Aryl Hydrocarbon Receptor Activation in Dendritic Cells by the Small Molecular Weight Compound (VAF347) Promotes FoxP3-expressing T Cell Differentiation

S94 cytokine and chemokines commonly linked to the development of cellular and pro-inflammatory antiviral responses. The current standard treatment consists in combination regimens of Pegylated interferon-alpha plus Ribavirin and the impact in the host immune response is still poorly known. In the present study we profiled 27 cytokines, chemokines and growth factors involved in the innate and adaptive responses to the virus in the serum of 27 hepatitis C virus infected patients before and after 12 weeks of combined treatment. Hepatitis C virus infection induced most importantly the secretion of chemokines and cytokines participating of Th1 responses (MIP-1α, IP-10, TNF-α, IL-12p70, IL-2), but also of cytokines involved in the development of Th17 responses (IL-6, IL-8, IL-9 and IL-17). Hepatitis C virus infection induced also the secretion of two pro-fibrotic factors (FGF-b, VEGF). Combined treatment with Pegylated interferon-alpha plus Ribavirin down-modulated the secretion of key Th1 and Th17 pro-inflammatory mediators and also of pro-fibrotic growth factors as early as 12 weeks after treatment instauration. Correlation studies demonstrated that decrease in the levels of these mediators was significantly associated one to each other, pointing to a coordinated effect of the treatment on their secretion. This is the first work reporting a role of Th17 immunity in viral hepatitis. These results open the opportunity to evaluate the impact of the treatment with Peg-INF-α and RBV on the prevention of the immune-driven tissue damage in the infected patients. doi:10.1016/j.clim.2010.03.281

F.60. Allogeneic Blood Cell Engraftment after Cadaveric Multivisceral Organ Transplant Nauman Salim, Rodrigo Vianna, Stephen Dlouhy, Dean Hawley, Andrew Lobashevsky, Ryan Des Jean, Muhammad Mujtaba, Daniel Smith, Magdalena Czader, Byron Batteiger, Joseph Tector, Robert Nelson. Indiana University School of Medicine, Indianapolis, IN Mixed donor hematopoietic cell microchimerism occurs in some recipients of liver and multivisceral transplants. Conceivably, the number of passenger hematopoietic progenitor cells in a multi-organ graft may be sufficient to permit donor hematopoietic cell engraftment; however, immunosuppressive conditioning would be an expected requirement for full donor engraftment to occur. A 60 year old male underwent cadaveric multi-visceral (liver, stomach, small intestinal and kidney) transplantation at Indiana University Hospital and experienced autoimmune hemolytic anemia, multiple bacterial infections including disseminated Nocardiasis, cutaneous graft-versus-host reaction and pancytopenia. He was treated with transfusion support, granulocyte colony stimulating factor, cyclosporine and antibiotics for several months. Upon white blood cell recovery, autoimmune anemia recurred and his blood type converted from recipient A-positive to donor A-negative nineteen months post transplant. Bone marrow examination 20 months post transplant revealed 80% cellularity, active erythropoiesis, increased left shifted granulopoiesis and

Abstracts adequate megakaryopoiesis. Marrow mononuclear cell chimerism by short tandem repeat (STR) analysis revealed 98% donor DNA. This report provides evidence that mesenchymal cells capable of hematopoiesis reside in somatic organs in sufficient numbers to effect lymphohematopoietic reconstitution under certain circumstances in multivisceral transplant recipients. doi:10.1016/j.clim.2010.03.282

F.61. Targeting of Peptide Coupled Dendrimers to Human Dendritic Cells leads to Autologous T Cell Activation Philipp Gert, Mario Assenmacher, Peter Jähn. Miltenyi Biotec, Bergisch Gladbach, Germany Targeted delivery of vaccines to dendritic cells (DC) has been shown to be an effective approach to induce antigenspecific T cell immunity in vitro and in vivo. Peptides are used for ex vivo pulsing of DC, but not suitable to target dendritic cells in vivo. To overcome this limitation we have covalently attached 15 mers peptides covering the whole CMV pp65 amino acid sequence onto polyamidoamine (PAMAM) dendrimers and coupled them to an anti biotin antibody. Using this construct we were able to specifically target large amounts of peptides via CD36 biotin antibody to blood DC for effective stimulation of autologous CMV specific T cells analyzed by intracellular interferon gamma production. Appropriate activation of targeted DC with Toll like receptor ligands proved to be essential for effective T cell stimulation. Peptide pulsed DC were not sensitive to the antigen processing inhibitor chloroquine, whereas PAMAMpeptide targeted DC were and failed to stimulate T cells. Moreover T cell stimulation with PAMAM-peptide alone showed a substantial CD4 T cell response, but only weak CD8 response. Taken together this demonstrates that targeted PAMAM-peptides are internalized and processed before presentation via MHC class II molecules. In vivo targeting of PAMAM-peptides may represent a promising perspective for effective induction of DC derived immune responses against defined antigen epitopes. doi:10.1016/j.clim.2010.03.283

F.62. Aryl Hydrocarbon Receptor Activation in Dendritic Cells by the Small Molecular Weight Compound (VAF347) Promotes FoxP3-expressing T Cell Differentiation Kevin Goudy 1, Linda Kenins 2, José Carballido 2, MariaGrazia Roncarolo 1. 1San Raffaele Scientific Institute, Milan, Italy; 2Novartis Institutes for Biomedical Research, Basel, Switzerland The small molecular weight compound VAF347, an agonist of the aryl hydrocarbon receptor (AHR), has potent regulatory effects capable of inducing tolerance to alloantigens.

