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An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Pre-clinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of thirteen C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevantNrasdriver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.SIGNIFICANCENRAS-activating mutations are the second most common genetic driver event in cutaneous melanoma, occurring in 15% to 25% of cases. With few therapeutic options beyond immunotherapy, patients with NRAS-mutant melanoma have a poorer prognosis. Pre-clinical mouse models that mimic the high mutational burden of human NRAS-mutant melanomas are lacking in the field. Here, we describe a series of NRAS-mutant melanoma cell lines, derived from ultraviolet (UV)-induced, spontaneous tumors. These lines permit the study of targeted, NRAS mutant-specific, immune, and combination therapies in C57BL/6J mice. With the release of this resource, we hope to catalyze new therapeutic approaches for NRAS-mutant melanoma.

West Nile Virus (WNV) is an arbovirus of the Flaviviridae family that causes annual outbreaks of encephalitis across North America. RIG-I like receptor (RLR) signaling through the central adaptor Mitochondrial antiviral signaling protein (MAVS) is essential for promoting immunity against WNV infection. While MAVS has been associated with type I interferon (IFN) induction during virus infection, here we describe a novel, cell-intrinsic role for MAVS in regulating antigen-specific CD8+ T cell responses. MAVS is expressed in CD8+ T cells and remains constant during the differentiation of CD8+ T cells. Generation of an effective CD8+ T cell response against WNV is essential for limiting virus replication in neurons, mitigating pathology in the brain, and protecting against lethal outcome. To understand how MAVS impacts CD8+ T cell responses during WNV infection, we utilized the WNV mouse model and adoptively co-transferred WT and Mavs−/− CD8+ T cells into congenically marked WT mice. We found that compared to WT cells, Mavs−/− antigen-specific CD8+ T cells displayed reduced accumulation of antigen-specific CD8+ T cells, reduced IFN-γ production and reduced formation of memory precursor cells during virus infection. At the peak of brain infection, Mavs−/− antigen-specific CD8+ T cells displayed highly divergent transcriptomes, including altered metabolic profiles, compared to WT CD8+ T cells. Further mechanistic studies found that after TCR stimulation of CD8+ T cells, MAVS promotes oxidative respiration and remodeling of mitochondrial morphology. Our findings identify a novel, non-canonical role for MAVS in the regulation of antigen-specific CD8+ T cell responses and mitochondrial function during neuroinvasive viral infection.

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8 + T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8 + T cells behave similarly to endogenous CD8 + T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8 + T cells within the spleen and brain. We found that red-pulp WNV-I CD8 + T cells were more effector-like than white-pulp WNV-I CD8 + T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8 + T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69 + and CD103 + ), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8 + T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection.

Background and Aim Hepatocyte growth factor (HGF) is a pleiotropic cytokine related with cell proliferation and survival; however, its role in viral hepatitis is not elucidated. In this study, we studied HGF immune role in viral hepatitis. Methods Mice received hydrodynamically delivered HGF plasmid or control plasmid and then infected with adenovirus, and parameters of immune-mediated liver damage were evaluated. We studied dendritic cell (DC) activation in the presence of HGF. T cells collected from infected mice were restimulated with virally infected DC to measure cytokine production in vitro. Results HGF ameliorated the liver inflammation during viral hepatitis as alanine transferase, intrahepatic lymphocytes, and splenocyte counts were diminished by HGF. Lower histological scores of liver pathology were observed in the HGF group. DC from the HGF group expressed reduced CD40. The hepatic expression and serum concentration of IL-12p40 were diminished in HGF-transfected mice. In vitro experiments with DC confirmed that HGF diminished CD40 expression and IL-12p40 production. The expression and serum levels of IFN-γ, IL-6 and CXCL9 were significantly decreased in the HGF group. HGF overexpression diminished the expression and concentration of IL-10 and TGF-β. The frequency of PD-1+Tim-3+ in CD8 T cells was decreased by HGF overexpression. Moreover, T cells in the HGF group at day 14 secreted more IFN-γ and TNF-α than those in the control group when restimulated with virally infected DC. Conclusion HGF modulated DC activation and T cell priming, thereby limiting the immune-mediated damage in the liver. However, viral clearance was not compromised by HGF.

