PS2-081 Cytokines production in mice bearing breast tumor treated with pulchellin

Abstract / Cytokine 56 (2011) 83–87 cell surface after TNF-apha treatment. In aadition, presence of these molecules was confirmed in supernatants using western blot analyses. These results indicated the complexity of events on cell membrane, including association between LDH release and shading of membrane molecules following TNF-alpha mediated processes. Shedding of cell surface molecules mostly precede apoptosis induction and caspase activation. doi:10.1016/j.cyto.2011.07.239

PS2-077 Association of inhibition of tumor growth with intratumoral hematopoiesis induced by IL-33 Juyang Kim a, Wonyoung Kim b, Hyun Ju Kim b, Hye-Jeong Choi c, H.R. Cho a,d, B. Kwon a,b, a Biomedical Research Center, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea, b School of Biological Sciences, University of Ulsan, Ulsan, Korea, c Department of Pathology, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea, d Department of Surgery, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea Immune surveillance by a functional immune system is a well-established mechanism for regulation of tumor growth. In this study, we have shown that c-kit+ Sca-1+ IL-7Ra+ could be an important component responsible for tumor immunosurveillance. Local production of IL-33 by lymphoma cells induces recruitment of c-kit+ Sca-1+ IL-7Ra+ into tumor masses. In response to IL-33, c-kit+ Sca-1+ IL-7Ra+ can proliferate, produce high levels of Th2 cytokines, and be differentiated into eosinophils in an IL-5-dependent manner. Eosinophils in turn exhibit a potent anti-tumor activity. The ability of IL-33 to induce the emergence of a c-kit+ Sca-1+ IL-7Ra+ population in tumors identifies a link between the Th2 cytokine family member and extramedullary hematopoiesis, and suggest a previously unrecognized innate immune pathway involving hematopoietic progenitor cells which mediated type 2 innate immunity leading to antitumor activity.

doi:10.1016/j.cyto.2011.07.240

PS2-078 Interferon Regulatory Factor-1 promotes NK cell-mediated suppression of lung metastasis through DNAM1/CD155 interaction A. Ksienzyk a,b,c, Berit Neumann a, Katja Finsterbusch, Ramya Nandakumar a, Martina Grashoff a, Rainer Zawatzky b, Günter Bernhardt c, Hansjörg Hauser a, Andrea Kröger a, a Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection research, Braunschweig, Germany, b Division of Viral Transformation Mechanisms, German Cancer Research Center, Heidelberg, Germany, c Institute of Immunology, Hannover Medical School, Hannover,Germany The ineffectiveness of standard cancer therapies makes immunotherapy an attractive alternative. IFN-c, a key cytokine in tumor-suppression, was shown to increase visibility of tumor cells and to improve immune surveillance. Using a mouse model of pulmonary metastases we show that local IFN-c treatment inhibits metastases formation and development. When IRF-1, a transcription factor that is induced by IFN-c, inflammation and infections, is blocked by shRNA in the tumor cells, the IFN-c effect on metastases is abolished. Forced expression of IRF-1 inhibits metastases in a similar way as IFN-c. IRF-1 has no influence on the survival of tumor cells in the circulation or infiltration into lungs. IRF-1 inhibits metastases formation by attraction and activation of NK cells since depletion of NK cells abolishes its protective effect. NK cell cytotoxicity assays revealed that tumor cells expressing IRF-1 are better targets of NK cells than non-expressing tumor cells. Moreover, NK cells isolated from lungs inoculated with IRF-1 expressing tumor cells exhibit a higher cytotoxic potential. NK-cell cytotoxicity induced by IRF-1 is independent of perforin and granzyme but is mediated by DNAM-1. Thus, the IRF-1 pathway represents a signaling pathway that mediates crosstalk between the micro-environmental components and tumor cells and represents a potential target for tumor therapy.

