BACKGROUND & AIMS Interleukin (IL)-17–producing CD4+ helper T cells (Th17) mediate mucosal immunity and are involved in the pathogenesis of inflammatory bowel disease (IBD). They are believed to arise from the same precursor population as regulatory T (Treg) cells, but little is known about how these T-cell subsets interact under chronic inflammatory conditions. We studied Th17 cells and Treg cells isolated from intestinal lamina propria of patients with inflammatory bowel disease (IBD) to investigate their role in pathogenesis. METHODS FoxP3 expression (a marker of Treg cells) and IL-17 production were assessed in CD4+ lamina propria lymphocytes (LPLs) isolated from IBD patients and healthy subjects. IL17+FoxP3+ and IL-17+ CD4+ T-cell clones were generated by limiting dilution. An in vitro suppression assay was performed to assess the functional capacity of derived T-cell clones. RESULTS IL17+FoxP3+ T cells were identified in inflamed intestinal mucosa of patients with Crohn’s disease (CD), but not in patients with ulcerative colitis (UC) or healthy controls. These cells shared phenotypic characteristics of Th17 and Treg cells, and showed potent suppressor activity in vitro. Transforming growth factor-® was necessary and sufficient to induce development of a IL17+ FoxP3+ cell population in CD4+ LPLs derived from patients with UC. CONCLUSIONS The inflammatory environment in the intestinal mucosa of patients with CD contributes to the generation of a distinct population of Treg cells that are FoxP3+ and produce IL-17. These cells are likely to arise during differentiation of Th17 and Treg cells. Specific microenvironmental cues from tissues are likely to determine their commitment to either lineage and affect the balance between regulation and inflammation in the intestine.

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-A g7 lacking a canonical aspartic acid residue at position 57 are associated with coeliac disease 1,2 and type I diabetes 3,4 . However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues 5,6 on the basis of their spacing to proline residues 7 . The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation 8 and that T-cell responses against native gluten peptides are found 9,10 , particularly in children 11 . Here we show that 57 polymorphism promotes the recruitment of Tcell receptors bearing a negative signature charge in the complementary determining region 3 (CDR3 ) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3 . Thus, the lack of a negative charge at position 57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the Tcell response against dietary gluten. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA specific assessment of the role of MHC class II alleles in antigen-specific T-cell responses is possible in coeliac disease, because it is caused by a single and well-defined antigen, gluten, which is present in wheat, rye and barley (reviewed in ref. 12). Gluten is rich in glutamine and proline, and the high content of proline renders gluten resistant to intestinal enzymatic digestion, explaining the persistence of intact peptides for immune recognition. Notably, despite the presence of few negatively charged amino acid residues in gluten and the expression of other MHC alleles potentially capable of presenting gluten peptides, all gluten-specific CD4 + T cells isolated from the intestinal mucosa of adult patients are restricted by HLA-DQ2 or HLA-DQ8 (refs 10, 13 and 14), and most recognize a dominant peptide with a deamidated Gln-that is, a negatively charged Glu residue 5,6 .To study the T-cell response to native and deamidated gluten peptides (Fig. 1a), we immunized humanized HLA-DQ8 mice with the native 24-amino-acid gluten peptide 219-242 from the 2 (AJ133612) gliadin comprising the commonly recognized DQ8--I epitope (QGSFQPSQQ) 10 , or with a peptide deamidated at positions 229 and 237, the known ...

The Notch-1 signaling pathway is responsible for homeostatic tight junction expression in vitro, and promotes barrier function in vivo in the RAG1-adoptive transfer model of colitis. In this study, we sought to determine the role of colonic Notch-1 in the lymphoepithelial crosstalk in health and disease. We utilized in vivo and in vitro knockdown to target the expression of Notch-1. We identified that epithelial Notch-1 is required for appropriate activation of intestinal epithelial cells at steady state and upon inflammatory stimulus. Notch-1 expression modulates mucosal chemokine and cytokine secretion, and FoxP3 and effector T-cell responses. We showed that epithelial Notch-1 controls the immune function of the epithelium through crosstalk with the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways that, in turn, elicits T-cell responses. Overall, epithelial Notch-1 bridges innate and adaptive immunity in the gut. Our findings highlight an indispensable role for Notch-1-mediated signaling in the intricate epithelial-immune crosstalk, and validate that epithelial Notch-1 is necessary and sufficient to support protective epithelial proinflammatory responses.

Normal intestinal epithelial cells (IECs) could act as non-professional antigen presenting cells, selectively activating CD8+ suppressor T cells. An epithelial cell surface glycoprotein, gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical in this process. Purification and sequence analysis of mAb B9 reactive material revealed amino acid sequence homology with CEACAM5. We demonstrate that CEACAM5 has properties attributed to gp180, such as CD8α binding and activation of CD8-associated Lck. CEACAM5 is the only CEACAM member interacting with CD1d through the B3 domain. Its N-domain (recognized by B9) is required for CD8α binding. Removal of the N-domain glycosylated residues reduces B9 recognition, CD8α binding affinity and activation of LcK. Therefore conformational changes in CEACAM5 glycosylation site are critical for its interaction with CD8α. CEACAM5-activated CD8+ T cells acquire the ability to suppress the proliferation of CD4+ T cells in vitro in the presence of IL-15 or IL-7. We provide new insights into the role of CEACAM5 and define its specific immunoregulatory properties amongst the CEACAMs expressed on IECs. We suggest that unique set of interactions between CEACAM5, CD1d and CD8 render CD1d more class I-like molecule, facilitating antigen presentation and activation of CD8+ suppressor regulatory T cells.

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)–induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.