Figure 3. Tim-1 expression or defects affects the balance between regulatory and inflammatory cytokines in B cells that subsequently alter T cell responses.

A) Purified splenic CD19⁺ B cells from WT or Tim-1^−/− mice were cultured in the presence of anti-IgM ((Fab’)₂ fragment) for 24 h. Total RNA was isolated, and relative expression (mean ± SEM; n = 5) of Tim-1, IL10, IL12, IL6, and IL1b mRNA was measured by realtime PCR. * P < 0.01. B) WT total CD4⁺ T cells (10 x 10⁶/mouse) were co-transferred together with WT or Tim-1^Δmucin CD19⁺ B cells (20 x 10⁶) into Rag1^−/− mice. One day after, mice were immunized with MOG35-55/CFA to induce EAE. At the peak of disease, splenic Tim-1⁺ and Tim-1⁻ CD19⁺ B cells were purified from WT and Tim-1^Δmucin groups of mice. Total RNA was isolated, and relative expression (mean + SEM; n = 5) of Tim-1, IL10, IL12, IL6, and IL1b mRNA was measured by realtime PCR. * P < 0.01. C) WT naïve CD4⁺ T cells were cultured with splenic CD19⁺ B cells purified from WT or Tim-1^−/− IL-10^GFP/+ mice in the presence of anti-CD3 under Th0 (no cytokine), Th1 (IL-12 + anti-IL-4), Th2 (IL-4 + anti-IL-12/anti-IFN-γ), Th17 (TGF-β1 + IL-6), Tr1 (TGF-β1 + IL-27), and iTreg (TGF-β1) conditions. After culture for 4 days, production of indicated cytokines in T cells and IL-10 (GFP⁺) in B cells was measured by flow cytometry after intracellular cytokine staining. Representative of 5 independent experiments was shown. D) WT CD4⁺ naïve T cells were cultured with Tim-1⁺ or Tim-1⁻ B cells purified from WT in the presence of anti-CD3 under Th17, Tr1, and iTreg conditions. After culture for 4 days, production of indicated cytokines in T cells was measured by flow cytometry after intracellular cytokine staining. Representative data from 3 independent experiments are shown.