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The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity may be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-c , IL-13 and IL10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-c . The MCPyV antigen tended to induce stronger IFN-c responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-c responses. IFN- c being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance. doi:10.1016/j.cyto.2011.07.242

PS2-080 Th17 cells activate NK cell mediated cytotoxicity to control tumour growth via IL-21 Neil A. Marshall, Anna Marie Corcoran, Kingston H. G. Mills, IRRG, School of Biochemistry and Immunology, Trinity College Dublin The role of IL-17 and CD4+ T-cells that secrete IL-17 (Th17 cells) in the prevention or enhancement of anti-tumour immunity remains unclear. Reports in the literature indicate both pro and anti-tumour effects of IL-17-secreting cells. We and others have detected significant populations of IL-17-secreting T cells within the microenvironment of various murine and human tumors, indicating a function for such cells in tumour immunity. We report here on a novel role for IL-21 derived from Th17 cells in the activation tumour cell killing by NK cells. We first established that IL-17 has a protective role in anti-tumor immunity through the demonstration that IL-17-/- mice have increased rates of tumour growth in vivo. We found that Th17 cells mediated tumour cell cytotoxicity in a non-specific manner via the activation of NK cells. Although unable to mediate direct tumour cell cytotoxicity, in vitro polarised Th17 cells activated NK cell cytotoxicity. We found that IL-17 and IL-21 promoted activation and tumour killing by NK cells. Neutralisation of IL-21 with a specific antibody resulted in loss of expression of CD107a and loss of cytotoxic activity from Th17 activated NK cells. In addition, recombinant IL-21 enhanced expression of cytotoxic markers on NK cells and enhanced their ability to kill tumour cells in vitro, in a manner seen with Th17 activated NK cells. Interestingly IL-17, although required for the activation of NK cells in co-culture experiments with Th17 cells, was not able to directly activate NK cells. Using an immunotherapeutic approach involving a TLR agonist and PI3 kinase inhibitor in the B16 melanoma model in mice, we found that tumor regression was associated with significantly increased numbers of T-cells co-producing IL-17 and IL-21, and with increased numbers of tumour infiltrating NK cells expressing high levels of cytotoxic markers. In addition, tumour bearing IL-17-/- mice displayed a profound defect in both the numbers and activation of NK cells infiltrating into tumours. Furthermore, infusion of recombinant IL-21 into tumours delayed tumour growth in vivo due to increased infiltration of NK cells into the tumour mass. These studies have revealed a novel role for Th17 cells and IL-21 in the control of tumour cell growth that might be exploited for the rational design of anti-tumour therapies. doi:10.1016/j.cyto.2011.07.243

doi:10.1016/j.cyto.2011.07.241

PS2-079 T-helper cell-mediated proliferation and cytokine responses against recombinant merkel cell polyomavirus-like particles Arun Kumar a, Tingting Chen a, Sari Pakkanen b, Anu Kantele a,c, Maria SöderlundVenermo a, Klaus Hedman a,d, Rauli Franssila a, a Departments of Virology, Haartman Institute, University of Helsinki, b Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, c Division of Infectious Diseases, Helsinki University Central Hospital, d Helsinki University Central Hospital Laboratory Division

PS2-081 Cytokines production in mice bearing breast tumor treated with pulchellin Djamile Cordeiro de Matos, Livia Carolina de Abreu Ribeiro, Lucas Souza Ferreira, Marcela Bassi, Marisa Campos Polesi, Lucas Colombo, Iracilda Zeppone Carlos, Laboratory of Clinical Immunology, Universidade Estadual Paulista Júlio de Mesquita Filho, Araraquara, Brazil Breast cancer is the most frequent type in women in the United States and the second cause of death between the types of cancer. In Brazil, 49,240 new cases of breast cancer were estimated for 2010, and in 2008 there were 11,860 deaths in women due to breast cancer. Pulchellin is a ribosome inactivating proteins (RIPs) type

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Abstract / Cytokine 56 (2011) 83–87

