department of Surgery, School of Medicine, University of Belgrade, Belgrade, Serbia
5
Department of Pathophysiology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia
2

PRESENTATION

348

Background: As numerous signaling molecules regulate effector functionsof peripheral blood lymphocytes (PBL) that have an important antitumor
activity the aim of this study was to analyze their level in metastaticmelanoma (MM) patients compared to healthy controls (HC).
Methods: Peripheral blood mononuclear cells (PBMC) of 33 MM and 26
HC were analyzed for the level of perforin, interferon-regulatingtranscription factor-1 (IRF-1), DAP10 and Src homology 2 domaincontaining tyrosine phosphatase-1 (SHP-1) by reverse transcriptase
polymerase chain reaction (RT-PCR), level of phosphorylated signaltransducers and activators of transcription (pSTATs), STAT-1, STAT-4 andSTAT-5, by Western blot and IFN-gamma (IFN-γ) production by enzyme
linked immunosorbent assay (ELISA). The expression of activating
NKG2D and inhibitory killer immunoglobulin-like receptors (KIRs),
CD158a, and CD158b, on PBL, CD3-CD56+ NK cells and CD3+CD8+
cytotoxic T cells (CTL), as well as the percentage of CD14+HLA-DR- cells
in PBL were estimated by Flow cytometry.
Results: MM patients, compared to HC, had signiﬁcantly lower level ofcytotoxic molecule perforin and decreased IFN-γ production, as well as
lower level of pSTAT-1, -4, -5 and IRF-1 signaling molecules in their
PBMC. Furthermore, MM patients have decreased expression of activating
NKG2D receptor on PBL and NK cells and low level of its DAP10 signaling
molecule contrary to no changes in NKG2D expression on CTL and
CD158a and CD158b KIR expression on all investigated cells. Increased
percentage of immunosuppressive CD14+HLA-DR- myeloid-derived
suppressor cells (MDSC) was estimated in MM patients.
Conclusions: The altered signaling molecules in peripheral blood
lymphocytes of metastatic melanoma patients could represent biomarkers
of impaired cytotoxic and immunoregulatory function of these cells
indicating melanoma associated immunosupression that may facilitate
tumor progression. Furthermore, these molecules could be used asparameters for the evaluation of standard or developing immunotherapy inpatients with advanced melanoma.

84

Decreased interferon γ production in CD3+ and CD3-CD56+ lymphocyte subsets
in metastatic regional lymph nodes of melanoma patients
Ana Vuletić

As lymphogenic dissemination is very common in melanoma, regional
lymph nodes (LN)s represent ﬁrst immunological barriers to tumor invasion
and play a complex role in antitumor immune defense. In this sense, themost prominent role is the presentation of tumor-derived antigens to naïve T
cells and generation of cell-mediated adaptive immune response.
Since tumor micro-environment affects immune cell function in this study
we have evaluated the ability of T cells and NK cells in metastatic (involved)
and non-metastatic regional LNs to produce interferon γ (IFNγ), a
pleiotropic cytokine that regulates adaptive antitumor immune response.
Our results show reduced IFNγ production in both T and NK lymphocyte
subsets and decreased prevalence of T cells in metastatic regional LNs of
melanoma patients. The decrease of IFNγ production in T cells was more
pronounced with increased number of involved regional LNs indicatingtumor-induced functional impairment of both T and NK cell lymphocyte
subsets in involved regional LNs. Therefore, low IFNγ production inmetastatic LNs may represent an obstacle in adaptive cell-mediatedantitumor immune response and hence may enable tumor progression.

PRESENTATION

Department of Experimental Oncology, Laboratory for Immunology, Institute of Oncology and Radiology of Serbia, Belgrade,
Serbia49

85

Tumor cell lines K562 and HEPG2 decrease human natural killer cell cytotoxicity
and increase expression of inhibitory receptor TIGIT
Vera Abramova

PRESENTATION

Laboratory of Molecular Immunology and Immunobiotechnology, M.A. Aitkhozhin Institute of Molecular Biology andBiochemistry, Almaty, Kazakhstan