Antiphospholipid (aPL)/anti-β2 glycoprotein I (anti-β2GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2′) on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-β2GPI monoclonal antibody (E7) and of a constructed dimeric β2GPI I (dimer), which in vitro mimics β2GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (−/−) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (−/−) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.

West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. Currently, there are no vaccines or therapeutics approved for use in humans. Virus-specific CD8+ T cells are essential in mediating protective immunity during WNV infection. However, the mechanisms that promote protective CD8+ T cell immunity against WNV are not well understood. In this study, we characterized a novel transgenic mouse encoding CD8+ T cells to the immunodominant epitope located within the WNV NS4b protein (WNV-I). Through adoptive transfer studies in mice, we evaluated the expansion, migration, and functional properties of WNV-I CD8+ T cells during infection. In the spleen, WNV-I CD8+ T cell numbers increased on day 7 post-infection followed by contraction in cell numbers through day 15 post-infection. We observed a robust infiltration of virus-specific CD8+ T cells to the brain on day 7 concomitant with an increase in virus replication within the CNS. Interestingly, we observed clearance of infectious virus from the brain, however, WNV-I CD8+ T cells remained elevated in the brain through day 15 post-infection. Functionally, WNV-I CD8+ T cells secrete both IFN-γ and TNF-α, with peak responses in the spleen and in the brain on days 7 and 11 post-infection, respectively. Furthermore, Rag-1−/− mice that received WNV-I CD8+ T cells had lower viral burden in spleens and serum at day 7 of infection. Thus, WNV-I cells are functionally active and can mediate protective immunity against WNV infection. Further studies are underway to better understand how CD8+ T cells mediate WNV clearance in the central nervous system.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine related with cell proliferation and survival; however, its role in viral hepatitis is not elucidated. In this study, we studied HGF immune role in viral hepatitis.Mice received hydrodynamically delivered HGF plasmid or control plasmid and then infected with adenovirus, and parameters of immune-mediated liver damage were evaluated. We studied dendritic cell (DC) activation in the presence of HGF. T cells collected from infected mice were restimulated with virally infected DC to measure cytokine production in vitro.HGF ameliorated the liver inflammation during viral hepatitis as alanine transferase, intrahepatic lymphocytes, and splenocyte counts were diminished by HGF. Lower histological scores of liver pathology were observed in the HGF group. DC from the HGF group expressed reduced CD40. The hepatic expression and serum concentration of IL-12p40 were diminished in HGF-transfected mice. In vitro experiments with DC confirmed that HGF diminished CD40 expression and IL-12p40 production. The expression and serum levels of IFN-γ, IL-6 and CXCL9 were significantly decreased in the HGF group. HGF overexpression diminished the expression and concentration of IL-10 and TGF-β. The frequency of PD-1(+) Tim-3(+) in CD8 T cells was decreased by HGF overexpression. Moreover, T cells in the HGF group at day 14 secreted more IFN-γ and TNF-α than those in the control group when restimulated with virally infected DC.HGF modulated DC activation and T cell priming, thereby limiting the immune-mediated damage in the liver. However, viral clearance was not compromised by HGF.

Molecules containing damage-associated molecular patterns play an important role in many pathogenic processes. In this study, our aim was to investigate the role of IL-33, a damage-associated molecular pattern molecule, in adenovirus (Ad)-induced liver inflammation. Ad-infected mice exhibited a steadily increased IL-33 and its receptor IL-1R–like 1 expression in the liver during the first week of infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase levels and the number of Councilman bodies in the liver. Attenuated liver injury by IL-33 correlated with an increase in T regulatory cells but with a decrease in macrophages, dendritic cells, and NK cells in the liver. IL-33 enhanced both type 1 (IL-2 and IFN-γ) and type 2 (IL-5 and IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α expression in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. We found that in addition to its direct effects, IL-33 strongly induced novel nuocytes in the livers and spleens of infected mice. When cocultured with nuocytes, hepatic T cells and macrophages expressed lower levels of TNF-α. The IL-33–treated mice also demonstrated a slight delay, but no significant impairment, in eliminating an intrahepatic infection with Ad. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in acute viral hepatitis. Its direct immunoregulatory functions and ability to induce novel nuocytes further suggest to us that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.

