Hilary Clark

The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells

Here we have identified a surface protein, TIGIT, containing an immunoglobulin variable domain, a transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif that was expressed on regulatory, memory and activated T cells. Poliovirus receptor, which is expressed on dendritic cells, bound TIGIT with high affinity. A TIGIT-Fc fusion protein inhibited T cell activation in vitro, and this was dependent on the presence of dendritic cells. The binding of poliovirus receptor to TIGIT on human dendritic cells enhanced the production of interleukin 10 and diminished the production of interleukin 12p40. Knockdown of TIGIT with small interfering RNA in human memory T cells did not affect T cell responses. TIGIT-Fc inhibited delayed-type hypersensitivity reactions in wild-type but not interleukin 10–deficient mice. Our data suggest that TIGIT exerts immunosuppressive effects by binding to poliovirus receptor and modulating cytokine production by dendritic cells.

Genome-wide association study for Crohn's disease in the Quebec Founder Population identifies multiple validated disease loci

Genome-wide association (GWA) studies offer a powerful unbiased method for the identification of multiple susceptibility genes for complex diseases. Here we report the results of a GWA study for Crohns disease (CD) using family trios from the Quebec Founder Population (QFP). Haplotype-based association analyses identified multiple regions associated with the disease that met the criteria for genome-wide significance, with many containing a gene whose function appears relevant to CD. A proportion of these were replicated in two independent German Caucasian samples, including the established CD loci NOD2 and IBD5. The recently described IL23R locus was also identified and replicated. For this region, multiple individuals with all major haplotypes in the QFP were sequenced and extensive fine mapping performed to identify risk and protective alleles. Several additional loci, including a region on 3p21 containing several plausible candidate genes, a region near JAKMIP1 on 4p16.1, and two larger regions on chromosome 17 were replicated. Together with previously published loci, the spectrum of CD genes identified to date involves biochemical networks that affect epithelial defense mechanisms, innate and adaptive immune response, and the repair or remodeling of tissue.

Immune response in silico (IRIS): immune-specific genes identified from a compendium of microarray expression data

Immune cell-specific expression is one indication of the importance of a genes role in the immune response. We have compiled a compendium of microarray expression data for virtually all human genes from six key immune cell types and their activated and differentiated states. Immune Response In Silico (IRIS) is a collection of genes that have been selected for specific expression in immune cells. The expression pattern of IRIS genes recapitulates the phylogeny of immune cells in terms of the lineages of their differentiation. Gene Ontology assignments for IRIS genes reveal significant involvement in inflammation and immunity. Genes encoding CD antigens, cytokines, integrins and many other gene families playing key roles in the immune response are highly represented. IRIS also includes proteins of unknown function and expressed sequence tags that may not represent genes. The predicted cellular localization of IRIS proteins is evenly distributed between cell surface and intracellular compartments, indicating that immune specificity is important at many points in the signaling pathways of the immune response. IRIS provides a resource for further investigation into the function of the immune system and immune diseases.

Deconvolution of Blood Microarray Data Identifies Cellular Activation Patterns in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a complex spectrum of cellular and molecular characteristics including several dramatic changes in the populations of peripheral leukocytes. These changes include general leukopenia, activation of B and T cells, and maturation of granulocytes. The manifestation of SLE in peripheral blood is central to the disease but is incompletely understood. A technique for rigorously characterizing changes in mixed populations of cells, microarray expression deconvolution, has been applied to several areas of biology but not to SLE or to blood. Here we demonstrate that microarray expression deconvolution accurately quantifies the constituents of real blood samples and mixtures of immune-derived cell lines. We characterize a broad spectrum of peripheral leukocyte cell types and states in SLE to uncover novel patterns including: specific activation of NK and T helper lymphocytes, relationships of these patterns to each other, and correlations to clinical variables and measures. The expansion and activation of monocytes, NK cells, and T helper cells in SLE at least partly underlie this diseases prominent interferon signature. These and other patterns of leukocyte dynamics uncovered here correlate with disease severity and treatment, suggest potential new treatments, and extend our understanding of lupus pathology as a complex autoimmune disease involving many arms of the immune system.

