PIVAC-1919th International Conference on Progress in Vaccination Against Cancer

O r g a n i z e d b y• The Cancer Immunology and Immunotherapy Center,

St. Savas Cancer Hospital• The Hellenic Society of Immuno-Oncology

7th–9th June 2019 Hellenic Pasteur Institute, Athens, Greece

S u p p o r t o r C o n t a c tFor further details please mail to • baxevanis@ciic.gr• info@scep.gr

Please visit the website https://pivac19.eu/

ScientificProgram

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WELCOME LETTERDear colleagues and friends

On behalf of the organizing committee I would like to welcome you in Athens for the 19th Progress In Vaccination Against Cancer (PIVAC-19). This is the 2nd PIVAC meeting taking place in Athens (the first was back in 2005, again at the Hellenic Pasteur Institute) and the 3rd in Greece (PIVAC-17, in Loutraki).

We have made a scientific program to combine basic, translational, and also clinical research in the field of Cancer Immunology and Immunotherapyand for this we have invited speakers with outstanding experience in these topics for addressing important issues in the field, including (i) therapeutic vaccines, immune checkpoint inhibitors, T cell therapies, targeted therapies, (ii) prognostic and predictive biosignatures, immune resistance and (iii) tumor microenvironment and antitumor immunity.

During the meeting there will be plenty of time for fruitful discussions between speakers and participants to encourage the emergence of new collaborations and moving the field of cancer immunotherapy forward.

We do hope that you will have a pleasant stay in Athens. Last but not least, we would like to thank all those who were involved in the organization of this event and of course our sponsors for supporting this meeting and the participants for joining us.

On behalf of the Organizing Committee

Constantin N. Baxevanis

SILVER SPONSOR

BRONZE SPONSOR

SPONSORS

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SPECIAL THANKS TO

ANTONIOS AND IOANNIS ANGELICOUSSIS FOUNDATION

«The Antonios and Ioannis Angelicoussis Foundation was established in February 2013. It is a charitable Foundation and it is regulated under private law. The Foundation allocates funds to support non-profit orga-nizations in their sustainable projects -especially child-care units-, educational, cultural and ecclesiastical institutions as well as public hospitals through medical equipment donations».

PAVLIS FOUNDATION Theodoros Pavlis & Kalliopi ProkopakiP u b l i c B e n e f i t O r g a n i z a t i o n f o r H e a l t h a n d E d u c a t i o n

Pavlis Foundation is a non-profit organization engaged in public benefit through the areas of health and education, Pavlis Foundation is a grant donnated by the surgeon Theodoros Pavlis in 2015. Dr Theodoros Pavlis devoted his whole life for the benefit of cancer patients.

Ησιόδου 12, 106 74, Αθήνα email info@pavlisfoundation.gr www.pavlisfoundation.gr

AEGEAS AMKE

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FRIDAY JUNE 709.00-09.30 Registrations

Νewstrategiesforimmunestimulation Chair: P.T. Straten

09.30–10.00 New strategies to reprogram dysfunctional tumor-infiltrating T cells in paediatric high-risk neuroblastoma D. Fruci, Immuno-Oncology Laboratory, Ospedale Pediatrico Bambino, Rome, Italy

10.00–10.30 Adjuvants enhancing cross-presentation by dendritic cells: the key to in situ cancer vaccines? G. Adema, Radiotherapy & OncoImmunology laboratory, Dept. of Radiation Oncology Radboud UMC, Nijmegen, The Netherlands

10.30-11.00 The exercise of TAMing the immune system P.T. Straten, Center for Cancer Immune Therapy, Department of Hematology, University Hospital Herlev, Denmark

11.00 -11.30 C o ffe e b re a k

Immunomodulationviaspecifictargeting Chair: S. Lucas

11.30-12.00 The amino acid transporter xCT in breast cancer (stem) cells F. Cavallo, Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy

12.00-12.30 New generation of personalized cancer vaccines C. Gouttefangeas, Group leader at the Institute for Cell Biology, Department of Immunology, Tübingen, Germany

12.30-13.00 Targeting GARP on human Tregs: a novel approach for the immunotherapy of cancer? S. Lucas, de Duve Institute, UCLouvain, Brussels, Belgium

13.00-15.00 L u n c h

ImmunotherapiesduringImmunesenescence Chair: D. Kletsas

15.00-15.30 The immune system ages like everything else: what does this mean for cancer immunotherapy? G. Pawelec, 1. Department of Immunology, Tübingen University, Tübingen, Germany

2. Cancer Solutions Program, Health Sciences North Research Institute, Sudbury, Ontario, Canada

15.30-16.00 NK cell immunosenescence and NK cell-based immunotherapy in AML patients R. Solana, Maimonides Biomedicine Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba, Spain

16.00-16.30 Interplay between cellular senescence and cancer D. Kletsas, Institute of Biosciences and Applications, NCSR “Demokritos”, Athens, Greece

Poster Session I16.30-17.45 Oral presentations of selected posters (10 min each)

SATURDAY JUNE 8 PotentiatingCancerImmunotherapies Chair: M. Nishimura

09.00-09.30 Dissecting T-cell responses in vaccinated, long-term surviving patients E.M. Inderberg, Department of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital,

Oslo, Norway

09.30-10.00 Adoptive T-cell therapy in academia – Oslo experiences G. Kvalheim, MD, PhD, Professor emeritus Gunnar Ikke sensitivt, Oslo, Norway

10.00-10.30 Epitope Spreading to Enhance the Effectiveness of TCR Transduced T Cell M. Nishimura, Research, Department of Surgery, Stritch School of Medicine Loyola University Chicago, USA

10.30-11.00 C o ffe e b re a k

Poster Session II11.00-12.30 Oral presentations of selected posters (10 min each)

12.30 – 14.30 L u n c h

Immunebiomarkersandimmunotherapiesfor hematologicaldiseases Chair: G. Vassilopoulos

14.30–15.00 Next generation sequencing (NGS) – personalized medicine in haematology A. Madrigal, Royal Free Hospital and UCL Cancer Institute, UCL Country Ambassador for Mexico

15.00–15.30 New treatments for leukaemia A. Madrigal, Royal Free Hospital and UCL Cancer Institute, UCL Country Ambassador for Mexico

15.30 – 16.00 Immunotherapeutic targets for the treatment of adult B-cell acute lymphocytic leukaemia B.A. Guinn, Department of Biomedical Sciences, University of Hull,UK

16.00-16.30 Biomarker-guided risk stratification in chronic lymphocytic leukemia K. Stamatopoulos, Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, GREECE

16.30-17.00 Immune checkpoint inhibitors for hematological diseases G. Vassilopoulos, Haematology Laboratory, Biomedical Research Foundation Academy of Athens (BRFAA),

Athens, Greece

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SUNDAY JUNE 9 Immuneescapeandimmunesuppression Chair: P. Foukas

09.00–09.30 Cancer immune escape: MHC expression in primary tumors “versus” metastatic lesion F. Garrido, Departamento de Bioquimica, Biologia Molecular III e Inmunologia,Facultad de Medicina, Universidad de Granada, Spain

09.30–10.00 Role of miRNAs and RNA-binding proteins for the immune escape of tumors B. Seliger, Institute of Medical Immunology, Martin - Luther - University Halle -Wittenberg, Halle, Germany

10.00-10.45 C o ffe e b re a k

10.45-11.15 Myeloid cells assist tumor progression by molecular mechanisms either dependent or independent from adaptive immunity V. Bronte, Immunology Section, Department of Medicine, Verona University Hospital

11.15-11.45 How do myeloid-derived suppressor cells (MDSC) survive in the inhospitable tumor microenvironment? S. Ostrand-Rosenberg, Department of Pathology, Huntsman Cancer Institute, University of Utah, Salt Lake City, USA

11.45-12.15 Dynamic interactions within the tumor microenvironment P. Foukas, 2nd Department of Pathology, National and Kapodistrian University of Athens, Attikon University Hospital, Athens, Greece

Special Lecture12.15-12.45 Assessment of Antibody Functional Affinity Using ImmunSpot® G. Kirchenbaum, Fridolin Frankeand Paul V. Lehmann Cellular Technology Ltd, USA

12.45-14.30 L u n c h

15.00-20.00 Excursion

ABSTRACTSI n v i t e d S p e a k e r sNewstrategiestoreprogramdysfunctionaltumor-infiltratingTcellsinpaediatrichigh-riskneuroblastomaValeria Lucarini1, Ombretta Melaiu1, Adele De Ninno2, Luca Businaro2, Doriana Fruci1

1 Immuno-Oncology Laboratory, Department of Pediatric Hematology and Oncology, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy2 Institute of photonic and nanothecnology, CNR, Rome, Italy

IntroductionNeuroblastoma (NB) is the most common cancer in infancy and early childhood accounting for 15% of paediatric cancer deaths. Survival of high-risk NB patients remains below 50% in spite of the advance and intensive mul-tidisciplinary therapeutic approaches. Tumor microenvironment (TME) plays a crucial role in cancer progression. The presence of tumor-infiltrating T cells (TILs) and the absence of immunosuppressive elements have been associated with favourable prognosis of high-risk NB patients. Recently, a subset of intratumoral dendritic cells (iDC) has been found crucial for anthracycline-induced anticancer immune responses, suggesting that they might be exploited to improve NB therapy. Herein, we investigated the modulation of the immune infiltrate in NB murine models treated with chemotherapeutic drugs in combination with immune checkpoint blocking antibodies. Methods Luciferase-expressing NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were injected subcutaneously or orthotopically in syngeneic mice. Mice bearing established tumors were sacrificed and the function of TILs was evaluated by flow cytometry. The crosstalk between TILsand tumor cells was evaluated in culture systems resembling the native three-dimensional (3D) TME. Specifically, NB organoids were treated with chemotherapeutic drugs, alone or in combination with immune checkpoint receptors inhibitors (ICRi) and co-cultured with TILs. The ability of recall immune cells towards tumor spheroids was evaluated in microfluidic devices, and the functional status of TILs by flow cytometry.Results and ConclusionsTME was characterized by a consistent number of CD45+ immune cells, including T cells, NK cells, NKT cells, mac-rophages, neutrophils and iDCs. The orthotopic model showed a more immunosuppressive TME as comparedto the subcutaneous model, predominantly infiltrated by macrophages, myeloid-derived suppressor cells and T regulatory cells. Ex vivo experiments showed a reduction of organoid diameter treated with chemotherapeutic drugs, alone or in combination with ICRiand an increased recruitment of TILs in microfluidic devices.Moreover, flow cytometry analysis revealed an increased expression of IFNγ and TNFα, as well as a decrease of exhaustion markers such as Tbet and Tigit in CD8+ T cells. In vivo experiments are currently underway to confirm the ex vivo results.Overall, we provide insights for the study of a novel immunotherapeutic approach in NB.

Adjuvantsenhancingcross-presentationbydendriticcells:thekeytoinsitucancervaccines?Gosse J Adema; Radiotherapy & OncoImmunology laboratory, Dept. of Radiation Oncology RadboudUMC, Nijmegen, The Netherlands

Tumors can serve as their own antigenic vaccine “in situ” provided that appropriate tumor ablation approaches are combined with immune activating compounds such as adjuvants or checkpoint mAbs. One of the outstanding questions is which adjuvants optimally synergize with in tumor ablation. Our data now show thatsaponin-based adjuvants are most effective adjuvants able to synergize with tumor ablation. Subsequent functional studies revealed that these adjuvants induce an unprecedented level of DC cross-presentation in vitro and in vivo. The presence of Saponin adjuvant increased cytosolic translocation of antigen, resulting in proteasome-dependent cross-presentation in monocytic CD11b+ DC. Strikingly, specifically in this monocytic CD11b+ DC subset, sa-

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depression and anorexia, comorbidity factors commonly reported by cancer patients. Inhibition of this multi-tasking molecule is therefore a way to hinder different aspects of breast cancer: dissemination and metastasis outgrowth, resistance to treatment, immunosuppression and psychological stress.We have established xCT as a promising immunotherapeutic target for breast cancer. In murine modelsxCT immune-targeting using different vaccination strategies (DNA-, VLP-, and BoHV-4-based vaccines) impairs breast tumor growth and metastasis and increases chemotherapy sensitivity. Immunization in B-cell deficient mice showed no therapeutic response, demonstrating that vaccine-induced xCT antibody represented the major therapeutic effectors. In conclusion, xCT immune-targeting could represent a safe, add-on therapy for conventional and emerging front-line therapies for breast cancer. The widespread expression of xCT in various cancer types (pancreatic, gastroin-testinal, glioblastoma and colorectal) extends the applicability of xCT immune-targeting to many other neoplastic diseases.