Abstracts The mechanism by which VAF347 imparts its regulatory activity in mouse is partly attributed to the expansion of FoxP3-expressing regulatory T cell (FoxP3-Treg) that occurs through an unknown mechanism. In the present study, we determined that culture of naïve CD4+ T cells in the presence of VAF347 has no direct effect on FoxP3 expression under FoxP3-polarinzing conditions. Since dendritic cells (DC) express AHR and are potent stimulators of T cells that can induce FoxP3-induction, we reasoned to evaluate whether the increased frequency of FoxP3-Treg is a result of VAF347 modulated DC. Toward this aim, bone marrow derived DC were treated with varying concentrations of VAF347, pulsed with OVA 323-339 and exposed to purified, naïve OT-II CD4+ T cells. After three days of co-culture, FoxP3 expression in OTII cells occurred in a dose-dependent manner whereby DC cultured with the greatest concentration of VAF347 induced the highest percentage of FoxP3+ T cells (approximately three-fold greater than untreated). Accordingly, the amount of proliferating OT-II responding cells was also reduced in a dose-dependent fashion. Taken together, this data suggests that VAF347 can act indirectly to promote FoxP3-induction whereby VAF347 can promote the tolerogenic properties of DC to induce the prevalence of FoxP3-Treg. doi:10.1016/j.clim.2010.03.284

F.63. Evaluation of Biologics for Autoimmunity in Murine T Cell Dependent Delayed-type Hypersensitivity (DTH) Reaction to Methylated Bovine Serum Albumin (mBSA) Anneline Nansen, Helle Markholst, Claus Haase. Novo Nordisk A/S, Maaloev, Denmark The mBSA-induced DTH reaction is a T cell-mediated immune reaction that develops in two phases: in the sensitization phase, T cells are sensitized and antigenspecific effector T cells are formed. Next, in the elicitation phase, recall responses of these T cells are induced upon secondary challenge with antigen. The effector T cells release cytokines, including IFN-γ and IL-17 that trigger recruitment of inflammatory cells such as neutrophils and macrophages. The inflammatory response reaches a maximum at 24 hrs. The advantages of the model are that it is short (7 days + 1-2 days), highly reproducible and dependent on an antigen-specific T cell immune response. Moreover, the DTH reaction is only seen in the challenged footpad and draining LN, thus the contra-lateral, PBS-challenged footpad and draining LN serve as an intra-animal control. This sitedirected and local reaction makes the model well suited for mode of action (MoA) studies of drugs that interfere with or block T cell priming, cell-migration or inflammatory cytokines. We will present data on our robust mBSA-induced mouse DTH model which we validated using reference compounds of relevance for the treatment of RA e.g. etanercept (hTNFR2-Ig) and abatacept (hCTLA-4-Ig). MoA was confirmed by functional ex vivo analyses of footpad exudate cells and popliteal LN following challenge. Our results underscore the relevance of DTH models in the

S95 evaluation of biologic drug candidates of relevance to the treatment of autoimmune diseases. doi:10.1016/j.clim.2010.03.285

F.64. The CD4+CD25- Adoptive Transfer Colitis Model: A Tool to Evaluate Immunosuppressive Therapies in IBD Thomas Holm, Helle Markholst, Stine Kjellev. Novo Nordisk, Maaloev, Denmark Background: Several animal models are used as screening tools for the development of new and better drugs in IBD. The translational value of a given model is difficult to predict without extensive analysis of the experimental system and the underlying drivers of disease. Objective: The purpose of this study was to evaluate the effect of currently used IBD drugs and IBD drug candidates i.e. TNFR-Fc, anti-TNF-α, corticosteroids, flouroqinolones, cyclosporin, anti-IL-12p40, anti-α4β7 integrin, CTLA-4-IgFc, anti-IL-6 and Copaxone in a colitis model colitis by transfer of CD25 depleted CD4+ T cells. Material and methods: 300.000 highly purified CD4 +CD25- T cells from Balb/c mice were adoptively transferred to C.B-17 scid mice by i.p. injection. Disease development was examined 3 x week during a 4 week period, previously described in (Kjellev et al., 2006 Int Immunol). Results: Early administration of either: anti-TNF-α, antibiotics, anti-IL12p40, anti-α4β7 integrin, CTLA4-IgFc or anti-IL-6 effectively prevented the onset of colitis. In contrast, mice treated with TNFR-Fc, corticosteroids, cyclosporine, copaxone or control reagents, developed severe colitis. Antibiotics, antiIL-12p40 and CTLA-4-IgFc reversed established colitis, whereas anti-TNF-α and anti-α4β7 integrin did not. Knowing the advantages and limitations of this model may provide important knowledge for future efficacy and mode of action studies. Moreover, the finding that several well established IBD therapeutics, did not show efficacy in this model, stresses the need for a better understanding of disease pathways and drivers in experimental colitis models in general. doi:10.1016/j.clim.2010.03.286

F.65. Successful Outcomes Following Matched Unrealted Donor BMT for Wiskott Aldrich Syndrome Chu Ri Shin, Mi Ok Kim, Alexandra Filipovich. Cincinnati Children's Hospital and Medical Center, Cincinnati, OH Introduction: Wiskott-Aldrich Syndrome (WAS), once a fatal disorder, is now successfully treated with bone marrow transplantation (BMT). However matched unrelated donor (MUD) transplantation remains controversial due to historically poorer outcomes. Methods: We retrospectively analyzed outcomes following BMT for 47 WAS patients treated at our institution from the time period of 1990 to 2009. Patients were divided into 2 cohorts based on timing