OR.75. Identification of Cytotoxic Human Leukocyte Antigen (HLA)-DR-Restricted CD4+ T-Cell Epitopes from HSV-1 Glycoprotein B that are Frequently Recognized by Seropositive Asymptomatic Patients

Abstracts Rashmi Singh,1 Jay Bullard,1 Krystal Vonfeldt,1 Senait Assefa,1 Mamta Kalra,1 Anil Kaul,1 Robert Conrad,1 Khalid Khan,2 Harvey Sharp,2 Rashmi Kaul.1 1OSU-Center for Health Sciences, Tulsa, OK; 2University of Minnesota, Minneapolis, MN Increased systemic LPS levels may cause bacterial translocation and inflammation in the liver. Epidemiologic data of bacterial translocation, colonization and inflammation in normal livers is lacking. TLR4 is LPS receptor involved in liver homeostasis modulating innate and adaptive immunity by activation of NFκβ. We hypothesize liver homeostasis will decrease the ability of translocating commensals to activate TLR4 and NFκβ. Colonization by aerobic and facultative anaerobic bacteria in normal, primary biliary cirrhosis (PBC), and nonalcoholic steatohepatitis, NASH explant livers was studied and bacteria speciated by API system. TLR4 and pIkKα protein expression analyzed by western blotting and TLR4 mRNA levels by real-time RT-PCR. Incidence of positive culture among normals (12/14), PBC (5/ 9), and NASH (3/6) was 86%, 56% and 50%. Both gram positive and negative bacilli and cocci were isolated. Relative mRNA levels for TLR4 varied significantly (p b 0.0008). TLR4 mRNA levels in normals were comparable to PBC (p N 0.05) but lower compared to NASH (P b 0.0001). TLR4 protein levels in normals (0.39 ± 0.02, Mean ± SEM) were significantly lower than PBC (0.58 ± 0.06, p b 0.05) and NASH (0.59 ± 0.03). pIkKα protein levels in normals were low (0.83 ± 0.07) compared to PBC (1.04 ± 0.06) and NASH (1.04 ± 0.09) (p N 0.05). This is the first clinical evidence showing bacterial colonization in normal human livers comparable to cirrhotic livers without activation of TLR4 and IkKα/NFκβ in normals highlighting the importance of bacterial colonization in liver homeostasis. In a susceptible host or in a liver transplant patient the resident bacteria in the liver may lead to break in tolerance resulting in re-infections, fibrosis, cirrhosis or cancer. doi:10.1016/j.clim.2008.03.079

OR.73. Mycobacterium Tuberculosis Derived Toll-like Receptor 2 Ligand Modulate the Function of CD4+ T Cells Directly Xinchun Chen, Boping Zhou, Mingxia Zhang. Shenzhen Donghu Hospital, Shenzhen, China Recent studies showed that Toll-like receptor 2(TLR2), which is previously known to be expressed in innate immune cells, also expressed on activated CD4 T cells. The objective of this study was to investigate whether and how mycobacterium tuberculosis derived TLR2 ligand modulate the function of CD4 T cells in vivo and in intro. TLR2 expression of CD4 T cells from 33 cases of pulmonary tuberculosis and 79 healthy donors was analyzed by real-time RT-PCR and flow cytometry. Human-TLR2 transduced 293 cell line with IL-8 production as a reporter was used to determine TLR2 ligand activity in serum samples. The effect of BCG and mycobacterium tuberculosis derived TLR2 ligands (19 kDa lipoprotein, ESAT-6) on cytokine secretion and proliferation of CD4 T cell was assessed in vitro by ELISA and CFSE labeling, respectively. Our results showed that expression of TLR2 on

S31 activated CD4 T cells was significantly increased in patients with pulmonary tuberculosis compared to healthy donors (p b 0.05). Similarly, the TLR2 ligand activity was also significantly increased in circulation of patients with pulmonary tuberculosis. In vitro assay showed that 19 kDa lipoprotein, live BCG and ESAT-6 significantly enhanced the interferon-gamma secretion and proliferation of CD4 T cells activated by anti-CD3 and anti-CD28. Blocking TLR2 receptor by antibody against TLR2 abrogate these effects indicated that the dependence on TLR2. These results suggested that direct modulating function of CD4 T cells through TLR2 is a mechanism of immunopathogenesis of tuberculosis since mycobacterium tuberculosis contains high proportion of lipoprotein, the natural TLR2 ligand. doi:10.1016/j.clim.2008.03.080

OR.74. GCF2/LRRFIP1 Signals Influenza Infection and Drives a Type I Interferon Response Kathleen Sullivan, Asen Bagashev. CHOP, Philadelphia, PA Uptake of virus and the cell autonomous response to infection is carefully regulated to induce type I interferons which in turn, induce the establishment of an anti-viral state. We studied the role of a protein whose expression was found to induce type I interferon expression. LRRFIP1/GCF2 is recruited specifically to virus-containing early endosomes within a few minutes. It is also recruited to RNA-containing structures but not endosomes containing transferring or E. coli. Overexpression drives type I interferon expression at baseline and increases type I interferon transcripts after influenza infection. GCF2 co-immunprecipitates with p38, and Akt, proteins known to be involved in innate anti-viral responses. Inhibition of p38 abrogates colocalization of GCF2 with virus-containing endosomes and impairs phosphorylation of GCF2. GCF2 also colocalizes with sites of replicating virus and RNA suggesting an ability to recognize pathogen-associated patterns. Overexpression of GCF2 diminished the ability of the cells to support influenza infection. Taken together, these data suggest that GCF2 acts early in the viral entry process to signal viral infection and plays a role in initiating antiviral responses. Recruitment as early as 3 minutes after infection suggests that GCF2 is a very early sensor of viral infection. doi:10.1016/j.clim.2008.03.081