NewgenerationofpersonalizedcancervaccinesCécile Gouttefangeas1, Stefan Stevanović1, Hans-Georg Rammensee1

1University of Tübingen and DKFZ partner site Tübingen, Dept. of Immunology, Tübingen, Germany

T cells recognizing tumor-specific mutations have been identified in patients responding to checkpoint inhibition, and such neoantigens are now being incorporated in personalized vaccines. However, and even with a consequent number of non-synonymous mutations at the exome level, it appears that only a few mutated sequences will finally be presented onto tumor MHC-molecules for T cell recognition. In addition, targeting mutations with T cells is most probably not feasible for many tumor types with low mutational burden. Hence, identifying - patient individual -, tumor-specific, but non-mutated antigens is required for further development of cancer vaccines. We are specialized in the large-scale analysis of the HLA-ligandome from human primary tumor (and normal) tissues. We have been able to detectmutated peptides by mass spectrometry in certain tumors, but their frequency is much lower than the frequency of exome mutations. Instead, we reproducibly find HLA-ligands with germline, i.e. non-mutated, sequenc-es that appear as tumor-specific as mutated antigens by their virtual absence on normal tissues. Our vaccination approach builds on the great potential of such wildtype, tumor-specific peptides, as they could be targeted in many tumor types. Immunopeptidome and mutanome analyses were recently combined in a vaccine study for glioma patients developed by the EU-consortium GAPVAC. In addition to identifying patient-individual target peptides, we investigate specificity and (dys)function of T cells in cancer patients, and have also developed a new adjuvant for peptide vaccination. Overall aim is to increase clinical efficacy of cancer our vaccines.

TargetingGARPonhumanTregs:anovelapproachfortheimmunotherapyofcancer?Stéphanie Liénart1, Grégoire de Streel1, Romain Merceron2 Bas van der Woning3, Savvas N. Savvides2, Nicolas van Baren1 and Sophie Lucas1.1de Duve Institute, UCLouvain, 1200 Brussels, Belgium.2VIB Center for Inflammation Research, 9052 Ghent, Belgium.3argenx, 9052 Zwijnaarde, Belgium.

Abstract: Transforming growth factor- 1 (TGF- 1) is one of very few cytokines produced in a latent form, re-quiring activation to exert any of its vastly diverse effects on development, immunity, or cancer. Regulatory T cells (Tregs) suppress immune cells within close proximity by activating latent TGF- 1 presented by GARP to integrin αVβ8 on their surface. We solved the crystal structure of GARP: latent TGF- 1 bound to an antibody that stabilizes the complex and blocks release of active TGF- 1. This reveals how GARP exploits an unusual medley of interactions, including fold complementation by the N-terminus of TGF- 1, to chaperone and orient the cy-tokine for binding and activation by αVβ8. Our work further elucidates the mechanism of antibody-mediated blockade of TGF- 1 activation and immunosuppression by Tregs. Finally, we present analyses of GARP-express-ing Tregs in human melanoma samples, which suggest that targeting GARP on human Tregs with blocking anti-bodies could represent an novel approach for the immunotherapy of cancer.

ponins enhanced DC cross-presentation by lipid body induction. Both pharmaceutical and genetic interference with lipid body formation inhibited the SBA-induced cross-presentation in these DCs in vitro and in vivo. How lipid bodies affect DC cross-presentation, type I IFN production and other immune processes are just beginning to be studied in any detail.

TheexerciseofTAMingtheimmunesystemPer thor Straten, PhD, Center for Cancer Immune Therapy (CCIT), Department of Hematology, University Hospital Herlev & Department of Immunology and Microbiology, University of Copenhagen, Denmark

Cells of the immune system, e.g., CD8 T cells and Natural killer (NK) cells are capable of recognizing and killing cancer cells. Naturally elicitedanti-cancer immune responses lead to infiltration of immune cells into tumors and the presence of these cells impact on disease progression, i.e., overall survival of the patient. Two main obstacles seem to limit the therapeutic impact of the immune system on cancer progression: limited infiltration of immune cells to tumors, and immune suppressive mechanisms in the tumor microenvironment (TME). Thus, ways to in-crease immune cell infiltration in tumorsand unleash suppressed immune cells in the TME hold great promise in therapy. We recently demonstrated that voluntary wheel running showed over 60% reduction in tumor incidence and growth across several murine tumor models. Immune cell infiltration including T and NK cells was signifi-cantly increased in tumors from exercising mice, and depletion of NK cells blunted the exercise-dependent tumor control. Moreover, NK cells were engaged through an epinephrine-dependent mobilization, and blockade of this pathway blunted the exercise-dependent tumor inhibition. Together these results link exercise with improved immunological control of tumor progression, suggesting that exercise could be a beneficial partner for immuno-therapy. Along those lines we are initiating a clinical trial where exercise is combined with immunotherapy.T cells are carefully regulated by signals from the environment, either soluble factors interacting with surface receptors on T cells or via receptor-ligand interactions e.g., directly allowing cancer cells to block or inhibit func-tionality of T cells in the tumor. We have characterized a novel co-stimulatory pathway in CD8 T cells. The stimu-latory signal goes via activation induced surface expression of both the receptor as well as the soluble ligand. Importantly, increased signaling via this pathway leads to increased cytokine release and proliferation, whereas blocking of the pathways by mono-clonal antibody or siRNA technology leads to diminished production of TNF-α and INF-γ , as well as and reduced proliferation. Using the xCelligence technology we could further demonstrate that blocking of the pathway in tumor specific T cells led to reduced capacity to control cancer cell growth. Inter-estingly, cancer cells as well as cells of the innate immune system, e.g., macrophages express the same receptor on the surface which implies that cancer cells may hijack the ligand and thereby convert a co-stimulatory signal for the T cells into oncogenic signaling in the cancer cells. We believe this pathway plays an important role in suppression of CD8 T cell responses in the tumor microenvironment, which could be subject to therapeutic in-tervention.

TheaminoacidtransporterxCTinbreastcancer(stem)cellsFederica Cavallo, Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Italy

To meet their biosynthetic and bioenergetic needsand to maintain appropriate redox balance, cancer cells require an adequate supply of small nutrients that must be transported across the cell membrane by specific proteins that may therefore represent potential therapeutic targets. Among them, xCT (SLC7A11), the functional subunit of the cystine-glutamate antiporter system xc-, which expression correlates with reduced patients’ survival.xCT exchanges extracellular cystine with intracellular glutamate. Inside the cell, cystine is reduced to cysteine to synthetize glutathione, which protects the cell against oxidative insults that can derive from microenvironmental stress and radio/chemotherapy. Exported glutamate potentiates oncogenic signallingand promotes invasionby acting on selective receptors expressed by cancer cells. Indeed, increased xCT expression correlates with breast cancer invasiveness, epithelial to mesenchymal transition and the cancer stem cell phenotype. Moreover, xCT is also expressed by the immune cells (Treg cells, myeloid derived suppressor cells and neutrophils) co-opted by the tumor for its survival and progression towards metastasis. Finally, xCTis involved in glutamate-induced anxiety,

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inhibitory receptors such as TIGIT, TIM3, LAG3 and PD1 may represent novel strategies to increase NK function in cancer patients. Altogether, the identification of NK cell and tumour cell markers of resistance or susceptibility to the action of NK cells will contribute to identify those patients that will most likely benefit from NK cell-based immunotherapy.

InterplaybetweencellularsenescenceandcancerDimitris Kletsas, Institute of Biosciences and Applications, NCSR “Demokritos”, Athens, Greece

Stromal fibroblasts, the most abundant cell type of tumor microenvironment, regulate local homeostasis by se-creting extracellular matrix molecules and soluble mediators. When exposed to a classical genotoxic anticancer treatment, i.e. ionizing radiation, they can become senescent in vitro, in a p53-dependent mode. In addition, radiation provokes the accumulation of prematurely senescent stomal fibroblasts also in vivo. These cells express an inflammatory/catabolic phenotype, called Senescence Associated Secretory Phenotype (SASP) and they en-hance significantly the growth of cancer cells in vitro and in vivo. This effect seems to be largely dependent on the increased secretion of matrix metalloproteases. Senescent cells have also an altered expression of proteogly-cans, whose expression in the stroma regulates cancer progression. They also overexpress syndecan-1 (SDC1), a poor prognostic factor in cancer development, via a TGF-β-mediated autocrine loop leading to SDC1 upregula-tion due to the activation of the Smad pathway. This overexpression is further enhanced by invasive cancer cells via the paracrine action of TGF-β. In addition, they down-regulate the expression of decorin, the latter having an anti-tumorigenic effect. Interestingly, the autologous extracellular matrix deposited by senescent fibroblasts can promote senescent-like changes in young fibroblasts, further enhancing tumorigenesis. All the above indicate a role of senescent stromal cells as crucial modulators of the tumor microenvironment.

DissectingT-cellresponsesinvaccinated,longtermsurvivingpatientsElse Marit Inderberg, Department of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway

Whereas cellular anti-tumour immune responses have typically been attributed to CD8 T cells, CD4 T cells play a critical role in tumour elimination and the priming and maintenance of CD8 T-cell responses. Furthermore, CD4 T cells activate innate cells such as macrophages and NK cells to contribute to anti-tumour responses. The use of MHC class II-restricted CD4 T cells for adoptive immunotherapy has been limited due to i) a lack of well-characterized shared tumour antigens presented by MHC class II ii) the majority of tumour cells being class II negative and therefore not directly presenting antigen to CD4 T cells.We explore several strategies to expand the possibilities to target different types of solid cancer by T-cell based therapy.We have isolated tumour-specific T cells from several patients who experienced clinical benefit from treatment with cancer vaccines targeting universal tumour antigens and frequent neoantigens. After profiling of selected T cell clones, mainly CD4 T cells, we isolated their TCRs and expressed them in donor T cells. Functional assays revealed that the effector cells were efficiently redirected both in vitro and in vivo. This provides us with a panel of TCRs covering a range of HLA alleles and antigens which can be used for TCR therapy. The first TCR has now been tested in patients for the treatment of metastatic colon cancer.Combining HLA class I- and class II-restricted TCRs for T-cell redirection may provide a more potent therapeutic effect in adoptive T cell therapy. HLA class II-restricted TCRs may additionally have direct therapeutic value both in haematopoietic malignancies and in melanoma where tumour cells frequently express HLA class II. Important-ly, CD4 T cells and HLA class II TCRs therefore have the potential to orchestrate broad and long-lasting immune responses that enable cancer control.