Natural Killer (NK) cells lyse tumor cells lacking MHC-I and expressing
ligands for activating receptors, thereby participating in cancer immune
surveillance. However, their function and phenotype change dramatically inthe presence of tumors. The aim of the present study was to investigatechanges of both receptor phenotype and cytotoxic activity of NK cells cocultured with K562 and HepG2 cell lines in dual-chamber Transwell®
plates. NK cells were obtained from peripheral blood of healthy donors byimmunomagnetic separation. After co-culture with HepG2 or K562 inTranswells® for 48h, NK cells were assessed for cytotoxicity to K562 cells at
a 2:1 effector:target ratio and ﬂow cytometry analysis of phenotype.
According to MTT assay, NK cell cytotoxicity decreased by 20.8±3.1%(p=0.007) and 19.1±6.6% (p=0.038) relative to controls after co-culture
with HepG2 and K562 cells, respectively. No changes in the expression of
perforin, granzyme B, or CD107a were observed. Changes in the expressionof a majority (CD16, NKp30, NKp44, NKp46, CD69, DNAM-1, 2B4,
NKG2A) of NK cell receptors did not reach statistical signiﬁcance. TIGIT
expression increased on NK cells after incubation with K562 (12.0±5.7%,
p=0.015) and HepG2 cells (10.4±5.6%, p=0.042) compared to controls(4.7±3.2%). In conclusion, the observed ﬁndings demonstratethat tumor
cell derived soluble factors induce a decrease in NK cell cytotoxicity
accompanied by an increase in inhibitory receptor TIGIT expression.It is ofinterest to determine whether this phenomenon is recapitulated in cancer
patients and could represent a novel parameter in assessment of NK cellfunction.

50

86

Recombinantly produced Banana Lectin stimulates Humoral and Cellular Immune
Response in the Colon
Emilija Marinković1, Ivana Lukić1, Dejana Kosanović1, Aleksandra Inić-Kanada*, Marija GavrovićJankulović2, Marijana Stojanović1,*

Lectins are a broad group of bioactive molecules present in plant-based foodin appreciable amount. Their biological functions are associated with thecarbohydrate binding activities. They can bind the gut epithelium and,depending of their ﬁne speciﬁcity, affect digestive tract functioning in a
distinctive ways. We evaluated the inﬂuence of recombinantly produced
banana lectin (rBanLec) on the immune response in the colon of BALB/cmice. Local immune response was monitored up to 72h upon a rectal
administration of rBanLec (100 µl of 0.1, 1 or 10 µg/ml ; equal to 0.5, 5 and50 µg/kg body weight, respectively). Immunohistochemical analysis has
shown that rBanLec bound the mucosal surface of the colon and passed
through the epithelial barrier. Skewing of immune response toward
Th1/Th17 direction could be assigned as a general characteristic of rBanLec
stimulation in the colon but qualitative and quantitative characteristics ofthe established cytokine network depended on the applied rBanLec
concentration. rBanLec transitionally augmented secretion of IFNγ and
IL-17A, which was counterbalanced by increase in IL-10 production.
Signiﬁcant rise in the secretion of sIgA and IgG2b indicated the local
enhancement of TGF-β production as well. rBanLec promoted a transitionalrise in local NO concentration and MPO activity that coincided with thepeak in concentration of pro-inﬂammatory cytokines (48h upon theapplication).
Our results show that rBanLec, even in low dosage that could be taken in by
everyday nutrition, acts as a polyclonal stimulator affecting (in)directlyboth humoral and cellular immune responses in the colon. Its ability tostimulate balanced pro-inﬂamatory immune response in the colon and toenhance the antimicrobial activity implies that rBanLec could be a valuable
tool in prevention of a pathogen invasion via the colon.

87

PRESENTATION

1Institute of Immunology, Virology, Vaccine and Sera – Torlak, VojvodeStepe 458, 11152, Belgrade, Serbia*OCUVAC - Center of Ocular Inﬂammation and Infection; Laura Bassi Centers of Expertise; Center for Pathophysiology,
Infectiology and Immunology; Medical University of Vienna; Vienna, Austria
2
Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia

51

Lack of association between TNF gene polymorphism and the acute kidney rejection

V.Perovic1, D. Popadic1, R. Naumovic2,M.Kravljaca2, V.Pravica1
1

Institute of Microbiology and Immunology School of Medicine, University of Belgrade
Clinic of Nephrology, Clinical Center of Serbia