Background Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. Results We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. Conclusions Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Our aim was to investigate the role of IL-33, the damage-associated molecular pattern (DAMP) molecule, in acute viral hepatitis. C57BL/6 mice were i.v. infected with a recombinant adenovirus. IL-33 and its receptor ST2 expression increased steadily in the liver in the first week of the infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase (ALT) levels and the number of Councilman bodies. Attenuated liver injury by IL-33 correlated with an increase in T regulatory (Treg) cells but with a decrease in macrophages, dendritic cells and NK cells in the liver. IL-33 enhanced both type 1 (IL-2, IFN-γ) and type 2 (IL-5, IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. In addition to direct effects of IL-33, we found that IL-33 strongly induced nuocytes in the liver and spleen of infected mice. When co-cultured with nuocytes, hepatic T cells and macrophages expressed lower levels of TNF-α. In addition, the adoptive transfer of nuocytes led to lower serum ALT levels in the Ad-infected recipient mice. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in acute viral hepatitis. Its direct immunoregulatory functions and ability to induce nuocytes further suggest to us that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.

Abstract 147 Background: The pathogenic mechanisms mediated by antiphospholipid (aPL) antibodies are only partially understood. Tissue factor (TF) upregulation has been shown to play an important role and recent studies have demonstrated that mice deficient in complement C3, C5 or C5a receptor (R) are resistant to the thrombogenic effects of aPL antibodies. The complement membrane attack (C5b-9 MAC) complex has been shown to bind specific receptors, to induce TF expression and to exert procoagulant effect in various cell types (i.e. endothelial cells). However whether C5b-9 MAC plays a role in the pathogenesis of thrombosis in Antiphospholipid Syndrome (APS) is unknown. Here we addressed that question by studying the effects of human aPL antibodies on thrombosis and TF upregulation in C6 deficient (−/−) mice. Methods: C6 deficient (−/−) mice and the corresponding wild-type (WT) mice C3H/HeJ (C6+/+) were injected twice with 500γg/ml IgG isolated from a patient with APS (IgG-APS) or with the corresponding control IgG-NHS. Seventy-two h after the first injection the size of induced thrombi in the femoral vein, IgG anticardiolipin (ACL) activity in the sera were determined. TF activity was determined in homogenates of carotid arteries and in peritoneal macrophages using a chromogenic assay. Results: Thrombus sizes were significantly larger in WT mice C6 (+/+) treated with IgG-APS when compared with C6 (+/+) mice treated with IgG-NHS (p= Conclusions: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects and not only underscore the importance of complement activation by aPL antibodies, but also propose its inhibition as a possibly novel therapeutic target for thrombosis in APS . Disclosures: No relevant conflicts of interest to declare.