Theimmunesystemageslikeeverythingelse:whatdoesthismeanforcancerimmuno-therapy?Graham Pawelec, Department of Immunology, University of Tübingen Medical School, Tübingen, GermanyCancer Solutions Program, Health Sciences North Research Institute, Sudbury, ON, Canadagraham.pawelec@uni-tuebingen.de, grahampawelec@cantab.net, gpawelec@hsnri.ca

It is “common knowledge” that the human immune system deteriorates over time, and that this results in an in-creased frequency of and susceptibility to infectious disease, autoimmunity and cancer, and in poorer responses to vaccines. However, although there is a considerable weight of evidence supporting the idea that the elderly may deal poorly with novel infectious disease, evidence for the other claims is surprisingly weak. It is clear that the distribution of immune cell types (in peripheral blood) is skewed with age: more myeloid cells are present, fewer B cells and different proportions of naïve and memory T cells. However, disentangling the contributions of age per se from the result of a lifetime´s exposure to pathogens and other environmental challenges is no simple matter. This presentation will attempt to make some moves in this direction using longitudinal study data in human populations and considering the currently clinically highly relevant question of the impact of immune ageing on responses of cancer patients to immunomodulatory antibody therapy which is revolutionizing medi-cal oncology. In our own studies, in a recent series of 128 melanoma patients from 3 different centers, we have observed that benefit of anti-PD1 therapy, as assessed by OS, is influenced not by the age of the patient per se but by whether the patient was infected with Cytomegalovirus (CMV). While age itself had no significant impact (taking 70 years of age as the cutoff for stratification), patients who were CMV-infected tended to have superior OS (and the proportion of CMV-infected individuals in industrialized countries increases with age). This survival difference became statistically significant when patients were stratified according to relative percentages of natu-ral killer cells and CD8+ effector memory cells, together with their CMV serostatus. Thus, greater proportions of these cells in CMV-seropositive, but not CMV-seronegative, patients were associated with superior OS. This may be one mechanism by which cohorts of older melanoma patients seem to do better than younger patients on anti-PD-1 therapy.

NKcellimmunosenescenceandNKcell-basedimmunotherapyinAMLpatientsRafael Solana, Instituto Maimónides de InvestigaciónBiomédica (IMIBIC) - Reina Sofia University Hospital – University of Córdoba, Spain.

The incidence of some types of tumours has increased progressively in recent years and is expected to continue increasing in the coming years due in part to the aging of the population. Aging is associated with changes in the immune system involving both adaptive aswell as innate immunity. Immunosenescence refers to the deteriora-tion of the immune system function associated to aging. It has been proposed that the failure of tumorimmuno surveillance may be partly responsible for the age-associated increase in cancer incidence. Natural killer (NK) cells are innate lymphoid cells specialized in killing tumor cells as well as virus infected cells without the require-ment of prior sensitization. NK cells are also involved inregulating immune function as they produce several cytokines and chemokines. NK cell immunosenescence affects the frequency, phenotype, and subset distribution of human NK cells. A decreased expression of activating receptors is observed in the elderly that may contribute to the decline of NK cell function. Acute myeloid leukemia (AML) is a disease of older adults with a median age at diagnosis usually over 65 years old. NK cells in AML patients show alterations such as a reduced expression of several activating receptors that impair NK cell function and may result from the cell interaction between NK effectors and AML blasts. The levels of circulating microRNAs, potent post-transcriptional regulators of gene expression, are also altered by ageing or the presence of AML and they have been postulated as disease bio-markers in AML patients. NK cell-based immunotherapy emerges as a novel treatment for AML patients. The design of new therapies based on Natural Killer (NK) cells opens new possibilities especially for the treatment of elderly patients who are particularly susceptible to the toxicity of conventional chemotherapy treatments. The identification of key points (checkpoints) in the activation of NK cells that can be regulated by monoclonal antibodies has allowed the design of new therapeutic strategies based on NK cells. The first clinical trials block-ing KIR inhibitory receptors have shown little efficacy by inhibiting the maturation of NK cells. Blockade of other

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preventative interventions, measures or unique treatments to those groups of patients who will benefit, avoiding unnecessary costs and more importantly, secondary side-effects to those who will not respond or benefit from these treatments.Nowadays, with the use of next generation sequencing (NGS), it is possible to analyse unique multiple mutations that could be responsible for fatal conditions. Thus, in contrast with the PCR-SANGER technologies, where it is only possible to analyse one region with NGS, it is possible to analyse the whole genome, coding of the DNA regions (Exomer) and DNA regions of few genes of interest (Amplicon Panel). It also has enormous cost benefits, as for the same price per sample, NGS provides 12 times more information than that covered by SANGER technologies. The use of NGS has important diagnostic implications that range from genetic counselling for affected families, different options for treatment, early adoption of HSCT, cancer screening and avoidance of unnecessary treatments for affected patients. NGS impacts on the diagnosis in the WHO Classification, upon the diagnosis and the selection of specialised treatment. It may also impact on the course of the disease by the definition of response or referral, by the detection of minimal residual disease and to help to decide either to end or continue treatment.Indications for NGS in haematology, range from neoplastic conditions, such as acute myeloblastic leukaemia (AML), acute lymphoblastic leukaemia (ALL), MDS and multiple myeloma or non-malignant conditions, such as aplastic anaemias, hereditarycoagulations disorders, idiopathic cytopenias of uncertain significance, congenital haematological anaemias, and other conditions. A global review of the application of NGS inspecific haematologi-cal conditions will be discussed.

NewtreatmentsforleukaemiaJ Alejandro Madrigal, MD, PhD, FRCPath, FRCP, DScThe Anthony Nolan Research Institute, University College London, UK

Therapeutic manipulation of the immune system may overcome morbidity and mortality caused by a deficient capacity for adaptive immunity and by malignancies not eradicable by other therapeutic interventions, surgery radiotherapy or chemotherapy. Interventions to modulate immune reactivity may be valuable for treatment of autoimmune and inflammatory chronic disease and manoeuvres to provide adaptive immunity, active or adop-tive are of utmost importance to reduce the heavy burden of morbidity and mortality of patients and recipients. Different modalities of organs, tissues and cell transplants, and the efficiency of HSCT to treat haematological malignancies may be enhanced by interventions directed to provide adoptive or to generate endogenous anti-tumour immune reactivity. Thus, if cell therapy is considered as a use of cells as therapeutic agents, two large areas can be differentiated, one involving regeneration, substitution or replacement of functional cells (stem cell therapy) and the other, related to the use of immune cells to exploit their specific types of responses in the effector and suppressor directions (cellular immunotherapy). In the context of allogeneic HSCT this could lead to graft versus host disease (GvHD), graft versus leukaemia and graft versus infection. Another potential application outside of these areas is regenerative medicine in the context of degenerative diseases and/or autoimmunity. At the Anthony Nolan Research Institute, new strategies are being developed for optimising the use of allogeneic HSCT, based on the prediction and modulation of the immune response in order to reduce the risk of GvHD, generate selective anti-tumour immune responses and to provide protective immunity against opportunistic infections.Cord blood (CB) offers substantial logistical and clinical advantages over other sources of stem cells (SCs) such as (a) off-the-shelf access to the therapy with significantly faster availability; (b) higher frequency of less repre-sentative tissue types compared to bone marrow registries, since it is easier to target ethnic minorities; (c) lower risk of transmitting infections by latent viruses or inducing malignancies; (d) lack of donor attrition; (e) lack of risk to the donor; and finally, (f) an unlimited source of material for stem cell research.Large CB bank initiatives offer a unique opportunity to make available for research a high number of ethically collected, clinical grade CB units that are not suitable for clinical use when evaluated at the processing centre. Indeed, CB usage is increasing exponentially and a number of researchers have shown a wide range of therapeu-

AdoptiveT-celltherapyinacademia–OsloexperiencesG. Kvalheim (NO), MD, PhD, Professor emeritus Gunnar Ikke sensitivt, Οslo, Norway

Department of Cellular Therapy at Oslo University Hospital is one of the largest academic laboratories for cel-lular therapy in Europe. The facility has 42 employees involved in different clinical programs on hematopoietic and mesenchymal stem cells, pancreatic islets transplantation, therapeutic dendritic cell vaccines and adoptive T-cell therapy. Our translational research activity is currently focused on developing novel adoptive T-cell therapy against hematopoietic malignancies and solid tumors. Oslo University Hospital was elected to become one of the 7 centers in Europe to participate in the worldwide study by Norvartis on the CD 19 CAR T-cell therapy on pediatric ALL and adult high gradeNon-Hodgkin’s Lymphomas. The translational knowhow from Norvartis and the clinical experiences gained treating expected severe side effects after the CAR CD19 T-cells has been of great value for our hospital building up the infrastructure for future adoptive T-cell therapy programs. In the laboratory we have also learned how to optimize the cell collection and processing of the patient’s cells to obtain the best T-cell product for production of CAR CD19 T-cells. Our department have developed several different novel TCRs and CARs that have undergone preclinical testing and that remains to be tested in the clinic. Department of Cellular Therapy has developed clinical platforms for large scale T-cell production and also have available a GMP validated production of mRNA CARs and TCRs. In contrast to many we others we strongly believe that electroporation of mRNA CARS and TCRs administered in repeating dose escalations provide important clinical information about side effects and clinical efficacy. Fol-lowing these result the CARs and TCRs can be stabled expressed with virus transduction. The GMP guidelines for cell-based Advances Therapeutic Medicinal Product (ATMP) set the rules for how academia can make their own studies on Adoptive T-cell therapy. This will be discussed in details.

EpitopeSpreadingtoEnhancetheEffectivenessofTCRTransducedTCellM. Nishimura, Research, Department of Surgery, Stritch School of Medicine Loyola University Chicago, USA

The use of T cell receptor (TCR) and chimeric antigen receptor (CAR) gene modified T cells for adoptive transfer to cancer patients have had led to objective clinical responses. However, there are still several hurdles that must be overcome before gene modified T cells can benefit most cancer patients. These hurdles include but are not limited to severe adverse events due to off target killing of normal cells and relapse due to antigen loss/escape. We have addresses this problem by utilizing a local T cell delivery approach. Duval et al (2006) reported that intratumoral injection of a MART-1 TCR transducedallogeneic T cell line into tumor lesions. They found that many patients had local regression and in a few cases, untreated lesions had also regressed. The interpretation was the local tumor destruction induced cross priming and epitope spreading. The work presented in this conference is designed to improve the efficacy of this approach using the well characterized B16 animal melanoma model. Local delivery of TIL 1383I TCR transduced allogeneic T cells can mediate local regression of treated tumors and elicit systemic anti-B16 immunity as measured by ELISPOT in vitro and protection against subsequent B16 tumor challenges. The effects can be enhanced using checkpoint blockade. These findings open a new avenue for using genetically engineered T cells, even when the receptor used to engineer them would be inappropriate for use systemically.

NEXTGENERATIONSEQUENCING(NGS)–PERSONALIZEDMEDICINEINHAEMATOLOGYJ Alejandro Madrigal, MD, PhD, FRCPath, FRCP, DScThe Anthony Nolan Research Institute, University College London, UK

Personalised medicine should not be taken literally to mean ‘the generation of new medications or unique medi-cal technologies to treat each patient’. It refers to the capacity to treat and classify patients in different sub-populations according to their susceptibility to aquire, develop or process a particular disease, and also to un-derstanding disease prognostics and the unique response to specific treatments. Thus, it is possible to apply

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Biomarker-guidedriskstratificationinchroniclymphocyticleukemiaKostas Stamatopoulos, MD, Institute of Applied Biosciences, CERTH, Thessaloniki, Greece

Coordinator, Hellenic Precision Medicine Network in Oncology

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells, the most frequent adult leukemia amongst Caucasians. CLL is a prototype for cancers in which tumor initiation and progression are linked to the interplay between cell-intrinsic and cell-extrinsic mechanisms. The former include more general cancer-associated mecha-nisms e.g. activation of oncogenes and/or elimination of tumor suppressor genes. The latter relate to the cross-talk of CLL cells with the surrounding microenvironment through various receptors, including the B-cell receptor (BcR), which mediate signal transmission from the external milieu to the cell interior. The clinical course of CLL is highly variable, highlighting the need for recognizing disease subgroups with dis-tinct outcome. Traditionally, risk stratification in CLL is based on the Rai and Binet clinical staging systems; both systems have proved useful for estimating outcome and continue to be routinely used in disease evaluation. However, both have a limited ability to predict the clinical course at diagnosis for patients at an early clinical stage of the disease. Since most patients (~80%) are diagnosed at early clinical stages, clinical staging cannot dis-criminate patients with markedly different patterns of clinical evolution and outcome. Consequently, much effort has been made towards the identification of new prognostic factors which would better enable the stratification of patients to different treatment options. Many biomarkers have been identified from older, low-throughput and more recent, high-throughput studies and found to carry an independent prognostic value. However, issues exist regarding (i) the discordance between markers; (ii) their standardization; and, especially, (iii) their validation in the context of well-controlled, prospec-tive clinical trials. Thus, despite the plethora of potential biomarkers, presently IGHV gene somatic hypermutation (SHM) status and TP53 gene aberrations represent the only markers associated with therapy response thus far prospectively validated and considered essential for guiding treatment choices.

