The observation that 3B3-activated DCs produced IL-6 and IL-23 (Fig. 2C and D) at least partly explains the inhibition of Foxp3 induction, as blocking IL-6 and IL-23 in the Treg cultures restored Foxp3 expression and inhibited IL-17 production (Supporting Information Fig. 2). We have reported that i.p. injection of 3B3 worsened EAE in SJL mice immunized with PLP139–151/CFA emulsion 16. However, the systemic administration would allow the antibody access to many types of cells that express Tim-1 and thus could affect their function and the disease. Therefore, Afatinib to directly

assess a role for Tim-1 signaling on DC function, we immunized mice with PLP139–151/CFA emulsion containing anti-Tim-1. We reasoned that DCs, at the frontline of pathogen recognition, would most likely be the first major population affected by anti-Tim-1 in the emulsion. In this approach, anti-Tim-1 was not detectable in the sera from the mice (data not shown), indicating antibodies remained at the local administration sites. Interestingly, draining LN cells from mice treated with high-avidity anti-Tim-1 3B3 in emulsion showed both higher basal and Ag-dependent

proliferation in the responding T cells (Fig. 4A) and an increased frequency of IFN-γ- and IL-17-producing CD4+ T cells (Fig. 4B). The treatment consistently resulted in more severe and accelerated EAE compared with the control group (Fig. 4C and Table 1), while inclusion of low-avidity anti-Tim-1 RMT1-10 did not change the course of EAE (Supporting Information Fig. 3). These data suggest that the high-avidity anti-Tim-1 in the SCH727965 ic50 emulsion during the induction of EAE enhances the immunogenic Racecadotril function of DCs, which then increases the pathogenic Th1 and Th17 responses resulting in worsened disease in SJL mice. B10.S mice are congenic with SJL mice at the MHC level; however, in contrast to SJL mice, B10.S mice are resistant to EAE. Previous studies have suggested that EAE resistance in B10.S mice is in part

due to a lower APC capacity to stimulate proinflammatory T-cell responses against myelin self-antigens 20. Furthermore, B10.S mice express relatively high levels of myelin-specific Foxp3+ Tregs in their peripheral repertoire 21. Since inclusion of 3B3 anti-Tim-1 in CFA enhanced pathogenic Th1/Th17 responses and exacerbated EAE in disease-susceptible SJL mice, we asked whether the treatment would break tolerance and induce EAE in B10.S mice. In addition to having lower expression of MHC and costimulatory molecules 20, B10.S-derived DCs produced much less proinflammatory cytokines, such as IL-6, upon LPS treatment than SJL-derived DCs did. However, treatment with 3B3 anti-Tim-1 alone or together with LPS restored IL-6 production from B10.S-derived DCs to the level from SJL-derived DCs treated with LPS (Supporting Information Fig. 4). Next, B10.