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Final Report Summary - GUT TC PHENOTYPES (Regulation of Pathogenic CD4 T Cell Responses in Inflammatory Bowel Disease)

The mammalian gastrointestinal tract is the largest organ of the human body. It is the organ containing the largest number of immune cells and harbours a large and diverse population of commensal bacteria that exist in a symbiotic relationship with the host. In recent years, it has become increasingly clear that the composition of the gut microbiota and its interaction with the host immune system strongly influences the health of the host. Deciphering the complex interplay between the microbiota and both the genetic and environmental factors, and the impact of the microbiota on intestinal inflammation, is therefore of great biomedical importance.
One disease complex, in which maladaptation in this host-microbial dialogue is involved, is inflammatory bowel disease (IBD). Here, this maladaptation leads to an aberrant immune response in the gut, resulting in recruitment of various effector cell populations and inflammation of gut tissue. However, the exact aetiology of IBD remains uncertain, but it is thought to be a multifactorial disease that involves a complex interplay between genetic, environmental, microbial, and immune factors.
Cells of both the innate and adaptive arms of the immune response contribute to the cellular network that drives chronic intestinal inflammation through secretion of pro inflammatory cytokines. There is evidence that CD4+ T cells, monocytes/macrophages, NKT cells and innate lymphoid cells are key elements of the pathogenesis of IBD. Taken together, the aberrant response of the mucosal immune system, to the altered indigenous flora and other luminal antigens, drives intestinal inflammation and injury in in genetically susceptible individuals, with cytokines playing a central role in modulating inflammation.
As the aetiology of IBD remains unresolved and highly complex, clinical management requires lifelong pharmacotherapy with immunosuppressive and anti inflammatory drugs despite wide ranging deleterious side effects. Surgical intervention is often required due to refractory disease or complications. Biological therapies targeting certain inflammatory molecules have shown promising results. Antibody blockade of the cytokine TNF-a is now an integral part of IBD clinical management, however many patients remain non responders. Several other biological therapies (anti IL-17A, anti IFN-g, anti IL 12p40, anti integrin alpha4 beta7, anti CCR9) were recently introduced to the clinics or failed in recent clinical studies although they proved effective in other immune mediated disorders and others are currently still under clinical evaluation.

In this project, we combined mouse and human T cell immunology, mucosal immunology and animal models of disease as well as primary human tissue samples in order to identify microbial and inflammatory drivers that promote maladaptation and gut tissue inflammation in a multidisciplinary approach.
As a basis for the future development of novel and more specific therapies for IBD, this project aims at identifying the signals and molecular events that induce and sustain pathogenic CD4+ T cells in IBD. The project will address the following scientific questions:
1. What are the phenotypic and functional characteristics of mucosal T cell populations in healthy and IBD intestine including analysis of response to commensal and pathogenic intestinal bacteria? 2. What are the cell-extrinsic signals that promote and modulate mucosal CD4+ T cells toward pathogenic or protective phenotypes with particular emphasis on the role of IL 23 and IL-1 family members?
3. What are the signals that are involved in IBD pathogenesis and can these be over-ridden to promote homeostasis?

1. Isolation and characterization of CD4+ T cells from healthy donors and IBD patients using single cell analysis of cell surface molecules, signal transduction molecules, lineage-specific transcription factors and cytokines as well as gene expression profiling.
2. Analysis of the reactivity of circulating and gut-resident CD4 T cells by analysing the responses towards intestinal bacteria including pathogens.
3. Identification of targetable cytokine signals and pathogenic molecular pathways in IBD

Part 1: Gut Microbiota Induce Local And Systemic CD4 T Cell Responses In Healthy Individuals That Are Altered In Inflammatory Bowel Diseases
We established that for each bacterial strain tested, the healthy T cell repertoire contains specific cells at a frequency of 40–500 per million CD4+ T cells. Microbiota reactive CD4+ T cells were mainly of a memory phenotype, present in both blood and gut tissue, had a diverse TCRVbeta repertoire, and showed little clonotype sharing. These cells were functionally heterogeneous, produced barrier protective cytokines, and could stimulate intestinal stromal and epithelial cells through IL 17A, IFN γ, and TNF-a. Microbiota reactive CD4+ T cells were recruited to sites of inflammation and showed a phenotypic shift towards a Th17 phenotype in IBD. Thus, gut microbiota specific CD4+ T cells are part of the normal human T cell repertoire and do not necessarily indicate disturbed host microbiota interactions. T cell responses to commensals may therefore support local and systemic immune responses by generating a plethora of memory T cells reactive to pathogens.