ImmunecheckpointinhibitorsforhematologicaldiseasesG. Vassilopoulos (GR), Haematology Laboratory, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, Greece

The amplification of the p2.4 region on chromosome 9 was a well documented finding in classical Hodgkin’s lymphoma (cHL). The region was home to two important (apparently) molecules in cHL, JAK2 and PDL1/L2. The latter, programmed death 1 ligands, engage the PD1 receptor on T cells and control their level of activation. The PD1 receptor was cloned in an apoptosis screen of T-cells and plays a role in immune tolerance; engaged to its ligands, it can set brakes on T-cell activation. Now if we imagine that a tumor with a truck load of neo-antigens and an immunogenic phenotype, needs a way to dampen the host’s immune response, the PD-1 path would be a legitimate tool. In the case of hematological neoplasms, cHL is a winner in overexpressing PD1 ligands; this is augmented by activation of the JAK2 path and of the EBV proteins in the RS cells. As a result, the neoplastic RS cells survive and proliferate in an inflammatory tumor environment where the abundant T-cells fail to eradicate the malignant cells. In 2015, the registration trial for an anti-PD1 antibody was published; overall, in R/R cHL patients who have failed ASCT and antiCD30 treatment, one would expect an ORR of 75%, a CR of 20% and a median duration of response lasting 16 months. Based on these data, antiPD1 antibodies were approved “as monotherapy for the treatment of adult patients with relapsed orrefractory classical Hodgkin lymphoma (cHL) who have failed autologous stem cell transplant (ASCT) and brentuximab vedotin (BV), or who are transplant-ineligible and have failed BV”. In other types of lymphomas, antiPD1 antibodies have not been as successful. In R/R DLBCL one would expect short-living responses in the range of 20%; the treatment is more successful in primary CNS lym-phomas, in PMBCL and in Richter transformation of CLL. However, non of these data have resulted in expansion of their clinical indications. Another area where antiPD1 treatment has attracted attention is MDS; although not an immunogenic neoplasm per se, MDS are often caused by mutations in splicesome genes that eventually end up in producing aberrant RNAs and novel proteins that may evoke immune responses. The reported trials were not conceived under this rationale but on the fact that CD34+ cells of MDS patients had higher levels of PDL1

tic applications for these cells. There is now a real opportunity to use CB for non-haematopoietic transplants as well since non-haematopoietic SCs have been isolated from CB and the placenta. These cells can be grown and differentiated in various tissues including mesenchymal SCs, bone, cartilage and other lineages. They have an advantage over other sources of SCs, embryonic or induced pluripotent, because the supply is unlimited, they can be used in autologous or allogeneic situations. They need minimal manipulation and they raise no ethical concerns. Future studies will, in the near future, test the potential of CB cells for the treatment of several diseases including diabetes, arthritis, burns, neurological disorders and myocardial infarction. Recently, our group has focussed on CB, trying to define cell subpopulations useful for therapy in the context of a well-defined product, but also with minimally manipulated procedures. This could make possible the genera-tion of ‘off-the-shelf’ products to be tested clinically for application in regenerative medicine (using CBSCs after CD133+ selection) and tolerance (using natural naïve T regulatory cells after CD25+ selection). Obviously, this approach will require understanding and control of the potential immunoreactions produced by infusing alloge-neic cells from one or multiple donors to a particular patient. Altogether, knowledge acquired by basic research studies on SCs and immunomodulatory cells contained in CB, would help with the translational challenges before application of CB cells in adoptive cell therapy.Data will be presented on the identification and the isolation of CB SCs, regulatory T cells (Tregs) and NK cells with the objectives of targeting GVL, GVI, and immunomodulation in HSCT. In addition, the use of CAR-T cells for the treatment of haematological malignancies will also be covered during the presentation.

ImmunotherapeutictargetsforthetreatmentofadultB-cellacutelymphocyticleukaemiaLeah Cookseya, Stephanie Jordaensab, Stephanie Bonneyc, Laurence Orchardc, Viggo van Tendeloob, Ken Millse, Kim Orchardd, Barbara-annGuinna*

a Department of Biomedical Sciences, University of Hull, Hull, b Center for Oncological Research, University of Antwerp, Belgium c Cancer Sciences Unit, d Department of Haematology, Southampton University Hospitals Trust, Tremona Road, Southampton, England, d Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Northern Ireland.* B.Guinn@hull.ac.uk

BackgroundAcute lymphoblastic leukaemia (ALL) is a form of leukaemia characterized by excess lymphoblasts in the bone marrow. The most effective treatment to date is allogeneic stem cell transplant which can improve overall survival rates in part due to a ‘graft-versus-leukaemia’ effect. However few of the cancer antigens have been identified in adult B-ALL which can act as targets for immunotherapy.MethodsAntibody specific profiling was performed on sera samples from nine adult B-ALL patients and nine age and sex-matched healthy donor controls. Signals from 9,000 peptides were analysed on the ProScanArray using ProtoArray® Prospector v5.2 software. The mean value and standard deviation of each signal was calculated to produce a z-score and the six most promising antigens identified, ANOVA analysis was performed to determine the significance of the results. Results and ConclusionsAnalysis of the proto-array data showed clustering of frequently recognized antigens in patients compared to age and sex-mated healthy donor sera. Several promising targets were identified and were examined using litera-ture based research to determine the known functions of these proteins as well as their known roles in cancer. BMX, DCTPP1, GAK, MUC20, VGLL4 and WARS were chosen for further investigation based on their significant increase in detection in adult B-ALL patients compared with healthy donors and results from literature research. qPCR is being used to validate the expression of these protein in adult B-ALL; patient samples, cell lines and healthy volunteer samples are being used to determine if there is significant overexpression of these proteins, using 18S, GAPDH and TBP as reference genes. Despite the genetic heterogeneity associated with adult B-ALL, in terms of the proteins recognized by antibody responses adult B-ALL patients showed a surprisingly high level of similarity and we can differentiate patients and healthy donors, even at disease presentation, on the basis of sero-recognition of antigens.

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clinical relevance of miR-200a expression, since its overexpression was correlated with a reduced patients’ survival. Furthermore, miR-21 has been also identified as a negative regulator of HLA class I surface expression. In contrast, overexpression of miR-9 leads to upregulation of TAP1, tpn and β2-m in tumor cells. These data suggest that there exist immune modulatory miRNAs of HLA class I APM components, which negativelyand positively control their expression and are associated with the patients’ prognosis. For a more comprehensive understanding of miRNA-mediated regulation of HLA-G, the interaction of miRNAs with thecoding sequence (CDS) and the 3’UTR of HLA-G was analyzed. Using an RNA affinity approach in combination with small RNA sequencing, candidate miRNAs binding to the CDS and 3’UTR of HLA-G were identified. Transfection of candidate miRNAs revealed a downregulation of HLA-G protein levels. Luciferase reporter assays using the wild type and mutated version of HLA-G CDS and 3’ UTR, respectively, confirmed a direct interaction of these miRNAs-with HLA-G. In contrast, overexpression of some candidate miRNAs sharing the same seed sequence resulted in an up-regulation of HLA-G, while miRNA inhibitors showed opposing effects on HLA-G surface expression.Due to the high sequence similarity between HLA-G and classical MHC class I molecules, the impact of HLA-G regulating candidate miRNAs on HLA-ABC was determined demonstrating comparable outcomes for HLA-A surface expres-sion after miRNA transfection in HEK293T cells. In silico analysis using TCGA data sets for miRNA and MHC class I expression further validated the results, which were most pronounced in breast cancer and colon adenocarcinoma. Thus, a coordinated regulation of both non-classical and classical HLA class I antigens could be mediated by miR-NAs. Similar results were also obtained by identifying RBPs regulating HLA class I molecules using the similar approach. The heterogeneous nuclear ribonucleoprotein R (HNRNPR) binds HLA class I mRNAs in their 3’ UTR thereby enhancing their stability, consequently their expression and modulates the cytotoxic activity of NK cells. Thus HNRNPR, acts as a general positive regulator of classical and non-classical MHC class I antigens. In sum, there exist miRNAs and RBPs regulating the expression of diverse immune modulatory molecules, which have clinical relevance and might be used in the future as prognostic markers or even as novel therapeutic targets.

Survivalof thefittest:Howdomyeloid-derivedsuppressorcells (MDSC)survive in theinhospitabletumormicroenvironment?Suzanne Ostrand-Rosenberg1,2, Katherine Parker1, Daniel Beury1, and Lucas Horn1

1Dept. of Biology, University of Maryland Baltimore County; 2Dept of Pathology & Huntsman Cancer Institute, University of Utah

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and because of their potent im-mune suppressive activity, are significant contributors to the immune suppressive tumor microenvironment (TME) that facilitates tumor progression. For many solid tumors the TME is a hostile location for immune cells due to deficiencies in oxygen (hypoxia) and multiple factors produced by tumor cells and other cells of the stroma. The survival of tumor cells within this inhospitable milieu is partially governed by two mechanisms: (i) Expres-sion of the transcription factor Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) which turns on a collection of genes that attenuates oxidative stress; and (ii) The presence of High Mobility Group Box Protein-1 (HMGB1), a damage-associated molecular pattern molecule (DAMP) that induces tumor cell autophagy and protects against apoptosis. Because Nrf2 and HMGB1 promote tumor cell survival and MDSC produce high levels of reactive oxy-gen species (ROS), we have speculated that Nrf2 and HMGB1 may also facilitate MDSC survival within the TME. To test this hypothesis, we used Nrf2+/+ and Nrf2-/- BALB/c and C57BL/6 mice and pharmacological inhibitors of HMGB1. In vitro and in vivo studies demonstrated that Nrf2 increased the immune suppressive potency and the quantity of tumor-infiltrating MDSC by up-regulating their production of hydrogen peroxide and decreasing their rate of apoptosis. The decrease in apoptosis was accompanied by a decrease in the production of MDSC in the bone marrow demonstrating that MDSC levels are tightly controlled through a homeostatic mechanism. Pharmacological inhibition of autophagy increased the quantity of apoptotic MDSC indicating that autophagy increases MDSC half life. Inhibition of HMGB1 also increased the numbers of apoptotic MDSC and reduced MDSC autophagy. These results combined with our previous findings that HMGB1 drives the accumulation of MDSC, demonstrate that HMGB1 maintains MDSC viability by forcing them into an autophagic state. Collectively, these findings identify Nrf2 and HMGB1 as important factors that enable MDSC to survive in the TME.

and also on the lack of therapeutic tools for this particular disease. Results were disappointing when antiPD1 treatment was used as monotherapy but was rather significant when combined with hypomethylating agents (HMA) such 5-AZA or decitabine; HMAs seem to upregulate endogenous retroviruses in the malignant cells and lay the ground for the antiPD1 antibodies. Finally, there is a down side in the PD1/PDL saga; it has recently been reported that antiPD1 treatment both before or after allo-HSCT for cHL, can lead to a significantly increased risk of GVHD-related morbidity andmortality in this patient population. Overall, anti PD1 antibodies, have a place in the treatment of cHL but in the era of precision medicine, their exact target should be sought beyond the pres-ence of PDL1 on the tumor cell.

