Impaired Dendritic Cell Function in Ectodermal Dysplasia with Immune Deficiency is Linked to Defective NEMO Ubiquitination

Abstracts

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seen after sensitization with MOG35–55. As such, Aire gene deficiency would differentially alter autoimmune responses according to the presence or absence of the directed epitope in the thymus. doi:10.1016/j.clim.2007.03.205

Stem Cell/Immunodeficiency/HIV Friday, June 8 2:45 pm−4:45 pm OR.25 A Novel RAG-1 Null Mutant Causes T-B-NK+ SCID in Native Americans Morton Cowan, Professor, Pediatric Bone Marrow Transplant Division, University of California, San Francisco Children’s Hospital, San Francisco, CA Severe combined immunodeficiency disease (SCID) is the most serious inherited immunological deficit. Ubiquitous DNA double strand repair factors in the non-homologous ending joining (NHEJ) pathway resolve DNA double-strand breaks (DSB) during V(D)J recombination of T and B lymphocyte receptor genes. We have described a null mutation of the Artemis gene that has relatively high occurrence among Athabascan-speaking Native Americans, which results in a complete absence of T and B lymphocytes and increased cellular sensitivity to ionizing radiation causing radiosensitive-SCID (RS-SCID). Recently we identified a novel RAG-1 null mutation, R776W, in three related children from the Dine Indian Tribe in the Canadian Northwest Territories who manifest a classic T-B-NK+ SCID phenotype. We found a normal pattern of radiation sensitivity in skin fibroblast cell lines from these patients, indicating that the NHEJ pathway is intact. The impaired activity of this RAG-1 mutant in V(D)J recombination was revealed by an enhanced green fluorescent protein (EGFP) based assay. Additional studies of the RAG-1 null mutant will evaluate DNA binding and cleavage activity, hairpin formation in vitro as well as catalysis of other DNA strand transfer reactions such as transposition. Our current results suggest that the T-B-NK+ SCID phenotype is triggered by this novel RAG-1 mutant. doi:10.1016/j.clim.2007.03.206

OR.26 HIV-1 Infectivity Inhibited by Binding of the Green Tea Catechin, Epigallocatechin Gallate, to CD4 Christina Nance, Instructor, Baylor College of Medicine, Houston, TX, Edward Siwak, Assistant Professor, Baylor College of Medicine, Virology, Houston, TX, Aarthi Ram, Research Technician, Baylor College of Medicine, Pediatrics, Houston, TX, Van Willis, Research Technician, Baylor College of Medicine, Pediatrics, Houston, TX, Susan Westerfield, Research Technician, Baylor College of Medicine, Pediatrics, Houston, TX, William Shearer, Professor, Baylor College of Medicine, Pediatrics, Houston, TX

Binding of HIV-1 envelope glycoprotein, gp120, to the CD4 T cell receptor results in HIV-1 infection. Previously, we have presented evidence of high affinity binding of the green tea catechin, epigallocatechin gallate (EGCG), to the CD4 molecule at the gp120 binding pocket. We now present evidence that EGCG inhibits the binding of gp120 on human CD4+ T cells and PBMC and prevents the HIV-1 infectivity of human macrophages and PBMC. Binding studies utilized flow cytometry. HIV-1 infectivity was assessed by HIV-1 p24 EIA. Mtropic (R5) (strains SF162, Bal), T-tropic (X4) (strain IIIB), and dual tropic (R5X4) (strain 89.6) HIV-1 isolates were used. EGCG markedly inhibited the binding of HIV-1-gp120IIIB to CD4+ T cells in a dose-dependent manner at physiologically relevant levels (32% at 0.2 μM p b 0.05, 42% at 2 μM and 47% at 20 μM p b 0.01) and higher (55% at 50 μM and 71% at 100 μM p b 0.001). EGCG significantly inhibited the infectivity of human macrophages by M- and dual-tropic HIV-1 strains at 200–400 TCID50 in a dose-dependent manner. There was 100% inhibition of p24 production by EGCG (25–100 μM) (p b 0.0001), 95% at 12 μM, and 79% at 6 μM (p b 0.001). The response by human PBMC was similar. The control catechin did not alter gp120 binding nor inhibit HIV-1 infectivity. We conclude that EGCG is able to significantly reduce the attachment of gp120 to CD4, along with a decrease in the infectivity of HIV-1 to target cells. The competitive binding properties of EGCG for the CD4 binding sites by gp120 may translate to an HIV-1 preventative strategy. doi:10.1016/j.clim.2007.03.207

OR.27 Impaired Dendritic Cell Function in Ectodermal Dysplasia with Immune Deficiency is Linked to Defective NEMO Ubiquitination Stephan Temmerman, Postdoctoral Fellow, NIH-NIAID-LHD, Bethesda, MD, Chi Ma, Senior Biologist, NIH-NIAID-LHD, Bethesda, MD, Ashish Jain, PI, NIH-NIAID-LHD, Bethesda, MD NF-kappaB essential modulator (NEMO) regulates the activation of the transcription factors NF-kappaB. Alterations in NEMO cause ectodermal dysplasia with immunodeficiency (EDI), a disorder that is characterized by defects in innate and adaptive immunity as well as abnormal development of ectoderm-derived tissues. Because the biochemical mechanism by which NEMO mutations cause immune dysfunction remains undefined, we investigated the effect of a cysteine to arginine substitution found in the NEMO zinc finger domain on dendritic cell (DC) function. Following CD40 ligand (CD40L) stimulation of DCs from two EDI patients, we found that lysine 63-linked polyubiquitination of NEMO, which promotes activation of the IKK complex, was absent. We associated this defect with preserved RelA but absent c-Rel activity. Therefore, CD40L-stimulated EDI DCs failed to synthesize the c-Rel dependant cytokine IL12, displayed reduced cell aggregates and membrane extensions, and failed to support allogeneic lymphocyte proliferation. We utilized gene expression profiling to compare the genes induced by CD40L in normal DCs but not in EDI DCs. In this gene set, we identified a number

