Prostaglandin E2-dependent modulation of macrophages’ responses by colon cancer cells

Abstracts / Cytokine 48 (2009) 46–90 Most current immunotherapeutic approaches aim at inducing anti-tumor responses by stimulating the adaptive immune system, which is dependent on MHC-restricted ab T cells. However, loss of MHC molecules is often observed in cancer cells, rendering tumor cells resistant to these treatments. cd T lymphocytes constitute a promising alternative to the current immunotherapies because they are MHC-unrestricted and display strong in vitro cytotoxic activity against tumor cell lines of diverse tissue origin, and are capable of rejecting induced tumors in vivo. Despite great interest in cd T cells, the molecular mechanisms responsible for tumour cell recognition are still the object of much debate. In this study we aimed to determine the mechanism underlying hematological tumor cell recognition by V c 9V d 2 cells, the main cd T cell blood subset on human peripheral blood. Our biological system consisted of a large panel of human lymphoma and leukemia cell lines that we have recently characterized in terms of susceptibility to in vitro V c 9V d 2T cell-mediated killing. Here we show, using RNA interference, that cd T cell recognition and elimination of hematopoietic tumor is dependent malignant ULBP1 expression. Also, blockade of NKG2D, the receptor for ULBP1, significantly inhibit cd T cellmediated cytotoxicity. Importantly, we observed a large spectrum of ULBP1 levels is primary biopsies obtained from patients. We believe that these findings should be considered in upcoming clinical trials for hematological tumor based on cd T cell immunotherapy. doi:10.1016/j.cyto.2009.07.127

PP1-005 Prostaglandin E2-dependent modulation of macrophages’ responses by colon cancer cells Brandy M. Conner, Julia Ahn, Edith Chang, Alex Shnyra, Poster Presentation I Prostaglandin E2-dependent modulation of macrophages’ responses by colon cancer cells Brandy M. Conner 1, Julia Ahn 1, Edith Chang 2, Alex Shnyra 1, 1 Department of Pharmacology and Microbiology, Kansas City University of Medicine and Biosciences, Kansas City, MO, USA, 2 Department of Biochemistry, Kansas City University of Medicine and Biosciences, Kansas City, MO, USA Tumor-associated macrophages (M/) are reprogrammed by the microenvironment to adopt a distinct functional state that promotes growth and metastasis of solid tumors. We hypothesized that tumor-derived prostaglandin E2 (PGE2) modulates immuno-inflammatory responses of M/ controlled by Toll-like receptors (TLRs). To test our hypothesis we used HT-29 (Cox2low) and HCA-7 (Cox2high) human colon cancer cells and human THP-1 cells which were stably transfected with a reporter gene encoding a secreted alkaline phosphatase under the control of an NF-j B-inducible ESelectin promoter. In order to model the tumor microenvironment, M/ obtained from THP-1 by pretreatment with phorbol myristate acetate (PMA) were co-culture for 72 h with either HT-29 or HCA-7 cells. M/ ‘‘edited” by the tumor cells were then challenged with various TLRs-specific ligands including lipoarabinomannan (LAM, TLR2), dsRNA (poly I:C, TLR3), LPS O111:B4 (TLR4), recombinant flagellin (TLR5), thiazoloquinolone derivative (CL075, TLR7/TLR8), and type C CpG oligonucleotides (ODN M362, TLR9). Whereas Cox2low HT-29 cells moderately attenuated responses of the cells, PGE2-producing HCA-7 cells markedly suppressed TLRs-mediated activation of M/ derived from THP-1 cells. Furthermore, we used Oligo GEArray (Inflammatory Cytokines & Receptors Oligo GEArray, SABiosciences) to characterize regulatory role of the tumor microenvironment (PGE2) in modulation of macrophages’ activation programs at the transcriptional level. Comparative cDNA profiling of LPS-induced responses in M/ co-cultured with either HT-29 or HCA-7 cells has identified a cohort of differentially regulated genes across several major functional categories. Collectively, our data suggest that tumor-derived PGE2 may, at least in part, contribute to the microenvironment-controlled reprogramming of tumor-associated M/. doi:10.1016/j.cyto.2009.07.128

PP1-006 Exhibition of enhanced immuno-regulatory potential in human dendritic cells treated with gamma-irradiated colon cancer cells Sun Kyung Kim, Cheol-Heui Yun, Seung Hyun Han, Poster Presentation I Exhibition of enhanced immuno-regulatory potential in human dendritic cells treated with gamma-irradiated colon cancer cells Sun Kyung Kim 1, Cheol-Heui Yun 2, Seung Hyun Han 1, 1 Department of Oral Microbiology & Immunology, Dental Research Institute, and BK21 Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea, 2 Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea Dendritic cell (DC) is a potentially keen candidate for cell therapy in treatment of incurable diseases such as cancer since DC primed with tumor antigens has been shown to trigger the tumor-specific adaptive immunity and inhibit progression of