Cancerimmuneescape:MHCexpressioninprimarytumors“versus”metastaticlesionFederico Garrido, Dept Analisis Clinicos, Hospital Universitario Virgen de las Nieves; Dept. Bioquímica, Biología Molecular e Inmunologia, Facul-tad de Medicina, Universidad de Granada, 18014 Granada, Spain

Our laboratory has been HLA-typing tumour tissues for a long time and found that HLA class I loss is a frequent phenomenon. Primary tumours are HLA-I positive at early stages. T lymphocytes infiltrate tumour tissues, recog-nize and destroy HLA class I positive cancer cells (permissive phase I). This phase end with the total destruction of the tumour. Alternatively, HLA-I negative tumour cell variants can emerge. At this phase, tumours are het-erogeneous for HLA-I expression. HLA class-I loss variants are immunoselected “in vivo” by antitumor CTLs and escape T cell recognition and destruction during the natural history of tumor development. At the end, tumours are uniformly HLA class-I negative with T lymphocytes and other mononuclear cells surrounding the tumour nest (encapsulated phase II) (1). The transition from phase I to phase II will probably last for a short period of time. Metastatic lesions can have the same or different HLA-I phenotype as the original tumour harboring reversible (“soft”) or irreversible (“hard”) HLA class I defects. Immunotherapy is effective in eliminating HLA-I positive tumor cells and HLA deficient tumor cells with reversible/”soft” molecular lesions. I will discuss these different possibili-ties in the new era of immune checkpoint antibodies.

RoleofmicroRNAandRNA-bindingproteinsfortheimmuneescapeoftumorsMichael Friedrich, Marifili Lazaridou, Karthik Subbarayan, Anja Mueller, Diana Handke, Katharina Biehl, Ofer Mandelboim, Christine Stoehr, Arndt Hartmann, Simo-Jasinksi-Bergner, Eva-Maria Gonschorek, Barbara SeligerMartin Luther University Halle-Wittenberg, Institute of medical Immunology, Halle, Germany, Haddassah University, Jerusalem, Israel, University of Erlangen, Institute of Pathology, Erlangen, Germany

An escape from the immune surveillance frequently occurs in cancer patients and can be associated not only with a bad prognosis of patients with an impaired response, but also to a limited response to T cell-based immunothera-pies. The downregulation of HLA class I (HLA-I) antigens due to either dysregulated expression and/or function of components of the antigen processing and presentation machinery (APM) or upregulation of non-classical HLA class I antigens and co-inhibitory molecules, such as e.g. PD-L1 are tightly associated with tumor immune evasion. The molecular mechanisms of the aberrant expression of these immune modulatory molecules are diverse and at least partially mediated by post-transcriptional regulation, which substantially control gene expression. In this con-text, microRNAs (miRNAs) and/ or RNA-binding proteins (RBPs) are of key importance and their deregulation were linked with the development and progression of various cancer types. So far, the interaction of miRNAs and RBPs with HLA class I moleculesis only poorly investigated. Based on a dis-cordant mRNA and protein expression of HLA class I APM components, miRNAs and RBPs targeting specific HLA class I APM components were identified using the different experimental approaches. These include miR200a-5p, miR-21 and miR-9. MiR-200a-5p,which is in silico predicted to bind to the transporter associated with antigen pro-cessing 1 (TAP1) and has recently been associated with different cancers, is able to affect TAP1 mRNA and protein levels by binding to its 3’ UTR. This was accompanied by a downregulation of HLA class I surface expression and an increase of NK cell recognition as determined by a CD107 degranulation assay. In silico data demonstrated a

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P a r t i c i p a n t s

MOLECULARMECHANISMSOFBROMAMINET(BAT)INVARIOUSCANCERSStella Baliou1**, Markus Nagl2, Antony M. Kyriakopoulos3 and Vassilis Zoumpourlis1*.1. Biomedical Applications Unit, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece2. Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstr. 41, 6020 Innsbruck, Austria3. NASCO AD Biotechnology Laboratory, 11 Sachtouri str., 18536, Pireas, Greece.*Corresponding author: e-mail: vzub@eie.gr

Introduction: N-Bromotaurine is a physiological molecule which arises from the neutrophil -myeloperoxidase halide system of metabolism during inflammation and constitutes the reaction product of Taurine (Tau) with hypobromous acid (HOBr). N-Bromotaurine is a potent oxidant molecule, which exerts a complex anti-inflamma-tory action. However, due to the poor stability of the N-Bromotaurine molecule, an analogue molecule, named Βromamine T (BAT), was subsequently used. Methods: Cell proliferation experiments, colony formation assays, staining with CM-H2DCFDA dye, flow cytometry, Real-time PCR and western blot experiments highlighted the potential anti-cancer and anti-inflammatory effect of molecules. Α xenograft assay and an air-pouch model of inflammation induced by LPS constituted the in vivo experiments, delineating the functional significance ofmol-ecules. Results/Discussion: The tumor-suppressive effect of N-Bromotaurine was observed in various cancer cell types, including skin cancer. In the case of skin cancer, we showed that N-Bromotaurine can bypass the glucocorticoid receptor (GR)-resistance of cancer cells when used in combination with cis-platin. Cancer cell pro-liferation was suppressed with the use of BAT in breast, cervical, and colorectal cancer, while the anti-proliferative effect of BAT appeared to be superior to that elicited by taurine. In addition, flow cytometry and western blot experiments highlighted the intrinsic apoptotic pathway used by cancer cells following exposure to BAT or Tau. Additional experiments proved that BAT triggered oxidative burst to stimulate apoptosis. Following treatment of cancer cells with BAT, the phosphorylation of two main arms of the MAPK family (JNK1/2 and p38) were stimulated. This implied that BAT induced ROS accumulation, which triggered the phosphorylation of stress-related MAPK kinases, via eliciting pro-apoptotic signals in cancer cells. In parallel, we indicated that BAT exerted anti-inflammatory properties by suppressing mRNA expression levels of cytokines in LPS-induced macrophages and in LPS-induced mice with air pouch formation. More importantly, xenograft experiments showed that tumor formation was impaired after treatment of SCID mice with BAT or Tau. Our study has produced results that dem-onstrate that BAT is an emerging anti-proliferative agent with favorable efficacy.

INVESTIGATINGTHEROLEOFTGF-βINREGULATORYANDCONVENTIONALTCELLSUR-VIVALDURINGCONTINUOUSSTIMULATIONChristina Cunha1, Mariko Takami1,2,3, Makio Iwashima1,2

1 Department of Microbiology and Immunology, Loyola University, Chicago, IL, USA2 Van Kampen Cardio Pulmonary Research Laboratory, Loyola University, Chicago, IL, USA3 Department of Medical Immunology, Chiba University, Japan

BackgroundActivation induced cell death (AICD) occurs after expansion of antigen-stimulated T cells to reduce the number of these T cells and maintain immunological homeostasis. AICD of tumor-specific T cells can occur after continu-ous stimulation, and has been shown to prevent successful anti-tumor responses. Tumor-specific effector T cell responses can also be suppressed by Regulatory T cells (Tregs) in the tumor-microenvironment. A majority of Tregs are reactive to self-antigens and are constantly exposed to antigenic stimulation.Mechanisms controlling the balance between Tregs and conventional T cells during continuous stimulation remain largely unknown. We previously reported a form of activation-induced cell death, which is dependent on p53 (p53-induced CD28-dependent T-cell apoptosis, PICA). Under conditions that induce PICA, Tregs expand robustly while conventional

DymanicinteractionswithinthetumourmicroenvironmentPeriklis G. Foukas, 2nd Department of Pathology, National and Kapodistrian University of Athens, Attikon University Hospital, Athens, Greece

Cancer is a genetic disease and the intrinsic genomic instability facilitates the escape from cytotoxic or targeted therapies (1). Tumour tissues are complex structures where malignant cells interact with a variety of different non-tu-mour cell types that include cells from the innate and adaptive immune system, blood and lymphatic vessels, nerves, stromal cells such as fibroblasts and myofibroblasts. The mechanisms that regulate anti-tumour B and T cell responses are nicely depicted in cancer-immunity cycle (2). The release of tumour antigens constitutes a central part of the cycle. Antigens are captured by dendritic cells and transferred through afferent kymphatic vessels to lymph nodes for the priming and activation of peripheral immune cells which traffic back and infiltrate the tumour bed mediating tumour cell death upon recognition of tumour antigens. In nowadays many high-throughput platforms have been standardized and validated in order to characterize in depth the different steps of the cancer-immunity cycle and the complex interactions between evolving tumours and host im-mune responses (3). Genomic and transcriptomic analyses are now part of the routine clinical practice whereas pro-teomic and metabolomic approaches are currently considered rather experimental. We can use these different plat-forms in order to determine whether a tumour belongs to one of the 3 main cancer-immune phenotypes (4): Inflamed tumours with infiltration and expansion of CD8+ T cells, production of chemokines and IFN signalling that induces tolerance through the expression of PD-L1 and IDO; Immuno-excluded tumours with many CD8+ cells in the tumour stroma but with rare cells infiltrating tumour islands; Immuno-desert tumours that are essentially lymphopenic. The molecular mechanisms governing T cell engraftment in tumours are only partly understood. Not only the tumour vasculature, both blood and lymphatic neoangiogenesis, but also the constitutive and inducible level of expression of chemokines in the tumour microenvironment plays a key role in T-cell homing (5, 6, 7). Importantly, the anti-tumour immune barrier induces adaptive immune resistance mechanisms (8, 9) but also shapes the cancer genome evolu-tion (10), emphasizing not only the close interaction between the immune and genomic landscape but also the mul-tiple parameters that are needed to be taken into consideration for effective anti-tumour therapies.

AssessmentofAntibodyFunctionalAffinityusingImmunSpot®Greg A. Kirchenbaum, Fridolin Franke and Paul V. LehmannCellular Technology Ltd., Shaker Heights, OH, 44122 USA

Background: Studies of B cell immunity often relay upon serologic methodologies that primarily assess antibody specificity. However, functional affinity of the antigen-specific antibody repertoire is a key determinant of protective efficacy. Moreover, conventional assessments of serum antibody occur at the population level and may not accurately capture the spectrum of functional affinities. At present, detailed assessment of single B cell/antibody functional af-finity is labor-intensive and low-throughput. Therefore, we sought to evaluate the hypothesis that B cell Fluorospot would enable distinction between B cells with differential functional affinity since fluorescent intensity is directly pro-portional to antibody abundancein this assay. Furthermore, since antibodies with increased functional affinity for the antigen should be efficiently captured in close proximity to the secreting cell, whereas low-affinity antibody would be more prone to diffusion, the Fluorospot morphology is also anticipated to reflect upon differential functional affinity.Methods: To test our hypothesis, murine B cell hybridomas (P65C6-2, 36-65 and 36-71) secreting monoclonal antibodies (mAb) with differential affinity for the p-azophenylarsonate (Ars) hapten were utilized as model antibody-secreting cells (ASC) and resulting Fluorospots evaluated using the Fluoro-X™ suite.Results:Differential functional affinity of Ars-specific ASC resulted in highly reproducible Fluorospot morphology. Ad-ditionally, using an anti-idiotypic mAb (17-63) specific for the high-affinity anti-Ars mAb (36-71), unambiguous seg-regation of Ars-specific ASC was confirmed. Collectively, our data support the notion that fluorescent ImmunoSpot® assays can be used to assess the functional affinity of antigen-specific B cells, and that this platform is well-suited for detailed assessment of humoral immunity.Conclusions: Collectively, our data support the notion that fluorescent ImmunoSpot® assays can be used to as-sess the functional affinity of antigen-specific B cells, and that this platform is well-suited for detailed assessment of humoral immunit

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IMMUNOPROFILINGOFPSEUDOMYXOMAPERITONEIHedvig Vidarsdotter Juul1, Christin Lund-Andersen2, Annette Torgunrud Kristensen2, Torveig Weum Abrahamsen2, Kjersti Flatmark2, Else Marit Inderberg1

1Department of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway, 2Department of Tumor Biology, Institute of Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway

Pseudomyxomaperitonei (PMP) is a rare form of cancer which is caused by proliferation of mucin-producing tumor cells and is marked by accumulation of mucinous tumor tissue within the abdominal cavity. Large amounts of tumour in the abdomen results in distension and discomfort (often called “jelly belly”), and ultimately causes organ failure through compression if not treated. The majority of cases can be successfully treated by surgery and intraperitoneal chemotherapy. However, for the 30% of the patients for whom the standard treatment fails, no efficacious treatment exists and the prognosis is extremely poor.Very little is known about immune cell infiltration and thetumor microenvironment (TME) in these tumours. We therefore collected tumour biopsies from patients with PMP and from colon cancer patients with peritoneal me-tastases for comparison and performed immunoprofiling by mass cytometry (CyTOF) to investigate whether immunotherapy could be a treatment alternative for these patients.Single cell suspensions were analysed by high-dimensional single-cell mass cytometry (CyTOF) applying an an-tibody panel consisting of 37 markers for lymphocytes and tumour cells using the viSNE analysis tool to cluster the data from our samples into separate populations. Samples from eight non-PMP and four PMP patients were successfully analysed. Five out of eight non-PMP sam-ples had clearly distinguishable lymphocyte populations, while the remaining 3 samples consisted mainly of tumor cells. In the PMP samples 2/4 had clear lymphocyte populations, while the remaining two had infiltrating T-cell populations. The expression of memory and activation/exhaustion markers could be identified on CD4+ Tcells and CD8+ T cells, in particular PD-1 and TIGIT.Our results indicate that even in mucinous cancers like PMP, there are infiltrating populations of antigen-experi-enced T cells. These ongoing analyses are linked to testing of patient immune responses against tumourneoan-tigens found in PMP for the development of immunotherapy strategies.