II obtained from seeds of Abrus pulchellus. It is consisted of two dissimilar, disulfidelinked polypeptide chains. The A-chain has N-glicosilase enzymatic activity, and B-chain has lectin activity to b-D-galactose, a carbohydrate present in most mammals cells. We evaluated the production of IL-4, IL-10, TNF-a and IFN-g by cells stimulated or not obtained from mice bearing breast tumors treated with pulchellin 0.75 ug / kg of mice body (treatment 1) and 7.5 ug / kg of mice body (treatment 2) in the presence and / or absence of 0.2 M galactose. Although the treatment 1 has less antitumor effect (1% of growth inhibition) than the treatment 2 (5% of growth inhibition) when injected intratumorally, it has a greater responsiveness to stimulation of cells production of IFN-g, which stimulates functions of NK and TCD8+ cells, and anti-inflammatory cytokines such as IL-10, which inhibits the function of macrophages and dendritic cells, in an tumor environment this is interesting as macrophages and dendritic cells associated with tumor, which act by stimulating the tumor, would have inhibited their functions. Regarding the adding of galactose 0,2M in the treatment 1, there were a promotion of tumor growth by 18.7% and reduction of production of IFN-g (p < 0,01). The treatment 2 is uninteresting because it decreases the responsiveness of the cells to produce cytokines active against cancer (IL-4 and IFN-g, p < 0,01 and p < 0,001) and also cytokines that stimulate cancer (IL-10 and TNF-a, p < 0,001). However it is treatment 1 that could be interesting as an adjuvant in cancer treatment by acting on the immune system. Financial support: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) doi:10.1016/j.cyto.2011.07.244

PS2-082 Immunological effects of gene transfer with the sushi domain of IL-15Ra and a chimeric protein consisting of IL-15 fused to apolipoprotein A-1 M.C. Ochoa, J. Fioravanti, E.H. Duitman, J. Medina-Echeverz, A. Palazon, A. Arina, J. Dubrot, C. Alfaro, A. Morales-Kastresana, O. Murillo, S. Hervas-Stubbs, J. Prieto, P. Berraondo, I. Melero, CIMA of University of Navarra, Pamplona, Spain Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing IL-15 and Apo A-I (pApohIL15) that was tested by hydrodynamic injections into mice and co-administered with a plasmid encoding the sushi domain of IL-15Ra (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in the liver of IL-15Ra-/- mice. pApohIL15 + pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of Apo-IL-15 fusion protein holds promise for further development in combination with other immunotherapies. doi:10.1016/j.cyto.2011.07.245

PS2-083 Immunization with a novel dc-targeting lentiviral vector induces polyfunctional cd8 t cell responses and therapeutic anti-tumor immunity SH Robbins a,b, B Kelley Clarke a, JM Odegard a, SU Tareen a, N Van Hoeven a, DJ Campbell a, CJ Nicholai a, MM Slough a, C Vin a, D Baltimore b, SG Reed a, TW Dubensky Jr. a, a Immune Design Corp, Seattle, WA USA, b California Institute of Technology, Pasadena, CA USA Dendritic cells (DCs) are essential antigen presenting cells for the initiation and control of adaptive immune responses, therefore vaccines that deliver designated antigens directly to DCs in vivo are conceptually attractive. Immune Design Corp (IDC) is developing a vaccine platform around a novel class of non-integrating lentivectors that target DCs for the purpose of creating licensed immunotherapeutics. IDC’s vector platform, termed Dendritic Cell-targeting Non-Integrating Lentiviral Vector (DC-NILV), achieves DC targeting by utilizing a modified Sindbis virus envelope glycoprotein, SinVar1, which allows the transduction of DCs via the DC-SIGN receptor. Transduction of human DCs was highly efficient using DC-NILV produced in the presence of mannosidase I inhibitors which promote the generation of SinVar1 glycoproteins with terminal mannose residues. Integration-defective lentiviral vectors will facilitate regulatory approval for clinical evaluation of vaccine candidates given by direct administration. We utilized a redundant approach to render DC-NILV integration defective by combining a pol gene encoding a mutant Integrase (D64V) with a self-inactivating (SIN) vector backbone deleted in the 3’ LTR U3 region to the att site,

including the 3’ LTR-proximal polypurine tract. This composition favors formation of single-LTR reverse transcribed episomal dsDNA circles in infected DCs, which are not a template for chromosomal integration. Prime and prime-boost immunization regimens with 1e7 IU of DC-NILV encoding model antigens, such as LCMV gp33 and OVA, induced polyfunctional primary and secondary CD8 T cell immunity. A single immunization with 1e7 IU of DC-NILV encoding OVA induced a stable CD8 T cell memory population that provided 4 logs of protective immunity against challenge with vaccinia virus encoding OVA. DC-NILV encoding AH1A5, an endogenous rejection Ag for CT26 tumor cells, induced robust primary CD8 T cell cytokine responses and a single injection conferred 70% long-term survival in a therapeutic setting. Based on these results and methods for efficient large-scale production and purification, we are developing a DC-NILV vaccine candidate for a first-in-human evaluation in cancer doi:10.1016/j.cyto.2011.07.246