Abstract 5062 Purpose Systemic Lupus Erythematosus (SLE) and Antiphospholipid Syndrome (APS) are two closely related diseases. APL antibodies are present in 30-40% of SLE patients. Studies have indicated that certain cytokines, chemokines, tissue factor (TF), vascular endothelial growth factor (VEGF), soluble (s) E-selectin (sE-sel), tumor necrosis (TNF)-a, may be associated with APS and/or SLE. However, whether those biomarkers are elevated in plasma of APS patients with or without SLE is uncertain. Here we examined whether the levels of cytokines/chemokines in aPL antibody-positive patients positive are different from controls. Methods Twenty-two sera/plasma of patients with significantly and persistently elevated levels aPL antibodies (IgG and/or IgM aCL, and/or IgG and/or IgM anti-β2glycoprotein I (β2GPI) and/or positive lupus anticoagulant (LAC), demographic and clinical data were obtained from an on-going pilot clinical study (clinical trials.gov#NCT00674297). Patients that were on more than 10 mg prednisone/day, or on other immunosuppressive therapy, or on hydroxychloroquine or on statins were excluded. Thirty-two healthy donors with no evidence of autoimmune, infectious or inflammatory diseases were used as controls. .Levels of IL1b, IL6, IL8, TNF-a, VEGF, IP-10, sCD40L were measured in serum using a Millipore Millliplex” Multiplex Assay; titers of sE-sel, and sTF were detected by ELISA. The Kruskal-Wallis test was used to compare levels of biomarkers in aPL positive subjects vs. controls and Spearman tests to correlate levels of the biomarkers in the different subgroups of patients. Results Significant number of aPL-positive samples had elevated levels of IL1b, IL6, TNF-a, VEGF, IP-10, sCD40L, sTF, sCD40L and sE-sel and their titers were significantly different when compared to controls. APS /SLE patients (n=8) showed significantly higher levels of TNF-a compared to APS without SLE (n=6), to “asymptomatic” aPL positive (n=4), to “asymptomatic” aPL positive with SLE (n=2) or with “catastrophic” APS (n=2) patients (p=0.00630). Conclusions This study underscores the importance of identifying biomarkers of disease that may help to better understand pathogenic mechanisms, predict disease activity and possibly to better address treatment of patients with aPL antibodies. Disclosures No relevant conflicts of interest to declare.

Abstract 5066 Purpose Statins, in addition to lower cholesterol levels, have anti-thrombotic, anti-inflammatory and cardiovascular protective effects. Cardiovascular morbidity and accelerated atherosclerosis are important SLE features, but It is unclear whether statins have beneficial effects in these patients. Objectives To assess longitudinally the impact of statins on levels of biomarkers (cytokines/chemokines), SLAM-R and SDI. Methods Sera from 21 SLE patients (ACR criteria) from a multi-ethnic, multi-center cohort were assessed at baseline and at a subsequent visit after statin treatment started. Patients on >10 mg prednisone/day, or on other immunosuppressant were excluded (age range: 16-67; mean age: 44.6 yrs); 38% African American, 9% Caucasians and 4% Puerto Rican Hispanics; 86% female. Serum levels of IL1b, IL6, IL8, IFN- α,, IP-10, MCP-1. VEGF, sCD40L and TNF- α, were measured using a Millipore Millliplex™ Multiplex Assay and titers of soluble (s) E-selectin (E-sel), intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), were detected by ELISA. High sensitive C reactive protein (hsCRP) was measured by nephelometry. Changes in the levels of biomarkers were tested using a signed rank test. Changes in levels of biomarkers and those in SLAM-R or SDI scores were examined with Spearman correlations. Results SLAM-R and SDI scores were significantly decreased by statins (p=0.0199 and 0.039, respectively). IL1b, IL6, IL8, TNF-α, VEGF, IP-10, sCD40L and sVCAM-1 levels were reduced after treatment with statins in 28%, 28%, 52%. 43%, 57%, 42%, 52% and 62% of the subjects that had detectable levels of the biomarkers at baseline, respectively, but the overall reduction was not statistically significant (see Table). hsCRP was increased in 50% of the subjects at baseline (over the cut-off point) and reduced by 37% after treatment with statins. Importantly, the changes in IL-6 after treatment with statins correlated with changes in SLICC scores (p=0.036 Conclusions Significant number of SLE patients' samples showed a decrease in levels of some cytokines and chemokines and of hsCRP after statin treatment. Importantly, SLAM-R and SDI scores were significantly reduced by statins and the changes in IL-6 correlated with a significant decrease in organ damage accrual (SLICC). Disclosures No relevant conflicts of interest to declare.