CHEMICALINHIBITIONOF ENDOPLASMIC RETICULUMAMINOPEPTIDASE 1 AS ATOOL FORREGULATING THEIMMUNOPEPTIDOME OFCANCER CELLSDespoina Koumantou1, Eilon Barnea2, Adrian Martin-Esteban3, Zachary Maben4, Athanasios Papakyri-akou1, Paraskevi Kokkala5, Harris Pratsinis1, Dimitris Georgiadis5, Lawrence J. Stern4, Arie Admon2 and Efstratios Stratikos1,*1National Center for Scientific Research Demokritos, AgiaParaskevi, Athens 15310, Greece2Faculty of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel3Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas and Universidad Autonoma),Madrid,Spain4Department of Pathology, University of Massachusetts Medical School, Worcester01655, Massachusetts. 5Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Athens15771, Greece

Background: The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, is often limited by ineffective presentation of antigenic peptides that can elicit T-cell mediated anti-tumor cyto-toxic responses. Therefore, manipulating antigen presentation is an emerging approach for enhancing the im-munogenicity of tumors in immunotherapy settings. Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides that can bind onto Major Histocompatibility Complex class I molecules (MHC-I). We hypothesized that pharmacologically inhibiting ERAP1 in cells can regulate the global cellular im-munopeptidome.Methods: we treated the A375 melanoma cell line with a recently developed potent ERAP1 inhibitor and ana-lyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting the bound peptides and identifying them using capillary chromatography and tandem mass spectrometry.Results: Although the inhibitor did not negatively affect overall MHC-I presence on the cell surface, it induced

T cells undergo apoptosis. Resistance of Tregsto PICA is dependent on TGF-β signaling. We hypothesized that there are TCR-associated signaling differences between Tregs and conventional T cells that allow for Tregs to resist PICA.MethodsSplenic conventional T cells and tTregs were stimulated with plate-bound antibodies in the presence or absence of TGF-β, or a pharmacological TGF-β type I receptor kinase inhibitor. Expression of TCR-associated signaling proteins were examined by Western Blot. Cell survival was assessed by tryphan blue exclusion along with 7AAD and Annexin V analysis using flow cytometry. Results and ConclusionsHere, we report that the expression of RasGRP1 (Rasguanyl-releasing protein) is required for PICA, as conven-tional T cells isolated from RasGRP1-deficient mice become resistant to PICA. After continuous stimulation,Tregs express a substantially lower amount of RasGRP1 compared to conventional T cells. This reduced expression of RasGRP1 is dependent on TGF-β, as addition of TGF-β to conventional T cells reduces RasGRP1 expression. Conversely, RasGRP1 expression in Tregs increases when TGF-β signaling is inhibited. Together, these data show that RasGRP1 expression is repressed in Tregs by TGF-β signaling and suggests that reduced RasGRP1 expres-sion is critical for Tregs to resist apoptosis caused by continuous antigen exposure. Because tumor-specific T cell survival is so critical for anti-tumor responses, low RasGRP1 expression may be critical for conventional T cell survival in the tumor microenvironment. Additionally, Tregs may utilize TGF-β to maintain low RasGRP1 expres-sion, which could enable survival and prevent anti-tumor immunity.

CANCERCYTOTHERAPYVIAUSEOFUMBILICALCORD-DERIVEDMESENCHYMALSTEMCELLS Ioannis Christodoulou1, Maria Goulielmaki1 and Vassilis Zoumpourlis1*1 Institute of Biology, Medicinal Chemistry & Biotechnology (IBMCB), National Hellenic Research Foundation (NHRF), 48 VasileosKonstantinou Ave., 11635 Athens, Greece

* Corresponding author: e-mail: vzub@eie.gr

Background: Mesenchymal stem cells (MSC) encompass a heterogeneous population of cells with unique prop-erties, such as high proliferation rates and capability of differentiation intomultiple non-hematopoietic cell types, that can be isolated from both adult and fetal tissues. An additional, unique feature of MSC is their ability to mi-grate towards tumors within the body and to interact with the local supportive microenvironment, which rapidly gave rise to the so-called MSC-based cancer cytotherapy. Towards this purpose, both naïve (unmodified) and genetically modified MSC (GM-MSC) have been employed both in vitro and in vivo, thoughwith variable results.Methods: Based on the up to now published research on the field, we performed a small-scale meta-analysis using afour-step strategy: (1) compilation of a relevant publication library, (2) deconstruction of literature meth-odology and reported findings, (3) classification and organization of extracted experimental data, and (4) data consolidation and statistical analysis. According to the observations and conclusions, we evaluated the paracrine effects of various MSC populations on the proliferation and survival of four selected cancer cell lines in vitro. Sub-sequently, we examined the transcriptome of two cell lines by RNA microarrays in order to exploit the expression pathways and regulatory networks contributing to the observed anti-cancer activity. Results and Conclusions: Interpretation of the meta-analysis results led as to the deduction that the outcome of MSC mediated cancer cytotherapy approaches is largely dependent on various parameters. Furthermore, we have been able to highlight a set of optimal conditions, in which the tumor suppressive action of MSC predomi-nates. mRNA analysis of cancer cells revealed a significant target dependence of the anti-tumorigenic effects displayed by MSC, which are mediated by different pathways (metabolic, cell cycle and apoptosis regulation, in-nate immune response) and are initiated by the same MSC secretome-derived stimuli. Τhe research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 2400).

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IntegratingDNAdamageandimmuneresponseinpancreaticductaladenocarcinomaAlexandros Papalampros1*, MichalisVailas1*, Natasha Hasemaki1*, Niki V. Chouliari2, Menelaos G. Sa-maras2, Evangelos Felekouras1,Vassilis G. Gorgoulias2,3,4, Ioannis S. Pateras2

1.First Department of Surgery, Laikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece; 2.Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece; 3.Biomedical Research Foundation of the Academy of Athens, Athens, Greece; 4.Faculty of Biology, Medicine and Health Manchester Cancer Research Centre, Man-chester Academic Health Sciences Centre, The University of Manchester, Manchester, UK. *equal contribution

BackgroundPancreatic ductal adenocarcinoma (PDAC) has a poor prognosis; the 5- and 10-year reported survival rates are less than 10% and 5% respectively and by year 2020 is projected to be the second killer cancer in Europe. De-spite advances in oncology there has been little progress in the early diagnosis, prognosis and treatment of pa-tients with PDAC. Pancreatic resection is a treatment of choice for localized PDAC, although it is related with high morbidity and mortality rates even in specialized centers. Besides PDAC has a silent development and at early stages there is a high propensity for rapid dissemination, which reduces the number of patients that will benefit from surgical treatment. For advanced PDAC the limited effect of chemo- and radio- therapeutic modalities ren-ders PDAC incurable. Additionally, the clinical success of immunotherapy in first- and second-line treatment for several forms of cancer, has not been verified in PDAC. The results from the employment of immune-checkpoint inhibitors in PDAC are disappointing. Hence, it is important to understand the molecular mechanisms involved in pancreatic carcinogenesis in the context of dynamical interactions between cancer cells and the surrounding stroma. Within this context here we present an approach integrating two conservative homeostatic mechanisms in PDAC, namely DNA damage response (DDR) and immune response (ImR). MethodsFrom 30 patients with PDAC undergoing pancreaticoduodenectomy we collected tumor samples in accordance with the ethical standard. We employed in situ assays including immunohistochemistry and immunofluorescence to study the following: a) kinetic parameters and cellular senescence utilizing a novel biomarker called GL13 (SenTraGor®), b) γH2AX that accumulates upon double strand breaks and 8-oxo-G, a marker of oxidative DNA damage, c) immunophenotypic markers including CD3, CD8, Granzyme B (GZB), FOXP3 and CD163. Results A positive correlation is observed between the DNA damage markers and T lymphocytic infiltration. In terms of immunophenotypic analysis we found two distinct patterns: cases exhibiting high CD8+/FOXP3+ ratio and cases having low CD8+/FOXP3+ status, which comes in line with the literature. Of note, a significant percentage of CD8+ T cytotoxic lymphocytes infiltrating the tumor were negative for GZB, suggesting lytic dysfunction. ConclusionsAlthough preliminary, our approach demonstrates the DNA damage and immune response crosstalk in PDAC. The lack of detectable levels of GZB in tumor infiltrating CD8+ cells underlines that PDAC microenvironment compromises cytolytic activity which warrants further analysis.

VIRUSLIKEPARTICLE(VLP)VACCINETARGETINGTHETHIRDEXTRACELLULARDOMAIN(ECD3)OFxCTPROTEINFORMETASTATICBREASTCANCERTREATMENTV. Rolih1, E. Bolli1, L. Conti1, J. Caldeira2, N. Salameh2, F. Pericle2 and F. Cavallo1. 1 Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin. Via Nizza, 52 - 10126, Torino, Italy. 2 Agilvax, Inc. 5901 Indian School Rd NE. Albuquerque, NM 87110, USA.

Background Cancer stem cells (CSCs) are involved in the resistance mechanism to radio- and chemo-therapies and rep-resent a reservoir for relapse and metastatic evolution. The cystine-glutamate antiporter xCT (SLC7A11) is over-expressed on breast CSC (BCSC). It is characterized by 12 multi-pass transmembrane and 6 extracellular domains (named ECD1-6) with intracellular N- and C-terminals. xCT protein mediates the exportation of the glutamate and the importation of cystine that is converted to cysteine involved in glutathione (GSH) synthesis.

significant changes on the presented peptidomes, both at the qualitative and quantitative levels. Specifically, inhibitor treatment altered about half of the total 3204 identified peptides and about one third of the peptides predicted to be good ligands for MHC-I, affected length and sequence without however interfering with basic binding motifs. Surprisingly, the inhibitor enhanced predicted overall MHC-I binding affinity by reducing presenta-tion of sub-optimal long peptides and generating many high-affinity 9-12merssuggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes.Conclusions: Our results suggest that chemical inhibition of ERAP1 is a valid approach for manipulating the im-munopeptidome of cancer with potential applications in standalone or combination immunotherapy.