PS2-084 Dissection of kinase-dependent and -independent functions of Tyk2 in immunity to infection and tumor-surveillance Michaela Prchal a, Julianna Kobolák b, Ingeborg Teppner a, Barbara Wallner a, Christian Semper a, Caroline Lassing a, Eva Maria Putz c, András Dinnyés b, Thomas Kolbe d,e, Thomas Rülicke d,f, Marina Karaghiosoff a, Veronika Sexl c, Birgit Strobl a, Mathias Müller a,d, a Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Austria, b Molecular Animal Biotechnology Laboratory, Szent Istvan University, Gödöllö,Hungary, c Institute of Pharmacology and Toxicology , Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Austria, d Biomodels Austria, University of Veterinary Medicine Vienna,Austria, e Institute of Biotechnology in Animal Production, Department IFA-Tulln, University of Natural Resources and Applied Life Sciences, Tulln, Austria, f Institute of Laboratory Animal Science, Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Austria Tyrosine kinase 2 (Tyk2) is a member of the Janus kinase (JAK) family and has an important role in cytokine signaling. Tyk2-deficient mice show increased susceptibility to infectious diseases and impaired tumor surveillance. To study kinase dependence of Tyk2 functions in vivo we have generated kinase-inactive Tyk2 knockin mice. A lysine in the ATP-binding site of the kinase domain was replaced by glutamic acid (Tyk2K923E), a mutation already used for inactivation of several tyrosine kinases, including the JAKs. Here we show Tyk2 protein stability relies on its kinase activity in several cell types and in organs. Furthermore, efficient phosphorylation of signal transducers and activators of transcription (STATs) in response to interleukin (IL)-12 and interferon-b (IFNb) is dependent on the presence of kinase-active Tyk2 protein. Consistently, Tyk2K923E mice display similar susceptibility to viral infections as Tyk2-/mice. In contrast to these crucial requirements for Tyk2 kinase activity, we found kinase-independent functions in tumor surveillance. Wildtype and Tyk2K923E mice show higher resistance to subcutaneous MC38 colon adenocarcinoma cell-induced tumors than Tyk2-deficient mice. Although CD8+ T cells are the major effector populations for MC38 tumor growth control in wildtype mice, antibody depletion experiments revealed an important contribution of NK cells in Tyk2K923E mice. In vitro natural killer (NK) cell cytotoxicity is largely impaired in the absence of Tyk2, but partially restored in the presence of Tyk2K923E. Interestingly, Tyk2K923E and Tyk2 deficiency result in similarly increased lung melanoma nodule formation after systemic injection of B16F10 cells, which is known to be controlled by NK cells. Currently we investigate the impact of kinase-inactive Tyk2 on the composition and activation of tumor-infiltrating lymphocytes and on molecular mechanisms involved in T celland NK cell-mediated tumor surveillance. Taken together, our results indicate a crucial role for Tyk2 kinase activity for the host defense against virus infection, whereas kinase-independent Tyk2 functions contribute to immune surveillance in a tumorspecific manner. This work is supported by the Austrian Science Fund (FWF, SFB F28), the Austrian Federal Ministry of Science and Research (BM.W_Fa, GEN-AU II/III ‘‘Austromouse”), EU FP6 ‘‘Clonet” (MRTN-CT-2006-035468) and ‘‘MedRat” (LSHG-CT-2006-518240).

doi:10.1016/j.cyto.2011.07.247

PS2-085 Understanding anti-tumor immunity in breast cancer: Characterization the influence of B7-H4 in T cell responses to tumor antigens Ramtin Rahbar a,b, Albert Lin a,b, Magar Ghazarian a,b, Philipp Lang a,b, Alisha R Elford a,b,c, Woong-Kyung Suh a, Tak W. Mak a,b,c, Pamela S. Ohashi a,b,c,*, a Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, UHN, Toronto, Ontario M5S 1A8, Canada, b Department of Immunology, University of Toronto, Toronto, M5G 2M9, Ontario, Canada, c Department of Medical Biophysics, University of Toronto, Toronto, M5G 2M9, Ontario, Canada Corresponding author. Address: (Pamela S. Ohashi) Campbell Family Institute, Ontario Cancer Institute, Departments of Medical