OR.75. Identification of Cytotoxic Human Leukocyte Antigen (HLA)-DR-Restricted CD4+ T-Cell Epitopes from HSV-1 Glycoprotein B that are Frequently Recognized by Seropositive Asymptomatic Patients Aziz Alami Chentoufi,1 Alex Nguyen,1 Noureddine Berka,2 Ilham Bettahi,1 Steven Wechsler,1 Anthony Nesburn,1 Lbachir BenMohamed.1 1University of California Irvine, School of Medicine, Irvine, CA 92697-4375, CA; 2Calgary Laboratory Services, Calgary, AB T2L2K8, AB, Canada The identification of “protective” (i.e. “asymptomatic”) epitopes recognized by T cells from HSV-infected asympto-

S32 matic individuals is crucial to the development of an effective herpes vaccine. Using the PepScan epitopemapping strategy a library of 179 potential peptide epitopes (15-mers overlapping by 10 aa) was identified from the HSV-1 glycoprotein B (gB), that showed protection in both animal models and humans. Eighteen groups of 10 peptides each (G1 to G18) were used for T cell antigenicity pre-screening. CD4+ T cell responses to groups and later individual peptides were studied ex vivo in asymptomatic HSV seropositive patients (n = 30) using: (i) IFN-γ ELISpot; (ii) in vitro proliferation; (iii) CD69 expression and (iv) cytotoxic CD107a/b degranulation assays. Based on responder prevalence and magnitude of induced T-cell responses, two immunodominant “asymptomatic” gB peptide groups, G4 and G14, were identified. The human CD4+ T cell response to G4 and G14 was gender-dependent, with the strongest response found in asymptomatic women. Peptides gB161-175 and gB166-180 within G4 and peptides gB661-675 and gB666-680 within G14 showed the strongest HLA-DR-dependent CD4+ T cell induction. gB161-175, gB166-180 and gB661-675 elicited ex vivo CD107a/b degranulation of CD4+ T cells, suggesting that these epitopes contain at least one cytotoxic T-cell (CTL) epitope. Thus we identified three previously unrecognized HLA-DR-restricted cytotoxic CD4+ T cell HSV 1 gB epitopes, of which B166-180 appeared to be an the strongest immunodominant “asymptomatic” CD4+ T-cell epitope. These functional CD4+ T cell epitopes should be considered in the design of effective vaccine strategies against HSV-1 in humans. doi:10.1016/j.clim.2008.03.082

OR.76. BAY 41-2272 a Potential Pharmacological Tool for Treating Phagocyte Disorders Paulo-Vitor Pereira,1 Angela Falcai,1 Edgar Oliveira,1 Walmir Aragão-Filho,1 Josias Frazao,1 Otavio Marques,1 Paolo Errante,1 Edson Antunes,2 Antonio Condino.1 1University of Sao Paulo - Institute of Biomedical Sciences, Sao Paulo, Brazil; 2 State University of Campinas Medical School, Campinas, Brazil Phagocyte activation is critical for host defense against several pathogens. We have previously demonstrated that BAY causes a significant increase on the superoxide release and gp91phox gene expression in human THP-1 cells in addition to elevation of intracellular cGMP and cAMP levels (Eur J Pharmacol. 2007, 567:43–9). In this work we further investigated the effects of BAY 41-2272 (a non-nitric oxidebased soluble guanilate cyclase activator) in phagocytosis, superoxide release, and microbicidal activity of blood phagocytes and myeloid cell lines. We used PBMC, PMN and THP-1 cell line cultured with or not BAY 41-2272 (1 μM and 3 μM) for 1 h or 48 h at 37 °C. After treatment superoxide release was tested by superoxide dismutase-inhibitable cytochrome c reduction assay, phagocytosis was evaluated through co-culture with Zymosan particles and counting of ingested particles, and microbicidal activity was measured through co-incubation with enteropathogenic E. coli followed by counting the CFU recovered from ingested bacteria. All

Abstracts cell types showed a positive response to treatment. PBMC, PMN and THP-1 treated with BAY 41-2272 produced significantly more superoxide (about 50% more), and presented significantly more phagocytic (about 54% more) and microbicidal (about twice) activity than control group. We conclude that BAY 41-2272, especially at 3 μM, significantly activated phagocyte function and microbicidal activity. The potential of this drug for treating phagocyte disorders remains to be determined. doi:10.1016/j.clim.2008.03.083

Novel Regulatory Cells & Mechanisms Sunday, June 8 2:45 pm–4:45 pm OR.77. Impaired SLAM-SLAM Homotypic Interaction Between Invariant NKT Cells and Dendritic Cells Affects Differentiation of Regulatory NKT2 Cells in Non Obese Diabetic Mice Marika Falcone, Denis Baev, Ester Badami, Simone Caielli, Francesca Ronchi. San Raffaele Scientific Institute, Milan, Italy The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation towards a regulatory type 2 is unknown. We found that co-stimulatory signals provided by the surface receptor Signaling Lymphocytic Activation Molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. In addition, we demonstrated that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell specific antigen, aGalCer). In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induce intracellular signal) upregulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells towards a regulatory NKT2 type. Our results help to understand the mechanisms involved in the differentiation of regulatory NKT2 cells and could pave the way to design more efficient methods to induce regulatory NKT2 cells and to exploit their therapeutic potential for prevention and/or treatment of autoimmune diseases. doi:10.1016/j.clim.2008.03.084