ANEX-VIVOMODELSIMULATINGTHEBONEMARROWOFPATIENTSWITHMULTIPLEMY-ELOMAAndreas Metousis1, Konstantinos Papadimitriou1, Athanasios Velentzas1, Panagiotis Vitsos1, Ioan-nis Kostopoulos1, Paraskevi Miheli1, Anastasia Tsopanidou1, Panagiotis Pothos1,2, Nikolaos Orologas3, George Thyfronitis2, Efstathios Kastritis4, Evangelos Terpos4, Ioannis Trougakos1, Diana V. Portan5, Vassilis Kostopoulos5, Meletios-Athanasios Dimopoulos4, Ourania Tsitsilonis1

1Department of Biology, National and Kapodistrian University of Athens, Athens; 2Department of Biological Applications and Technology, University of Ioannina, Ioannina; 3SafeBlood BioAnalytica SA, Athens, Greece; 4Department of Clinical Therapeutics, “Alexandra” Hospital, School of Medicine, Na-tional and Kapodistrian University of Athens, Athens; 5Department of Mechanical Engineering and Aeronautics, Composite Materials Group, University of Patras, Patras 265 00, Greece

BACKGROUND: Multiple Myeloma (MM) is a B cell neoplasm of the Bone Marrow (BM), characterized by the pres-ence of BM-infiltrating malignant plasma cells. Nowadays 80% of newly diagnosed patients achieve complete remission, althoughmost of them relapse. The BM microenvironment plays a critical role in the pathophysiology of MM. Both Mesenchymal Stem Cells (MSCs) and regulatory T-cells (Tregs) have powerful immunosuppressive properties. Our aim was to reveal changes in various cell populations of the BM microenvironment, in order to shed light on their role in the onset and progression of MM.METHODS: After culturing human BM-derived MSCsin DMEM-15%FBS for ≥20 days, MSCs were stained with CFSE and seeded in 1x1cmPLA 3D-bioprinted, grid-like scaffolds with pore sizes of 60, 100, 60-70-80-90 and 60-80-100-120μm. The scaffolds were checked for the presence of adherent cells, using Fluorescence Microscopy (FM). For Scanning Electron Microscopy (SEM) experiments, one scaffold was fixed in 4% glutaral-dehyde, dehydrated in increasing ethanol concentrations, mounted on aluminum stubs and gold-coated, before imaging. Another scaffold was fixed in 4% formaldehyde and treated with 0.1% Triton X-100. Phalloidin solution and mounting medium containing DAPI were added, before imaging with Confocal Microscopy (CF). To identify Tregs, BM and Peripheral Blood (PB) samples from 26 MM patients were labeled with 12 fluorescent antibodies: CD3-FITC, CD4-APC/Cy7, CD25-APC, FoxP3-PE, CD8-PerCP/Cy5-5, Ki67-AmCyan, CD45RA-PE/Cy7, CD39-Pacific Blue, CD45RO-PerCP/Cy5-5, CTLA-4-Pacific Blue, CD127-AmCyan, HLADR-PE/Cy7 and analyzed with Flow Cytometry.RESULTS: The presence of MSCs in the scaffolds was first observed four days after seeding. Pore size of 100μm was proven the most favorable for MSCs adherence and proliferation. Both SEM and CF confirmed mesenchymal cell morphology. MSCs attach on and between PLA filaments, occupying the 3-D space, and reach 40% con-fluence within one month. Cells with immunophenotype CD3+CD4+CD25+FoxP3+, were identified as Tregs and counted. Treg percentages based on total nucleated cells acquired (~0.15%) do not differ between the PB and the BM of MM patients.CONCLUSIONS: 3-D culture of human MSCs on PLA scaffolds was proven feasible. Thus, we simulated the BM microenvironment of MM patients in order to study its interactions with other BM cell populations as well as with myeloma cells. The next step is to co-culture MSCs, sorted BM-derived Tregs and myeloma cells, to unravel the processes underlying their crosstalk.

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minal center and/or high endothelial venules. Subsequently those were counted and furthermore characterized as adjacent(aTLS, within the tumor IM) or as distal (dTLS,≤5mm from the IM within the non-neoplastic tissue). Clinical follow-up was available for 112 of these patients. Kaplan-Meier and log-rank analyses were performed for DFS and OS survival estimation. Results: The absence of peritumoral TLS was associated with longer DFS and OS. Moreover, not only the pres-ence of peritumoral TLS, but also their density has been proven to be prognostically crucial, since patients with numerous peritumoral TLS exhibited worst DFS and OS. By combining quantitative peritumoral TLS measure-ments with our previous immunological signatures (FCIS & UCIS), we generated two combined immunological signatures with reinforced prognostic significance (RFCIS&RUCIS) which also offered a great coverage of our patient population. Conclusions: In the present study, we demonstrate for the first time an association between spatial differential densities of intratumoral immune infiltrates with peritumoral TLS and clinical outcomes. Our observations fur-ther support the existence and importance of a dynamic interplay between the immune cells within the tumor microenvironment(TC,IM) and its surrounding counterpart(TLS).

THEROLEOFHLA-A*02ANDHLA-A*24 IN LOCALIZEDANDMETASTATICPROSTATECANCERSavvas Stokidis1, Sotirios P. Fortis1, Paraskevi Kogionou1, Marina I. Konstantellou1, Catherine Stavro-poulos-Giokas2, Sonia A. Perez1, Constantin N. Baxevanis1

1. Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece2. Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Greece

Background: Immune biomarkers for cancer prognosis and prediction, including prostate cancer (PCa) are emerging as essential tools for treatment, assessment and monitoring of the disease. Given the heterogeneity of PCa and the diversity of therapeutic treatments applied, intensive efforts have been recently initiated for the establishment of personalized approaches. The prognostic markers typically used so far, including tumor stage, Gleason score(GS), and the serum level of Prostate-Specific Antigen (PSA) are not sufficiently precise to accu-rately predict the risk of disease progression in PCa patients. Therefore, an accurate risk stratification tool at the time of diagnosis is the cornerstone of clinical decision, paving the way for precision medicine for the manage-ment of PCa. As HLA-alleles are essential for an effective antitumor response, their expression could serve as prognostic/predictive biomarkers. Studies in other cancer types (NSCLC&ovarian cancer) have demonstrated that HLA-A*02 allele expression is correlated with poor clinical outcome. Results from our previous phase I clini-cal trial of PCa patients vaccinated with a HER-2/neu hybrid peptide, suggested that HLA-A*24 allele expression conferred a better clinical outcome. Based on these datawe evaluate herein the prognostic relevance of both alleles in PCa.Methods: PCa patients (n=104) were enrolled from 06/2014-5/2019 to this combined retrospective/pro-spective study. 52 of these patients were initially diagnosed, before enrollment, with localized disease (LPCa) and 52 were already metastatic at first diagnosis (MPCa). All patients received standard medical treatment upon diagnosis and follow-up data, either retrospective or prospective, were recorded. Patients were HLA-genotyped for the A locus. Time to biochemical recurrence (BCR), metastasis (M), castrate resistance (CR) and overall-survival (OS) were evaluated.Results: Both, LPCa and MPCa patients expressing HLA-A*02 had a significant worse clinical outcome than those lacking this allele, especially when compared to HLA-A*24 expressors. Moreover, MPCaHLA-A*24+ patients had a significant, favorable, advantage in overall survival. Interestingly, a group of HLA-A*02+ LPCa patients at low or intermediate risk (GS<8) had similar clinical progression as HLA-A*02- high risk patients (GS≥8). Furthermore, MPCaHLA-A*02+ patients with a “low burden” (PSA≤100ng/ml) disease had a similar clinical progression as MPCaHLA-A*02- patients with “higher burden” (PSA>100 ng/ml). Conclusions: The data presented herein strongly support the unfavorable and favorable rolesfor HLA-A*02 and HLA-A*24 expression, respectively, for PCa prognosis. The precise mechanisms (e.g.allelic-loss and/or immune escape or resistance) underlying this HLA allelic difference requires further investigation.

GSH protects cells against oxidative stress and chemical insults. xCT expression is restricted to a few normal cell types, as astrocytes and subsets of macrophages. In breast cancer patients, high levels of xCT correlate with significant reduction in distal metastases-free and overall survival. Thus, xCT represents a viable target for cancer immunotherapy. Methods We have developed a virus-like-particle (VLP) vaccine targeting the 3rd ECD of human xCT protein (AX09-0M3). We evaluated the AX09-0M3 effect on two different metastatic mammary tumor mouse models. BALB/c mice were immunized intramuscularly with AX09-0M3 or MS2 wt (control) and two weeks later were transplanted with mammary tumor cells. Tumorspheres derived from TUBO cells (Her2+ cell line) were injected intravenously and mice were culled 21 days later. In second metastatic model, tumorspheres derived from 4T1 cells (triple nega-tive cell line) were injected subcutaneously and spontaneously gave rise to lung metastases. Mice were culled when the primary tumor reached a mean diameter of 10 mm. At sacrifice lungs were explanted, weighted and processed to count metastases. Results and Conclusion AX09-0M3 vaccine elicited a strong antibody response against xCT protein and these antibodies affected BCSC function and biology in vitro. AX09-0M3 significantly reduced the number of lung metastases in the TUBO mouse model and to a lesser extent in the spontaneous metastatic 4T1 model. . Moreover, AX09-0M3 vaccinated mice shown a delay in the tumor onset and a slowdown in the primary tumor growth. FACS analysis on immune cell infiltrate demonstrated that AX09-0M3 vaccine induces an increase in CD4+ and CD8+ T lymphocytes and NK cells in the metastatic lung. The same trend has been observed in the primary tumor. These data show that AX09-0M3 induced antibodies inhibit xCT activity, affect CSC biology and significantly reduce metastatic progression in preclinical models. We are now generating MoAb targeting xCT using AX09 VLP vaccine to better investigate the effects of xCT inhibition in human breast cancer models.

THEPROGNOSTICSIGNIFICANCEOFPERITUMORALTERTIARYLYMPHOIDSTRUCTURESIN BREAST CANCERMichael Sofopoulos1*, Sotirios P. Fortis2*, Christoforos K. Vaxevanis2, Nectaria N. Sotiriadou1, Niki Arnogiannaki1, Alexandros Ardavanis3, Dimitrios Vlachodimitropoulos4, Sonia A. Perez2, Constantin N. Baxevanis2

* Equally contributed as first authors1. Pathology Department, Saint Savvas Cancer Hospital, Athens, Greece2. Cancer Immunology and Immunotherapy Center, Saint Savvas Cancer Hospital, Athens, Greece3. 1st Medical Oncology Clinic, Saint Savvas Cancer Hospital, Athens, Greece4. Department of Forensic Medicine and Toxicology, National and Kapodistrian University of Athens, Greece

Background: Tumors and their surrounding area represent spatially organized “ecosystems”, where tumor cells and the immune contextures of the different compartments are in a dynamic interplay. Tumor immune-cell infiltra-tion is progressively becoming an important aspect for tumor characterization and clinical outcome prediction of cancer patients. We have previously demonstrated the importance of immune infiltration in breast cancer (BCa) based on the combined differential densities of CD8+ and CD163+ cells in the Tumor Center-TC and Invasive Margin-IM, resulting in the identification of a favorable and an unfavorable combined-immune-signature (FCIS and UCIS, respectively) as prognostic immune signatures. Nevertheless, FCIS and UCIS could cover only half of our total patient population analyzed. Tertiary-Lymphoid-Structures (TLS) could provide a “third-level” biomarker along with immune infiltrates in TC and IM. TLS are lymph-node-like structures sharing architectural and func-tional characteristics with secondary lymphoid organs and are often detected in the tumor microenvironment. Their presence has been associated with controversial clinical outcomes. We aimed to clarify their relation with prognosis in BCa patients alone and in combination with tumoral immune infiltrates. Methods: Formalin-fixed, paraffin-embedded tumor tissue samples from 167 BCa patients, with invasive ductal, non-metastatic BCa, not treated with neoadjuvant chemotherapy, have been used to detect and quantify the presence of TLS. The latter were identified morphologically as ovoid lymphocytic aggregates presenting a ger-

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and when the metastatic form of the disease is present, the prognosis is dismally poor. Therefore, we started to investigate CSPG4 role in both human and canine OSA to eventually propose CSPG4 DNA electrovaccination as an innovative comparative therapy for OSA treatment. Methods CSPG4 over-expression and its impact on OSA patients’ survival was evaluated in human and canine OSA setting. Functional in vitro experiments were performed to assess the anti-tumor potential of CSPG4 immune-targeting in human and canine OSA cell lines.Results and conclusionsWe found a strong correlation between CSPG4 over-expression and a worse prognosis in both human and canine OSA patients. The potentiality of CSPG4 immune-targeting for OSA treatment was demonstrated by the ability of anti-CSPG4 monoclonal antibodies (mAbs) to significantly inhibit canine CSPG4+ OSA in vitro proliferation, migration and osteospheres generation. In addition, anti- CSPG4 mAbs potentiated the anti-proliferative effect of doxorubicin. Interestingly, sera derived from canine MM patients enrolled in our previous clinical trials, were also able to in vitro inhibit OSA cells tumorigenic potential in both adherent and non-adherent conditions. Overall, these results provide the rationale to propose the adjuvant anti-CSPG4 electrovaccination for the treat-ment of canine CSPG4+ OSA, with a powerful translational impact for human OSA management.

