We also reported that Tim-4 could bind to Tim-1 and regulate T-cell responses

12. Interestingly, treatment with Tim-4-hFc fusion proteins did not change DCs function in terms of the expression of CD80, CD86, and MHC class II molecules (Supporting Information Fig. 7). However, Tim-4 also binds to PS 35, 36 and potentially another unknown receptor 38. Thus, without knowing whether DCs express other Tim-4-binding protein(s) Sunitinib manufacturer in addition to Tim-1, it is difficult to understand whether the effect of Tim-4-hFc on DCs is through Tim-1 and/or other pathway(s). These issues will only be clearly addressed using Tim-1 deficient mice, which just became available most recently 15. In summary, we show that Tim-1 plays different roles in the innate and adaptive Raf inhibitor immune responses. Since Tim-1 is constitutively expressed on DCs in the steady state, Tim-1 is readily available for crosslinking on DCs before it is even expressed on adaptive immune cells. The present study highlights the role of Tim-1 expressed on DCs in regulating the balance between effector and regulatory T cells and thus regulating immune responses. A better

understanding of the mechanism by which Tim-1 regulates DC and T cell responses will provide a target by which DC/T cell functions can be regulated so as to treat inflammatory diseases including autoimmune diseases, and to improve vaccination and tumor immunotherapy. SJL mice were purchased from The Jackson Laboratory. B10.S mice and 5B6 SJL mice transgenic for the PLP139–151-specific TCR 5B6 have been described previously 20. Foxp3/GFP ‘knock-in’ mice originally generated on the C57BL/6 background 26 were back-crossed for >10 generations onto the B10.S background. The mice were maintained, and all animal experiments were performed according to the animal protocol guidelines of Harvard Medical

School. PLP139–151 and OVA323–339 peptides were synthesized by Quality Controlled Biochemicals. Anti-Tim-1 antibodies 3B3 and RMT1-10 have been described previously 11, 16. Cytokines and antibodies Interleukin-3 receptor for FACS and ELISA were obtained from eBioscience, BD Biosciences, and R&D Systems. Different populations of immune cells were purified with MACS beads (Miltenyi Biotec). Naïve CD4+ T cells (CD4+CD62LhiCD25–) and DCs (CD11c+CD3−CD19−) were purified using a FACSAria cell sorter following MACS bead-isolation of CD4+ and CD11c+ cells, respectively. CNS-infiltrating mononuclear cells were isolated from mice with EAE as previously described 26, 27. Naïve CD4+ cells (1×106/well) were activated with either plate-bound anti-CD3/CD28 (1 μg/mL for both) or with PLP139–151 (25 μg/mL) plus syngeneic DCs (2×105/well) in the presence or absence of anti-Tim-1 (10 μg/mL).