PRESENTATION

2Introduction: Acute and chronic rejections remain an important cause ofgraft loss after renal transplantation. The inﬂammatory microenvironment
within the graft may play a role in the precipitation of rejection. Several
lines of evidence have implicated TNF in the early alloimmune response tosolid organ transplantation. Previous studies reported that tumor necrosis
factor (TNF) promoter polymorphism (-308 G/A) has inﬂuence on thefunctional immune response of transplant recipient outcome.
Methods: One hundred ﬁfty two patients who underwent renaltransplantation were included in this study. The genotyping was based on
the TaqMan method using commercially available probes. Genotype
frequencies were assessed for Hardy–Weinberg equilibrium using a chisquared goodness-of-ﬁt test. The statistical difference in genotype
distribution and allele frequencies in case subjects was analyzed using
Pearson's chi-square or Fisher's exact tests.
Results: The genotype frequency in group of transplanted patients with andwithout acute graft rejection was AA 1.0%, GA 26.5%, GG 72.4% and AA
2.0%, GA 22.4%, GG 75.5% respectively. This study showed that TNF-α
promoter polymorphism was not associated with the risk of acute rejection.
Conclusions: Our ﬁndings demonstrated no signiﬁcant association
between TNF-α gene polymorphisms and acute renal transplant rejection.
However, due to conﬂicting results from this and other studies, a multicentre collaborative study may be required to determine whether cytokine
genotypes are signiﬁcant, independent predictors of renal allograft
rejection.

52

88

Vitamin D insufﬁciency affects IL-10 mediated regulation of cellular immunity
O. Angelova1, M. Alexandrova1, N. Yancheva2, M. Muhtarova1, I. Elenkov2, T. Tchervenyakova2, M. Nikolova1
National Center of Infectious and Parasitic Diseases
Specialized Hospital for Active Treatment of Infectious and Parasitic Diseases “Professor Ivan Kirov”, Soﬁa

Background: Vitamin D (vitD) has well-established immunomodulatory
functions, at least in part associated with effector and regulatory immune
cell cytokines. Although vitD deﬁciency in HIV+ subjects is wellrecognized, current evidence for its impact on cellular immunity isrelatively weak.
Aim: To assess the effect of vitD insufﬁciency on the “ex vivo” cytokine
proﬁles (IFNg, TNFα, IL-4, IL-6, IL-10) and immune restoration of HIV+patients subjected to antiretroviral therapy (ART).
Materials and methods: Serum vitD levels (ng/ml, VIDAS 25OHVitD
BioMèrieux ELISA), CD4 absolute count (CD4AC cells/mkl) andrestoration rate (ΔCD4/months on ART), CD4/CD8 ratio (BDMultiSet,
FA C S C a n t o I I ) , a n d v i r a l l o a d ( V L , H I V- R N A c o p i e s / m l ,COBASAmpliPrep/ TaqManPCR) were determined in 60 HIV+ART+
patients. For 30 of them, “ex vivo” cytokine concentrations (pg/ml) wereTM
measured in peripheral blood plasma by ﬂow cytometry (CBA BD ).
Results: Patients fell into three groups according to vitD levels: A, deﬁcient(<20ng/ml, n=23); B, insufﬁcient (20-30ng/ml, n=35) and C, sufﬁcient(>30ng/ml, n=11). While CD4AC, CD4/CD8 and VL did not differ between
groups, a signiﬁcantly lower CD4AC restoration rate was observed in groupA (mean 6.4 vs.16.4/B), combined with decreased IL-10 level (mean 1.2
vs.1.6/B). After excluding patients with extremely low IFNg expression and
low CD4AC, considered as “exhausted”, decreased vitD was associatedwith higher levels of IL-6 (1.0/A, B vs.0.88/C); while vitD insufﬁciency
was distinguished by increased IL-10 level (1.7 vs.1.3/A), and decreased
mean IFNg/IL-4 ratio (45 vs.71/A). For all comparisons p<0.05.
Conclusion: VitD insufﬁciency is a critical stage contributing to increased
immune activation (IL-6), stimulated regulatory response (IL-10) andhence, suppression of Th1 response (IFNg/IL-4). A hallmark of advanced
vitD deﬁciency is the reduced regulatory potential (IL-10) bringing tofurther Th1 exhaustion and slower recovery of CD4 pool, regardless ART.
Our data propose that vitD supplementation could improve the effect of
ART when timely applied.

