CD161 DEFINES EFFECTOR T CELLS THAT EXPRESS LIGHT AND RESPOND TO TL1A-DR3 SIGNALING.

Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation.

Colonic bacteria express an ulcerative colitis pANCA-related protein epitope.

Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae and Escherichia coli. Isolation and partial sequencing of the B. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.

Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA.

Perinuclear anti-neutrophil cytoplasmic antibody (pANCA)(4)is a predominant serum marker of ulcerative colitis (UC), and a familial trait associated with disease susceptibility and disease associated MHC haplotypes. This study characterizes the pANCA antigen defined by representative UC-pANCA human monoclonal antibodies, Fab 5-3 and 5-2. Western blot analysis probed with Fab 5-3 revealed specific binding to a nuclear protein doublet (apparent MW=32-33 kDa) expressed in several cell types. Purification and tryptic peptide sequencing identified the protein as histone H1, and this specificity was confirmed by Fab 5-3 binding to purified H1. Rabbit anti-histone H1 immunostaining and Western blot analysis confirmed that the pANCA epitope is preferentially immunoaccessible in polymorphonuclear neutrophils (PMN). The epitope was localized to the COOH-terminal region by site-specific proteolysis, and recombinant deletants further localized binding activity for both Fab 5-2 and 5-3 to two non-overlapping segments (AA 69-171 and 172-226) associated with a recurring PKKAK motif. Serum IgG binding was detectable to these segments, but was not significantly correlated with pANCA titer or disease status. These findings indicate that histone H1 bears a recurring COOH-terminal epitope recognized by monoclonal ulcerative colitis-associated pANCA marker antibodies, but this epitope is not a predominant specificity of serum pANCA.

Identification of a novel mycobacterial histone H1 homologue (HupB) as an antigenic target of pANCA monoclonal antibody and serum immunoglobulin A from patients with Crohn's disease.

pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved approximately 32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.