Cancerassociatedfibroblastssuppressanti-tumorimmunityandpromotetumorgrowthAthina Varveri, Aigli-Ioanna Legaki, Themis Alissafi, Aikaterini Hatzioannou and Panayotis VerginisLaboratory of Immune Regulation and Tolerance, Biomedical Research Foundation, Academy of Athens, Athens, Greece.

Background:The advent of checkpoint blockade immunotherapy has revolutionized cancer therapeutics, yet such regimens are associated with low response rates in a sizable portion of cancer patients. The immunosuppressive and hypoxic networks of the tumor microenvironment (TME) impede the elicitation of potent anti-tumor immune responses and impair the success of immunotherapy. Among the constituents of the TME, cancer-associated fibroblasts (CAFs) represent a highly heterogeneous yet abundant cell population, quite resistant to chemo/radiotherapy. Although their tumor-promoting function is acknowledged, their role in shaping the anti-tumor im-mune response remains elusive.Methods:For tumor experiments, C57BL/6 mice were implanted with B16/F10 melanomacells; alpha smooth muscle actin (α-SMA) expression in the TME and peripheral blood was assessed with flow cytometry. In Treg induction experiments, CD4+FoxP3- cells were sorted and treated with murine tumor explant supernatants (TES) from CAFs depleted tumors (TESCAFsΔ) and FoxP3 transcription factor expression and Treg cell proliferation were assessed with flow cytometry. Autophagy was determined based on the expression of the PI3K/AKT/mTOR axis by flow cytometry as well as through the levels of LC3, Lamp-1 and p62 on NIH/3T3 fibroblastic cells (untreated vs treated with TES) by confocal microscopy.Results and Conclusions:Herein, we demonstrated that α-SMA+CAFs were enriched in the TME of mouse melanoma, while a clear α-SMA+ population was detected in circulation in later tumor stages. Specific ablation of αSMA+ CAFs resulted in sig-nificant regression of tumor growth, accompanied with elevated numbers of tumor infiltrating CD45+ cells. Fur-thermore, following CD4+FoxP3- naive T cells culture in Treg induction conditions with TES vs TESCAFsΔ, we found FoxP3 expression levels to be significantly downregulated in the presence of TESCAFsΔ. Finally, after validating that TES-treated 3T3 cells exhibited increased autophagy levels compared to untreated cells in vitro, we observed that the phosphorylated levels of AKT, mTOR and S6 in vivo were downregulated upon tumor progression. Collec-tively, our data bring into focus CAFs as an important cell population with immune-suppressive properties, char-acterized by increased autophagy and a potency to induce highly immunosuppressive populations, such as Tregs. Elucidation of how these processes unfold will provide a better mechanistic insight of how CAFs influence tumor immune response and ultimately lead to development of more efficacious immunotherapeutic approaches.

StudyofCD155andCD112expressionontumorcellsasligandsoftheTIGIT/DNAM-1axis in Natural Killer cellsIsabel Valhondo1, Beatriz Sanchez-Correa1, Fakhri Hassouneh1, Nelson Lopez-Sejas1, Carmen Mestre1, Miriam Blazquez1, Carlos Blanco1, Alejandra Pera2, Juan M. Bergua3, MariaJose Arcos3, Helena Bañas3, Esther Durán4, Corona Alonso5, Rafael Solana2,5,6, Raquel Tarazona1

1 Immunology Unit, University of Extremadura, Cáceres, Spain2 Immunology, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain3 Department of Haematology, Hospital San Pedro de Alcantara, Caceres, Spain4 Histology and Pathology Unit, Faculty of Veterinary, University of Extremadura, Cáceres, Spain5 Immunology and Allergy Unit, Reina Sofia University Hospital, Córdoba, Spain6 Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain

Natural killer (NK) cells are lymphocytes of the innate immune response characterized by their role in the de-struction of tumor cells. NK cell activation depends on a fine balance between activating and inhibitory signals mediated by different receptors. A family of paired receptors that interact with ligands of the family of Nectins and Nectin-like (Necls) molecules has attracted great interest. Thus, DNAM-1 (CD226) and TIGIT receptors share the same ligands, CD155 (Necl-5) and CD112 (Nectin-2) but exert opposite functions on T and NK cells. Where-as activation through DNAM-1 enhances NK cell-mediated cytotoxicity against a wide range of tumor cells, TIGIT mediated signaling exerts an inhibitory action on NK cells by diminishing IFN-γ production, as well as NK cell-mediated cytotoxicity. We have observed in a large panel of melanoma cell lines a higher expression of CD155 compared to CD112. In contrast, the expression of CD112 is greater than that of CD155 in the blasts of acute myeloid leukemia patients. We have also analyzed the expression of DNAM-1 and TIGIT in NK cells from healthy donors and acute myeloid leukemia patients. We observed a decreased expression of DNAM-1 in acute myeloid leukemia patients whereas the expression of TIGIT did not show statistically significant differences compared to healthy donors. The decreased expression of DNAM-1 may shift the balance in favor to the inhibitory receptor TIGIT contributing to the diminished NK cell-mediated cytotoxic capacity observed in these patients. Analysis of the TIGIT/DNAM-1 axis on human NK cells from solid cancer and leukemia patients will help to clarify the role of these paired receptors in cancer surveillance. Overall, it can be speculated that in cancer patients the TIGIT/DNAM-1 axis is altered with a shift to the inhibitory pathway, supporting that TIGIT represents a novel target for checkpoint blockade immunotherapy.

CSPG4IMMUNE-TARGETINGASAPROMISINGSTRATEGYFORTRANSLATIONALSTUD-IESINPRE-CLINICALMODELSOFCSPG4+TUMORSL. Tarone1, F. Riccardo1, S. Iussich2, D. Giacobino2, F. Gattino2, S. Ferrone3, P. Buracco2 and F. Cavallo1 1 Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy 2 Department of Veterinary Sciences, University of Turin, Grugliasco, Italy 3 Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States

Background Chondroitin Sulfate Proteoglycan 4 (CSPG4) is a highly glycosylated transmembrane proteoglycan with low ex-pression in healthy tissues, but high expression in a wide range of solid tumors. Since CSPG4 has been recognized playing a central role in several tumorigenic processes, its immune-targeting by means of DNA vaccination could represent an interesting opportunity to successfully counteract CSPG4+ tumors. Because of CSPG4 over-expression by both human and canine malignant melanoma (MM), the high homology between the human and canine CSPG4 orthologues and the recognized translational power of dogs as pre-clinical models for several human malignancies, we evaluated the efficacy of the adjuvant anti-CSPG4 DNA electrovaccination in client-owned dogs with surgically resected CSPG4+ MM. This approach demonstrated to be safe and effective in significantly prolonging canine patients’ survival as compared to conventionally treated controls, thanks to the induction of anti-CSPG4 antibodies. CSPG4 over-expression on tumor histotypes other than MM, prompted us to expand our research evaluating the potential of anti-CSPG4 immune-targeting in the osteosarcoma (OSA) setting. OSA is the most common malignant bone tumor among children and adolescents,

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DEVELOPMENTOFTGF-βBINDINGPROTEINAleksandra Vuchkovska1,2, Veronica Volgina1,2, Makio Iwashima1,2

1 Department of Microbiology and Immunology, Loyola University, Chicago, IL, USA2 Van Kampen Cardio Pulmonary Research Laboratory, Loyola University, Chicago, IL, USA

IntroductionCancer progression relies on avoiding immune surveillance and developing an immunosuppressive environment that hinders the anti-tumor immune response. While enhancing immune cell activation has proven to be a promis-ing immunotherapy for some patients, the treatment is still limited by factors that alter the immune function. One such immunomodulatory factor is transforming growth factor-β (TGF- β). In a cancer setting, TGF-β promotes the growth, invasion, and metastasis of cancer cells, and also aids in dampening the immune response. Although there are many studies analyzing the biology of TGF-β, there are still many unsolved issues regarding how TGF-β modulates immune responses. For example, there is no reliable reagent that detects cells that express active TGF-β. This limitation creates a technical hurdle in studying TGF-β mediated immune modulation under physi-ological conditions. MethodsTo address this challenge, we used phage-display technology to engineer a TGF-β binding protein. The protein was developed using a fibronectin type III domain (FN3) library. To isolate monobodies, we did several rounds of affinity selection. The specificity and functionality of the selected clones were evaluated using various techniques, such as ELISA, flow cytometry, microscopy and Western blotting. Results and conclusionsWe screened over 200 phage clones and identified Ph6, the highest affinity clone, to specifically recognize ac-tive, but not latent TGF-β. Ph6 blocked TGF-β mediated signaling, and also neutralized TGF-β biological activities, such as epithelial to mesenchymal cell transition. A number of studies suggest that active TGF-β is expressed on cell surfaces. However, the sequence of active TGF-β lacks a cell binding domain, raising the question how it is anchored to cell surfaces. Although several anti-TGF-β antibodies are commercially available, detection of cell surface bound active TGF-β remains challenging and is not well documented. To determine if our reagent is suitable for detection of active TGF-β on cell surfaces, we adapted Ph6 clone for flow cytometry. We found thatPh6 clone serves as a reliable reagent for detection of active TGF-β on cell surfaces. Altogether, our data suggests that we have a reagent that can both serve as a neutralizing and detection reagent for active TGF-β. We hypothesize that we can use the Ph6 reagent as a tool for evaluating TGF-β expression from cells and tissues from various pathological conditions. Currently we are working on adapting the Ph6 clone sequence of FN3 into a mammalian expression system.

General Ιnformation OrganizerThe cancer Immunology and Immunotherapy Center, St. Savas Cancer Hospital

Hellenic Society of Immuno-Oncology

OrganizingScientificCommitteeConstantin N. Baxevanis, GreeceVasileios Georgoulias, GreeceAthanasios Kotsakis, GreeceAlejandro Madrigal, United KingdomGraham Pawelec, GermanyBarbara Seliger, Germany

DateJune 7-9, 2019

MeetingWebsitehttps://pivac19.eu/

MeetingVenueHELLENIC PASTEUR INSTITUTE127, Vasilissis Sofias Avenue11521, Athens, GreeceTel.: (+30) 210 64 78800

Certificate of AttendanceCertificate of Attendance will be given to the participants at the end of the meeting

Official LanguageThe official Language of the Meeting is English

The Meeting Secretariat

Scientific | Cultural Events & Publications T +30 2810 222155, +30 210 7240039 F +30 2810 222156, +30 210 7240139 E info@scep.gr www.scep.gr

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NOTESNOTES

Scientific Ι Cultural Events and Publications T +30 2810 222155, +30 210 7240039 F +30 2810 222156, +30 210 7240139 E info@scep.gr www.scep.gr