89

PRESENTATION

1
2

53

In Vitro Effects of Binuclear (η6-p-cymene) Ruthenium (II) Complex Containing
Bridging Bis (nicotinate)-Polyethylene Glycol Ester Ligand on Differentiation
Pathways of Murine Th Lymphocytes
Miljana Momčilović1, Jana Blaževski1, Milica Vujičić1, Goran N. Kaluđerović2 and Stanislava Stošić-Grujičić1
1

Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany

PRESENTATION

2

T cell differentiation into distinct T-helper (Th) subpopulations is crucial ingoverning adaptive immune responses as well as some inﬂammatory andautoimmune disorders. In this study, the potential effect of the novel neutral
b in uclear ruthenium(II) comp lex [{RuCl 2 (η 6 -p-cym)} 2 -{(3py)COO(CH2CH2O)4CO(3-py)}] (4) on Th cell diffrentiation in mousesplenocytes was evaluated in vitro. It did not affect mouse splenocyte
viability (in concentration up to 50 μM), but signiﬁcantly reduced secretionof representative Th1 cytokine, IFN-γ induced by T cell mitogenconcanavalin A (ConA). Beside IFN-γ, 4 inhibited dose-dependently
expression and production of representative Th17 cytokine, IL-17, in thesecells. Otherwise, the production of anti-inﬂammatory cytokines IL-4 andIL-10 was up-regulated. Also, 4 signiﬁcantly increased CD4+CD25+FoxP3+
Treg cell frequency in the ConA-activated splenocytes. Moreover, 4
reduced expression of Th1 transcription factors, T-bet and STAT1, as well asof Th17-related protein STAT3, while the expression of Th2-relatedtranscription factor GATA3 remained stable. In conclusion, 4 modulates
mouse immune system cell functions in vitro by inhibiting T celldifferentiation towards pathogenic Th1/Th17 phenotype and inducing a
regulatory phenotype characterized by IL-10 and IL-4 production. The
ability of 4 to direct Th cell differentiation may provide novel therapeutic
opportunities for immune-inﬂammatory and/or autoimmune disorders.
This work was supported by the Ministry of Education, Science andTechnological Development, Republic of Serbia (grant no. 173013).

54

90

TRP channels overexpression contributes to inﬂammasome activation in clavicularcortical hyperostosis?
Lovro Lamot1, Mandica Vidović2, Marija Perica2, Lana Tambić Bukovac2, Kristina Gotovac1, Fran Borovečki1,
Miroslav Harjaček1,2
1

University of Zagreb School of Medicine, Zagreb, Croatia
Children's Hospital Srebrnjak, Zagreb, Croatia

Background: Clavicular cortical hyperostosis (CCH) is a sterile
inﬂammatory bone disorder of unknown etiology clinically characterizedby pain and/or swelling of the clavicle. It has been regarded as a variant ofchronic nonbacterial/recurrent multifocal osteomyelitis (CNO/CRMO) but
due to lack of other inﬂammatory sites and recurrence it could also be
regarded as a separate disease in the spectrum.
Objectives: Identiﬁcation of speciﬁc gene expression patterns in CCH
patients.
Methods: Total RNA was isolated from whole blood of 18 new-onset,
untreated CCH patients and 8 healthy controls. DNA microarray gene
expression was preformed in 5 CCH and 4 control patients along withbioinformatical analysis of retrieved data. Carefully selected differentiallyexpressed genes (TRPM2, TRPM3, TRPM7, CASP2, MEFV, STAT3,
EIF5A, ERBB2, TLR4, NLRP3, CD24, MYST3) where analyzed by qRTPCR in all participants of the study.
Results: Microarray results and bioinformatical analysis revealed 974
differentially expressed genes, while qRT-PCR analysis showedsigniﬁcantly higher expression of TRPM3 and TRPM7, and lower
expression of ERBB2 (Graph 1).
Conclusions: TRPM3 and TRPM7 are transient receptor potential (TRP)
proteins that act as multimodal sensor cation channels for a wide variety ofstimuli, one of which is environmental temperature that in the case of CCH
could be elicited by overuse of sterno-clavicular joint (SCJ) [1]. Altered
TRP channel function can have strong effects on a variety of cellular andsystemic processes, including the activation and the regulation of theinﬂammasomes, which are reported to be involved in CRMO pathogenesis[2,3]. ERBB2, third gene with signiﬁcant expression change, belongs to a
family of genes that encodes for cell surface growth factor receptors. ErbB
activation promotes protective cellular outcomes during inﬂammation,
hence lower expression of this gene could cause damage due toinﬂammation [4]. We hypothesize that CCH could be an autoinﬂammatory
disease induced by SCJ overuse, TRP channel overexpression,inﬂammasome activation and reduced protection during inﬂammation.

