W.O. CCL20 in the small intestine, facilitating the migration of these cells specifically to the small intestine Oxtriphylline via the CCR6/CCL20 axis. Moreover, we found that TH17 cells are controlled by two different mechanisms in the small intestine: first, they are eliminated via the intestinal lumen and simultaneously pro-inflammatory TH17 cells acquire a regulatory phenotype within vitroandin vivoimmune-suppressive properties (rTH17). These results identify mechanisms limiting TH17 cell pathogenicity and implicate the gastrointestinal tract as a site for control of TH17 cells. TH17 cells have been associated with the pathogenesis of several chronic inflammatory disorders including rheumatoid arthritis and multiple sclerosis2,7. To study the cellular and molecular mechanisms that control pathogenicity mediated by TH17 cells we first utilized the CD3-specific antibody treatment model. It is known that CD3-specific antibody treatment induces a cytokine storm and local inflammation mainly in the small intestine8. Despite this it has been validated as anin vivomodel of tolerization9and is now under study in human clinical trials10. By mimicking antigen, CD3-specific antibody treatment leads to activation induced cell death (AICD) of T cells11,12and consequently a systemic up-regulation of IL-69and transforming growth factor- (TGF-1) induced by phagocyte engulfment of apoptotic T cells13. In line with these publications, we found that CD3-specific antibody treatment induced an immuno-regulatory environment marked by simultaneous expression of TGF-1 and IL-6 (Fig.1a). The combination of these cytokines is important for the development of TH17 cellsin vitroandin vivoas it has been previously clearly established3,4. Accordingly, we found elevated levels of IL-17A in plasma of CD3-specific antibody treated animals compared to controls (Fig.1a). == Figure 1. Accumulation of TH17 cells in the small intestine after CD3-specific antibody treatment. == Mice were injected with CD3-specific antibody. (a) Plasma levels of TGF-1, IL-6 and IL-17A. (means.e.m.; n=4). (b) Flow cytometric analysis of IL-17A-eGFP expression (gated on CD4+TCR+events); numbers in quadrants indicate percent cells in each. (c) Immunofluorescence staining of frozen sections of the small intestine after CD3-specific antibody treatment (eGFP: green, CD4: red, and cell nuclei: DAPI). PROCR (Scale bar, 50m). Data are representative of at least three independent experiments. First, we aimed to investigate the source of IL-17A. It has been reported that a few hours after injection of CD3-specific antibody, there is a rapid disappearance of the majority of T cells from the circulation13,14. Surprisingly, in parallel with the disappearance of T cells from the periphery we found a concomitant increase Oxtriphylline in the percentage and the number of total T cells in the small intestine, in particular in the duodenum (Supplemental Fig.1ac). In a newly generated IL-17A-eGFP knock-in mice (Supplemental Fig.2ad and 3ac) injected with CD3-specific antibody, 5080% of the CD4+TCR+T cells located in the duodenum were expressing IL-17A (Figs.1bandSupplemental Fig.1d,e). The percentage and number of TH17 cells in the intestine decreased from the duodenum to the colon in a gradient-like fashion (Fig.1b). Detection of CD4+eGFP+T cells by immunofluorescence and two-photon-laser-scanning microscopy confirmed the high frequency of TH17 cells in the small intestinein situ(Fig.1candSupplemental Fig.4ac). Importantly, we also found TH17 cell infiltration in the duodenum when animals were injected with a therapeutic non-FcR-binding CD3-specific antibody15, although the frequency and numbers of the TH17 cells were lower compared to the FcR-binding antibody (Supplemental Fig.5a). Similar results were observed after antigen-specific stimulation when soluble myelin oligodendrocyte glycoprotein antigen (MOG) was administered to MOG-TCR transgenic mice (2D2 mice)16(Supplemental Fig.5b). Taken together these data suggest that the generation and the accumulation of TH17 cells in the small intestine was not restricted to the CD3-specific antibody treatment but was a general mechanism following strong TCR stimulation. Oxtriphylline We next wanted to identify the molecular signals important for the generation of TH17 cellsin vivoafter CD3-specific antibody treatment. Since IL-6 is known to be important for TH17 cell generation, we evaluated the importance of this cytokine.Il6/and wild type mice were treated with CD3-specific antibody. In theIl6/mice, only a very small population of TH17 cells (about 2%) could be found by flow cytometry in the small intestine (Supplemental Fig. 6a) and IL-17A was undetectable in the plasma (data not.