• Microbiota specific memory T cells exist in healthy adults but not in cord blood
• Microbiota specific T cells have diverse TCRVbeta and show minimal clonotype sharing
• These cells are functionally heterogeneous and produce barrier-protective cytokines
• These cells accumulate in inflamed gut tissue and show enhanced IL 17A production

Part 2: Oncostatin-M signaling is increased in inflammatory bowel disease and promotes intestinal inflammation by activating gut-resident stromal cells.
Inflammatory bowel diseases are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed cytokine pathways. Anti-tumour necrosis factor-alpha (TNF) antibodies are a mainstay therapeutic approach in IBD. However, many patients are non-responsive to anti-TNF agents, and identifying alternative therapeutic targets is an urgent priority. Here we show that the cytokine Oncostatin-M (OSM) and its receptor (OSMR) are highly expressed in the inflamed intestinal tissue of IBD patients and correlate closely with histopathological disease severity. Surprisingly, OSMR expression is restricted to non-hematopoietic, non-epithelial intestinal stromal cells, which respond to OSM by expressing a range of pro-inflammatory factors including CXCL1, CCL2, the CXCR3 ligands CXCL9/10/11, and ICAM-1. In an animal model of anti-TNF refractory IBD, genetic deletion or pharmacological blockade of OSM significantly attenuated colitis. High expression of OSM, OSMR, and OSM-induced stromal factors strongly predicts failure of anti-TNF therapy in human CD and UC patients, and these genes are expressed during experimental colitis in an OSM-dependent manner. OSM is thus a potential biomarker and therapeutic target for IBD, with particular relevance for anti-TNF refractory patients.

- OSM and OSMR are highly expressed in mouse colon tissue during colitis and correlate with inflammation severity
- OSM and its receptor (OSMR) are highly expressed in the intestinal mucosa of both UC and CD patients and correlate with disease severity
- High OSM expression in pre-therapeutic intestinal biopsies predicts non-responsiveness to anti-TNF therapy
- OSM knockout mice are protected from aggressive colitis
- CD4+ T cells require OSM to drive aggressive colitis
- OSM is a potential biomarker and therapeutic target for IBD

Inflammatory bowel disease represents a non-curable, recurrent disease which can significantly impair life quality of the patients, has a relevant economic impact and places a substantial burden on health care resources in Europe and industrial countries worldwide. Additionally, developing neoplasia leading to colorectal cancer is significantly increased in ulcerative colitis (UC) and most likely in Crohn's disease. Currently an estimated 50,000 to 68,000 new cases of ulcerative colitis and 23,000 to 41,000 new cases of Crohn’s disease are diagnosed annually throughout Europe.
The mucosal immune system drives intestinal inflammation and injury in IBD, with cytokines playing a central role in modulating inflammation. Biological agents targeted against leukocyte adhesion, Th1/Th17 polarization, T cell activation, nuclear factor-kappaB (NF-kB), and other miscellaneous therapies are being actively evaluated as potential therapies for the treatment of IBD. However, several of these drugs failed in IBD treatment. Therefore alternative agents, particularly those targeting the immunological memory that sustains intestinal inflammation, are required. The project focused on primary human microbiota-reactive CD4+ T cells characterisation and manipulation of pathological memory. Additionally we evaluated the role of novel cytokine in IBD pathogenesis. Therefore, the project is relevant for development of novel therapeutic targets and is particularly timely, because the field of biological therapies is rapidly developing as an integral part of the management of immune-mediated inflammatory diseases.

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