91

PRESENTATION

2

55

Effects of royal jelly fatty acids on proliferation and cytokine production by humanperipheral blood mononuclear cells
Dušan Mihajlović1, Dragana Vučević2, Sergej Tomić1, Ivan Rajković1, Miodrag Čolić1
1Medical Faculty of the Military Medical Academy , University of Defence in Belgrade , Belgrade , Serbia
Institute for Medical Research, Military Medical Academy, Belgrade, Serbia

PRESENTATION

2Royal jelly has been widely used as healthy food in humans due to thepresence of different biologically active components. Fatty acids, isolated
from royal jelly, have recently been investigated as potentiallyimmunomodulatory components. In this study, we investigated theimmunomodulatory effects of two fatty acids isolated from RJ, 10-hydroxytrans-2-decenoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10DDA), using a model of phytohaemagglutinin (PHA)-activated humanperipheral blood mononuclear cells (PBMCs). We demonstrated that bothfatty acids at higher concentrations (500 μM) inhibited the proliferation of
PBMCs and the process was followed by a decrease in the production ofinterleukin-2 (IL-2). 10-HDA at concentration of 500μM down-regulated
the production of IL-1β and tumor necrosis factor-α by stimulated PBMCs,
whereas the same dose of 3,10-DDA had no effect on the levels of thesecytokines. Regarding T-helper (Th) cytokine proﬁle, higher concentrationof 10-HDA, in contrast to the lower one (50 μM), inhibited both Th1 andTh2 response, whereas Th17 response was not signiﬁcantly modulated, as
judged by the levels of interferon-γ, IL-5 and IL-17A in culture
supernatants, respectively. Lower concentration of 3,10-DDA stimulated
Th1 and Th17 responses and inhibited IL-10 production, whereas the higher
dose augmented the Th2 response. In conclusion, our results showed asigniﬁcant, dose-dependent, immunomodulatory effect of RJ fatty acids invitro, which was also associated with their structure.

56

92

Compound A, a Selective Glucocorticoid Receptor Antagonist, has Immunomodulatory
and Cytoprotective Effects In Vitro
Tamara Saksida, Ivana Nikolić, Milica Vujičić, Ivana Stojanović and Stanislava Stošić-Grujičić

Autoimmune type 1 diabetes (T1D) is characterized by progressive
destruction of insulin producing beta cells mediated by various proinﬂammatory mediators that leads to hyperglycemia. Chronic elevation ofblood glucose impairs beta cell function and leads to beta cell death(glucotoxicity). Compound A (CpdA) is a selective glucocorticoid receptor
antagonist that exerts anti-inﬂammatory and immunomodulatory activities.
Since we have recently shown that CpdA protects C57BL/6 mice fromdeveloping of T1D induced by multiple low doses of streptozotocin, the aimof this study was to examine the direct in vitro effects of CpdA on multiple
immune system components and glucotoxicity in beta cells. We cultured
spleen cells (SC), lymph node cells (LNC) and peritoneal cells (PC)obtained from C57BL/6 mice in the presence or absence of CpdA (0.1 – 10μM) for viability assay (MTT test), or 10 μM CpdA for cytokine production(ELISA); SC and LNC were stimulated or not with 1 μg/ml concanavalin
Concanavalin A for 48 h, and PC were stimulated or not with 5 μg/ml
Lipopolysaccharide (LPS) for 24 h. Also, we exposed the rat insulinoma
INS-1 cells to high glucose (20 mM) in the presence or absence of CpdA (10μM) and determined cell survival by Real Time Cell Analysis. CpdA
treatment signiﬁcantly reduced the secretion of IL-1β, TNF, IL-6 and IL-10
by PC. In SC and LNC CpdA reduced IFN-γ and IL-17 and up-regulated IL4 secretion. Moreover, CpdA preserved INS-1 cells from high glucose
induced toxicity. In conclusion, our results indicate that CpdA has direct
immunomodulatory effects on activated macrophages and lymphocytes invitro and also protects beta cells from glucotoxicity.
Supported by the Ministry of Education, Science and Technologicaldevelopment, Republic of Serbia (grant no. 173013).