Regional Variation in Gene Expression in the Healthy Colon is Dysregulated in Ulcerative Colitis

Objective: To investigate differential intestinal gene expression in patients with ulcerative colitis and in controls. Design: Genome-wide expression study (41 058 expression sequence tags, 215 biopsies). Setting: Western General Hospital, Edinburgh, UK, and Genentech, San Francisco, USA. Patients: 67 patients with ulcerative colitis and 31 control subjects (23 normal subjects and 8 patients with inflamed non-inflammatory bowel disease biopsies). Interventions: Paired endoscopic biopsies were taken from 5 specific anatomical locations for RNA extraction and histology. The Agilent microarray platform was used and confirmation of results was undertaken by real time polymerase chain reaction and immunohistochemistry. Results: In healthy control biopsies, cluster analysis showed differences in gene expression between the right and left colon. (χ2 = 25.1, p<0.0001). Developmental genes, homeobox protein A13 (HOXA13), (p = 2.3×10−16), HOXB13 (p<1×10−45), glioma-associated oncogene 1 (GLI1) (p = 4.0×10−24), and GLI3 (p = 2.1×10−28) primarily drove this separation. When all ulcerative colitis biopsies and control biopsies were compared, 143 sequences had a fold change of >1.5 in the ulcerative colitis biopsies (0.01>p>10−45) and 54 sequences had a fold change of <−1.5 (0.01>p>10−20). Differentially upregulated genes in ulcerative colitis included serum amyloid A1 (SAA1) (p<10−45) the alpha defensins 5 and 6 (DEFA5 and 6) (p = 0.00003 and p = 6.95×10−7, respectively), matrix metalloproteinase 3 (MMP3) (p = 5.6×10−10) and MMP7 (p = 2.3×10−7). Increased DEFA5 and 6 expression was further characterised to Paneth cell metaplasia by immunohistochemistry and in situ hybridisation. Sub-analysis of the inflammatory bowel disease 2 (IBD2) and IBD5 loci, and the ATP-binding cassette (ABC) transporter genes revealed a number of differentially regulated genes in the ulcerative colitis biopsies. Conclusions: Key findings are the expression gradient in the healthy adult colon and the involvement of novel gene families, as well as established candidate genes in the pathogenesis of ulcerative colitis.

Gene Expression Patterns of Th2 Inflammation and Intercellular Communication in Asthmatic Airways

Asthma is canonically thought of as a disorder of excessive Th2-driven inflammation in the airway, although recent studies have described heterogeneity with respect to asthma pathophysiology. We have previously described distinct phenotypes of asthma based on the presence or absence of a three-gene “Th2 signature” in bronchial epithelium, which differ in terms of eosinophilic inflammation, mucin composition, subepithelial fibrosis, and corticosteroid responsiveness. In the present analysis, we sought to describe Th2 inflammation in human asthmatic airways quantitatively with respect to known mediators of inflammation and intercellular communication. Using whole-genome microarray and quantitative real-time PCR analysis of endobronchial biopsies from 27 mild-to-moderate asthmatics and 13 healthy controls with associated clinical and demographic data, we found that asthmatic Th2 inflammation is expressed over a variable continuum, correlating significantly with local and systemic measures of allergy and eosinophilia. We evaluated a composite metric describing 79 coexpressed genes associated with Th2 inflammation against the biological space comprising cytokines, chemokines, and growth factors, identifying distinctive patterns of inflammatory mediators as well as Wnt, TGF-β, and platelet-derived growth factor family members. This integrated description of the factors regulating inflammation, cell migration, and tissue remodeling in asthmatic airways has important consequences for the pathophysiological and clinical impacts of emerging asthma therapeutics targeting Th2 inflammation.