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the malignancy. In order to maximize the capacity of DCs to effectively induce antitumor immunity, we investigated whether c-irradiated tumor cells could activate and modulate phenotypic and functional changes in the DCs. When human DCs were stimulated with c-irradiated HT-29, a human colon cancer cell-line, there was an increase in the surface expression of DC maturation markers including co-stimulatory molecules (CD80 and CD86), MHC class I and II proteins, and CD205 with a coincident decrease in the expression of CD206. Furthermore, it is evident that the irradiated cancer cells were phagocytosed by DCs. Interestingly, the irradiated HT-29 did not induce the expression of inflammatory cytokines (TNF- a 8nd IL-12) in DCs but increased the expression of a chemokine, IL-8, and anti-inflammatory cytokine, IL10. In addition, DCs primed with c-irradiated HT-29 activated autologous T-lymphocyte as determined by the analysis of CD69 expression on the T-cells while T-cell proliferation was rather attenuated under the same condition. Collectively, these results suggest that c-irradiated cancer cells could activate and induce the maturation of DCs that essentially required for the subsequent adaptive immunity in cancer therapy. doi:10.1016/j.cyto.2009.07.129

PP1-007 Development of recombinant vesicular stomatitis virus for use as an oncolytic vector in cancer therapy Joshua Heiber, Jinhee Hyun, Masatsugu Obuchi, Glen N. Barber, Poster Presentation I Development of recombinant vesicular stomatitis virus for use as an oncolytic vector in cancer therapy Joshua Heiber 1, Jinhee Hyun 2, Masatsugu Obuchi 1, Glen N. Barber 1,2, 1 Department of Microbiology and Immunology, University of Miami, Miami, FL, USA, 2 Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Oncolytic virotherapy is emerging as a new treatment option for cancer. Vesicular Stomatitis Virus (VSV) has a number of inherent characteristics that give it the ability to be used as an oncolytic vector. It has a low morbidity and a simple 11kb genome, encoding 5 genes N, P, M, G, and L that can be genetically manipulated. VSV also preferentially replicates in and kills transformed versus non-transformed cells. The replication and killing in transformed cells is facilitated by acquired defects in innate signaling pathways or translation regulation systems. Our lab has focused on engineering new recombinant VSVs (rVSV) that retain oncolytic ability while being safer to surrounding normal cells. Trans-genes of interest, like the type 1 interferon (IFN) IFN-b that modulate the innate and adaptive immune responses, can be incorporated in the virus. This leads to the generation of a more attenuated virus in healthy cells that can still exhibit potent oncolytic activity. In this study the murine IFN-b gene was inserted between the G and L proteins of wild type VSV. After generation of the VSV-IFN-b virus, our analysis confirmed that the virus efficiently expresses the IFN-b trans-gene. Additionally, the IFN-b expressed by VSV is functional and can be used to pre-treat healthy cells leading to protection from viral infection. VSV-IFN-b is also able to replicate and kill tumor cells with the same efficiency as control rVSV or VSV-GFP. In vivo, VSV-IFN-b was able to protect mice better than control rVSV from tumor development after challenge from a number of different tumor lines and also led an increase in the number of IFN-c secreting T-cells compared to VSV-GFP. The generation of safe and effective oncolytic vectors is a necessary step for the development of oncolytic virotherapy as a viable treatment option. Currently, VSV-IFN-b is finishing toxicity studies in rhesus macaques and is poised to begin a phase 1 clinical trial at the Mayo Clinic for liver cancer by the end of 2009 or early 2010. doi:10.1016/j.cyto.2009.07.130

PP1-009 A transgenic animal model for the investigation of the role of type I interferons in T lymphocyte biology Nadia Kavrochorianou, Maria Evangelidou, Michael Tovey, George Thyphronitis, Sylva Haralambous, Poster Presentation I A transgenic animal model for the investigation of the role of type I interferons in T lymphocyte biology Nadia Kavrochorianou 1,4, Maria Evangelidou 2, Michael Tovey 3, George Thyphronitis 4, Sylva Haralambous 1, 1 Laboratory of Transgenic Technology, Hellenic Pasteur Institute, Athens, Greece, 2 Laboratory of Molecular Genetics, Hellenic Pasteur Institute, Athens, Greece, 3 Laboratory of Viral Oncology, CNRS, Institut Andre Lwoff, Villejuif, France, 4 Department of Biological Applications and Technologies, University of Ioannina, Ioannina, Greece Type I interferons (IFNs) exhibit a wide range of biological activities and are major immunomodulators acting on virtually all immune cells. Among them, IFN a/b is already used for the treatment of diverse diseases including cancers, virus infections, multiple sclerosis, and are proposed as an immune adjuvant in vaccine protocols. How the therapeutic activity of type I IFNs relates to their effects on different cell types remains, however, largely unknown. Therefore animal models with tissue spe-