PRESENTATION

Institute for Biological Research “Siniša Stanković”, Department of Immunology, University of Belgrade, Belgrade, Serbia

5793Focused ion-beam scanning electron microscopy and particle induced X-ray emission
as novel methods for the analysis of gold nanoparticles internalization by humandendritic cells
Sergej Tomić1, Rebeka Rudolf2, Primož Pelicon3, Ivan Anžel2, Miodrag Čolić1,41

Medical Faculty of the Military Medical Academy, Military Medical Academy, University of Defense, Belgrade, Serbia
Institute for Mechanical Engineering , University of Maribor, Maribor, Slovenia
Microanalytical Centre, Jožef Stefan Institute, University of Ljubljana, Ljubljana, Slovenia
4
Medical Faculty, University of Niš, Niš, Serbia
2

PRESENTATION

3

58

Immunological effects of gold nanoparticles (GNPs) greatly depend on
their size, mechanisms and levels of their internalization by dendritic cells(DCs), key antigen presenting cells and crucial regulators of T cells
development. However, common methods for the internalization analyses
often have limited spatial and depth resolution, approximative quantity
detection and neglect the cell-to-cell variability in nanoparticles
internalization. Therefore, we applied for the ﬁrst time in such studies
focused ion beam/scanning electron microscopy (FIB/SEM) and particle
induced X-ray emission (PIXE) analysis, due to their great potential for 3D
reconstruction of cell cross-sections at electron microscope scale and theprecise intracellular quantiﬁcation of elements, respectively. Confocal
microscopy showed that GNPs are internalized by DCs predominantly via
dynamin-dependent mechanisms, which was conﬁrmed by FIB/SEM.
However, cross-sectioning by FIB/SEM revealed that Dynasore-treatedDCs contain higher amounts of smaller GNPs intracellularly compared to
the larger ones, suggesting additional mechanisms of their internalization.
Furthermore, FIB/SEM, similarly as transmission electron microscopy(TEM), revealed that in addition to endosomal localization of GNPs,
smaller GNPs are more often found outside the endosomes. Analysis of
GNPs internalization by light microscopy and ﬂow cytometry indicatedhigh variability in the capacity of DCs to internalize GNPs, and that higher
percentage of DCs internalizes larger GNPs. Similarly, PIXE-based
quantiﬁcation by the thin sample approximation showed that the mass of
GNPs internalized by DCs increase with GNPs size. However, when themass of gold was recalculated to the number of GNPs per DCs, we observed
an inverse proportion, i.e. the number of intracellular GNPs increase with
their decreased size. These results suggest that FIB/SEM and PIXE
facilitate and complement other methods for the analysis of nanoparticles
internalization, but they also provide new information allowing one to avoid
the misinterpretation of the results.
Part of the results was published in PLoS one 2014, 9: e96584.

94

Polymorphism of interleukin-23 receptor in Serbian patients with inﬂammatory
bowel disease
Irena Vukovic1, Dragana Mijac2, Djordje Culaﬁc2, Dragan Popovic2, Vera Pravica1, Milos Markovic1
1

Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade, Serbia
Institute of Gastroenterology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia

Inﬂammatory bowel disease (IBD) that includes Crohn's disease (CD) andulcerative colitis (UC) is a group of disorders of unknown etiology,
characterized by chronic inﬂammation of the gastrointestinal tract in thegenetically predisposed individuals. The growing body of evidence
supports a potential role of the pro-inﬂammatory cytokines like interleukin(IL)-23, a key modulator in differentiation of Th17 lymphocytes, in thepathogenesis of IBD. Also, genome wide association studies (GWAS)
indicate that an uncommon coding variant of IL-23 receptor (rs11209026)
confers strong protection against both types of IBD.
The aim of this study was to determine whether single-nucleotide
polymorphism (SNP) of the gene encoding the IL-23 receptor is associatedwith IBD in Serbian population.
Genomic DNA samples from 206 IBD patients (106 CD, 100 UC) and 259
healthy volunteers were evaluated for IL-23R SNP (rs11209026) using
TaqMan genotyping assay. Comparison between genotype and allele
frequencies in different groups was performed using the Pearson chi-square
test (p<0.05 was considered statistically signiﬁcant).
The frequency of the rs11209026 A allele was lower in CD (1%, p<0.01) andUC patients (2%, p<0.05), when compared with controls (6.4%). The
frequencies of GG and GA genotypes were 98% and 2% in CD (p<0.01)
while 97% and 3% UC patients (p<0.05).
This study supports the association of IL23R polymorphisms as potential
protective factor against development of IBD in evaluated population andmay represent a novel approach to therapy of Th17-mediated diseases.

PRESENTATION

2

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Immunomodulatory effects of frog skin derived antimicrobial peptides

Pantic JM1, Radosavljevic GD1, Jovanovic IP1, Mechkarska M2, Arsenijevic NN1, Conlon JM3, Lukic ML1
1

Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Serbia
Department of Biochemistry, Faculty of Medicine, UAE University, UAE
3
SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, U.K.

PRESENTATION

260

In order to protect animal against invading microorganism host-defense
peptides secreted by epithelial cells exhibit various antimicrobial andimmunoregulatory effects. We have already provide evidence that peptides
derived from frog skin may have additional effects on human pathogens andalso in higher concentrations unwanted effects on human cells (i.e. platelets
and erythrocytes aggregations). The potential immunomodulatory effects
of frog skin peptides have been initially studied in vitro using lymphoidcells from the spleen and peritoneal cavity. In these studies we have shownthat Plasticin-L1, Frenatin 2D, Pseudohymenochirin-1Pb and -2Pa,
Frenatin-2.1S, -2.2S and -2.3S have immunostimulatory effects asevaluated by the production of proinﬂammatory cytokines such as TNF-α,
IL-1β, IL-12 and IL-23, while Esculentin-2CHa, Hymenochirin-1B,
Tigerinin-1R, -1M and -1V preferentially stimulates production of IL-10 byperitoneal cells, alone or with concomitant stimulation with LPS. In order totest whether immunostimulatory effect of frenatin 2.1S may be reproducedin vivo, we analyzed the effects of intraperitoneal injections of this peptide
on the cellular makeup in the peritoneum and spleen, 24 hours after
injection, in both Th1 type C57BL/6 and Th2 type BALB/c mice. The dataobtained strongly indicate that frenatin 2.1S enhances the activation stateand homing capacity of Th1 type lymphocytes (CD3+CXCR3+ cells) andNKT cells in the peritoneal cavity of both C57BL/6 and BALB/c mice.
Further we showed the activation of antigen presenting cells as well as highpercentage of M1 macrophages. Injection of frenatin 2.1S in the presence orabsence of LPS increased peritoneal B cells of B1a phenotype thus
contributing to an inﬂammatory milieu. Frenatin 2.1S also facilitatedaccumulation of NK cells in peritoneal cavity, particularly CD69+ NK cells,
indicating activation status of these cells. We suggest that
immunostimulatory effect of frenatin 2.1S may be considered as therapeutic
approach in disease states, such as cancer and infection, in which anenhanced inﬂammatory response may be beneﬁcial.
Key words: antimicrobial peptides, peritoneal cells, Th1 lymphocytes, NK
cells, macrophages
Funding: This work was supported by grants from the Serbian Ministry of
Science and Technological Development (175069, 175071 and 175103)(Belgrade, Serbia).

96

This Symposium is sponsored by:

IMMUNOLOGICALSOCIETY OF SERBIAEFIS European Federationof Immunology Societies

Serbian Society of Immunology

Center for molecular medicine
and stem cell research,
Faculty of Medical Sciences,
University of Kragujevac, Serbia