Association of IFIH1 and other autoimmunity risk alleles with selective IgA deficiency

To understand the genetic predisposition to selective immunoglobulin A deficiency (IgAD), we performed a genome-wide association study in 430 affected individuals (cases) from Sweden and Iceland and 1,090 ethnically matched controls, and we performed replication studies in two independent European cohorts. In addition to the known association of HLA with IgAD, we identified association with a nonsynonymous variant in IFIH1 (rs1990760G>A, P = 7.3 × 10−10) which was previously associated with type 1 diabetes and systemic lupus erythematosus. Variants in CLEC16A, another known autoimmunity locus, showed suggestive evidence for association (rs6498142C>G, P = 1.8 × 10−7), and 29 additional loci were identified with P < 5 × 10−5. A survey in IgAD of 118 validated non-HLA autoimmunity loci indicated a significant enrichment for association with autoimmunity loci as compared to non-autoimmunity loci (P = 9.0 × 10−4) or random SNPs across the genome (P < 0.0001). These findings support the hypothesis that autoimmune mechanisms may contribute to the pathogenesis of IgAD.

Functional Studies on the IBD Susceptibility Gene IL23R Implicate Reduced Receptor Function in the Protective Genetic Variant R381Q

Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohns disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23RR381 and IL23RQ381 haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23RQ381 was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23RQ381 positive donors. Our study shows conclusively that IL23RQ381 is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD.

Characterization of Intestinal Gene Expression Profiles in Crohn's Disease by Genome-wide Microarray Analysis

Background: Genome‐wide microarray expression analysis creates a comprehensive picture of gene expression at the cellular level. The aim of this study was to investigate differential intestinal gene expression in patients with Crohns disease (CD) and controls with subanalysis of confirmed CD susceptibility genes, associated pathways, and cell lineage. Methods: In all, 172 biopsies from 53 CD and 31 control subjects were studied. Paired endoscopic biopsies were taken at ileocolonoscopy from five specific anatomical locations including the terminal ileum (TI) for RNA extraction and histology. The 41,058 expression sequence tags were analyzed using the Agilent platform. Results: Analysis of all CD biopsies versus controls showed 259 sequences were upregulated and 87 sequences were downregulated. Upregulated genes in CD included SAA1 (fold change [FC] +7.5, P = 1.47 × 10−41) and REGL (FC +7.3, P = 2.3 × 10−16), whereas cellular detoxification genes including‐SLC14A2 (FC‐2.49, P = 0.00002) were downregulated. In the CD TI biopsies diubiquitin (FC+11.3, P < 1 × 10−45), MMP3 (FC+7.4, P = 1.3 × 10−11), and IRTA1 (FC‐11.4, P = 4.7 × 10−12) were differentially expressed compared to controls. In the colon SAA1 (FC+6.3, P = 5.3 × 10−8) was upregulated and thymic stromal lymphopoietin (TSLP) (FC‐2.3, P = 2.7 × 10−6) was downregulated comparing noninflamed CD and control biopsies, and the colonic inflammatory CD signature was characterized by downregulation of the organic solute carriers‐SLC38A4, SLC26A2, and OST alpha. Of CD susceptibility genes identified by genome‐wide association scan IL‐23A, JAK2, and STAT3 were upregulated in the CD group, confirming the dysregulation of Th17 signaling. Conclusions: These data characterize the dysregulation of a series of specific inflammatory pathways highlighting potential pathogenic mechanisms as well as areas for translation to therapeutic targets. (Inflamm Bowel Dis 2010)

Negative regulation of autoimmune demyelination by the inhibitory receptor CLM-1

Multiple sclerosis and its preclinical model, experimental autoimmune encephalomyelitis, are marked by perivascular inflammation and demyelination. Myeloid cells, derived from circulating progenitors, are a prominent component of the inflammatory infiltrate and are believed to directly contribute to demyelination and axonal damage. How the cytotoxic activity of these myeloid cells is regulated is poorly understood. We identify CMRF-35–like molecule-1 (CLM-1) as a negative regulator of autoimmune demyelination. CLM-1 is expressed on inflammatory myeloid cells present in demyelinating areas of the spinal cord after immunization of mice with MOG35-55 (myelin oligodendrocyte glycoprotein) peptide. Absence of CLM-1 resulted in significantly increased nitric oxide and proinflammatory cytokine production, along with increased demyelination and worsened clinical scores, whereas T cell responses in the periphery or in the spinal cord remained unaffected. This study thus identifies CLM-1 as a negative regulator of myeloid effector cells in autoimmune demyelination.