S14 of molecules that are likely to be critical to the maturation and function of DCs. In contrast, downstream NF-kappaB activity, DC maturation, and NEMO polyubiquitination were normal in EDI DCs following stimulation with the TLR4 ligand. These results show for the first time that CD40 and TLR4 use different signaling pathways to ubiquitinate NEMO, and offer insight into how a mutation in the zinc finger domain of NEMO leads to pathway specific defects in NF-kappaB signaling and thus immune deficiency. doi:10.1016/j.clim.2007.03.208

OR.28 Treatment of HIV-infected Patients with Vitamin D-binding Protein Derived Macrophage Activating Factor (GcMAF) Eradicates HIV-Infection Nobuto Yamamoto, Director, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, Masumi Ueda, Immunology, Chief, Microbiology, Philadelphia, PA, Charles Benson, Head, Infectious Diseases, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA Serum vitamin D-binding protein (known as Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Since Nagalase is the intrinsic component of gp120, serum Nagalase activity is the sum of enzyme activities expressed in both HIV virions and envelope proteins released from HIVinfected cells. Because of Nagalase being an HIV viral protein and immunogenic, serum Nagalase was already complexed with anti-HIV immunoglobulin G (IgG) in patient blood stream. These antibodies, however, were largely not neutralizing antibodies. The IgG-bound viral proteins retained Nagalase activity that deglycosylates Gc protein. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effect in humans. Macrophages activated by administration of GcMAF (100 ng/patient) develop a large amount of Fc receptors as well as enormous variation of receptors that recognize IgG bound and unbound HIV virions. Thus, macrophages activated by GcMAF preferentially phagocytize IgG-bound HIV virions via Fc receptor mediation. Cells latently infected with HIV are unstable and spontaneously release the virions at a high rate. After less than 18 weekly administrations of 100 ng GcMAF for twenty-one nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV infection.

doi:10.1016/j.clim.2007.03.209

Abstracts

OR.29 Impaired In Vitro Regulatory T Cell Function Associated with Wiskott-Aldrich Syndrome Marsilio Adriani, Visiting Fellow, NIH/NHGRI, Bethesda, MD, Joseph Aoki, Visiting Fellow, NIH/NHGRI, Bethesda, MD, Reiko Horai, Postdoctoral Fellow, NIH/NHGRI, Bethesda, MD, Akihiro Kon, England Postdoctoral, Fellow, Bethesda, MD, Angela M. Thornton, Staff Scientist, NIH/NIAID, Bethesda, MD, Martha Kirby, Senior Research Assistant, NIH/NHGRI, Bethesda, MD, Stacie M. Anderson, Senior Research Assistant, NIH/NHGRI, Bethesda, MD, Richard M. Siegel, Investigator, NIH/NIAMS, Bethesda, MD, Pamela L. Schwartzberg, Investigator, NIH/NHGRI, Bethesda, MD, Fabio Candotti, Senior Investigator, NIH/NHGRI, Bethesda, MD Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency characterized by thrombocytopenia, eczema, recurrent infections and high incidence of malignancy. This disease is caused by mutations of the WAS protein (WASP) gene, a key regulator of actin polymerization. WAS patients show high incidence of autoimmune disorders (∼40–70%) the occurrence of which, however, does not correlate with the severity of the other classical hallmarks of the disease. A central question in understanding the pathophysiology of WAS is why immunodeficient patients develop symptoms suggestive of hyperactivation of immune compartments, such as eczema and autoimmune diseases. Since defects in CD4+CD25+ regulatory T cells (Treg) have been associated with autoimmunity, we examined the presence and function of these cells in WAS patients and Waspdeficient mice. Wasp-deficient patients and mice develop Treg cells that express Foxp3 with no detectable differences in frequency compared to controls. However, in vitro suppression assays showed that these Treg cells have impaired inhibitory function in vitro, which, in mice, could only be partially rescued by pre-activation with exogenous IL-2. The demonstration of impaired in vitro suppressor function in WASp-deficient Treg cells suggests that defective regulatory Tcell function may be an important factor contributing to immune dysregulation in WAS. doi:10.1016/j.clim.2007.03.210

OR.30 Regulatory T Cell Abnormalities Associated with Aberrant CD4+ T Cell Responses in Patients with Immune Reconstitution Disease (IRD) Nabila Seddiki, Research Fellow, Centre For Immunology and National Centre in HIV Epidemiology and Clinical Research (UNSW), Darlinghurst Up to 30% of patients with HIV commencing antiretroviral therapy (ART) late in the disease restore a pathogen-specific cellular immune response that is immunopathological and causes disease referred as immune reconstitution disease or IRD. The immunopathogenesis of IRD is not well understood and is likely to be a consequence of an aberrant reconstitution of certain T cell subsets. In a cross-sectional study, using HIV patients who developed mycobacterial IRD, we show that in comparison to those starting ARTand not developing IRD, CD4+ T cells specific for MTB and M. avium complex (MAC) antigens, produce high levels of IFN-g and IL-2 (Pb 0.01) and proliferate strongly when compared to controls. We postulated that deficits in regulatory T cells (Tregs) numbers may play a central role in