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. 2015 Feb 15;194(4):1602-8.
doi: 10.4049/jimmunol.1402632. Epub 2015 Jan 12.

Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation of tissue inflammation

Sheng Xiao  1 , Craig R Brooks  2 , Raymond A Sobel  3 , Vijay K Kuchroo  1
Affiliations

Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation of tissue inflammation

Sheng Xiao et al. J Immunol. .

Abstract

T cell Ig and mucin domain (Tim)-1 identifies IL-10-producing regulatory B cells (Bregs). Mice on the C57BL/6 background harboring a loss-of-function Tim-1 mutant showed progressive loss of IL-10 production in B cells and with age developed severe multiorgan tissue inflammation. We demonstrate that Tim-1 expression and signaling in Bregs are required for optimal production of IL-10. B cells with Tim-1 defects have impaired IL-10 production but increased proinflammatory cytokine production, including IL-1 and IL-6. Tim-1-deficient B cells promote Th1 and Th17 responses but inhibit the generation of regulatory T cells (Foxp3(+) and IL-10-producing type 1 regulatory T cells) and enhance the severity of experimental autoimmune encephalomyelitis. Mechanistically, Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. Treatment with ACs reduces the severity of experimental autoimmune encephalomyelitis in hosts with wild-type but not Tim-1-deficient Bregs. Collectively, these findings suggest that in addition to serving as a marker for identifying IL-10-producing Bregs, Tim-1 is also critical for maintaining self-tolerance by regulating IL-10 production in Bregs.

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Figures

Figure 1
Figure 1. Tim-1Δmucin mice at 16-18+ months of age develop splenomegaly and multi-organ inflammation
A) Representative images of organs from 16-18+-month old WT and Tim-1Δmucin mice. B&C) Representative flow cytometry plots showing CD4+ T cell phenotypes in spleens (B) and livers (C) isolated from 16-18+-month old WT and Tim-1Δmucin mice (n = 10). D) Representative histopathology of livers and lungs from 16-18+-month-old WT and Tim-1Δmucin mice. There are massive mononuclear cell infiltrates in the Tim-1Δmucin mice. Hematoxylin and eosin stain, 15x.
Figure 2
Figure 2. Tim-1 and BCR or IL-21 signaling together strongly promoted B cell IL-10 production while a defect in Tim-1 signaling in B cells reduced IL-10 production
Purified splenic CD19+ B cells from 2-3 month-old WT, Tim-1Δmucin or Tim-1−/− mice were cultured in the presence of anti-Tim-1 (clone 5F12), (Fab’)2 fragment anti-IgM or both without (A) or with IL-21 (B). After 3 days, IL-10 production in culture supernatants was measured by ELISA. * P < 0.01; ns, not significant. C) Representative flow cytometry plots showing Tim-1 expression by splenic CD19+ B cells from WT and Tim-1−/− mice after 3-day culture in the presence of IL-21. n ≥ 3 per group.
Figure 3
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-1Δmucin 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-1Δmucin 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-10GFP/+ 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.
Figure 4
Figure 4. Effect of Tim-1 expression or defects in B cells on EAE and T cell responses
A) WT total CD4+ T cells (10 × 106/mouse) were co-transferred together with either WT or Tim-1−/− CD19+ B cells (20 × 106) into Rag1−/− mice; the recipients were then immunized with MOG35-55/CFA to induce EAE. Mice were scored daily for clinical signs of EAE (left panel; n =10 per group). * P < 0.05. On day 20, CNS-infiltrating mononuclear cells were isolated and examined for the frequencies of IFN-γ+, IL-17+, and Foxp3+ cells in CD4+ T cell gates by flow cytometry after intracellular staining (middle panel; n = 5). CNS-infiltrating CD4+ T cells were also isolated and measured for their IL10 mRNA expression by realtime PCR (right panel; ** P < 0.01; n = 4 per group). B) WT total CD4+ T cells (10 × 106) were co-transferred together with WT (20 × 106), Tim-1-−/− (20 × 106), or Tim-1−/− (20 × 106) plus WT Tim-1+ (2 × 106) B cells into Rag1−/− mice; the recipients were then immunized with MOG35-55/CFA to induce EAE. Mice (n = 8-10 per group) were scored daily for clinical signs of EAE. * P < 0.05.
Figure 5
Figure 5. Effect of apoptotic cells on WT and Tim-1−/− B cells and the development of EAE
A) WT, Tim-1Δmucin and Tim-1−/− B cells from 2-4 month-old mice were incubated with CMFDA-labeled apoptotic WT thymocytes (AC) for 30 min, and analyzed by flow cytometry. WT and Tim-1Δmucin B cells had comparable Tim-1 expression. Gating strategy for Tim-1 staining is shown in the left panel. n = 3-5 per group. B) WT and Tim-1−/− B cells were cultured with unlabeled AC for 3 days, and IL-10 production in culture supernatants was then measured by ELISA. * P < 0.001; ns, not significant; n = 5. C) WT total CD4+ T cells (10 × 106/mouse) were co-transferred together with either WT or Tim-1−/− CD19+ B cells (20 × 106) into Rag1−/− mice. Apoptotic WT thymocytes (30 × 106/mouse) were injected one day before immunization with MOG35-55/CFA for EAE induction. Mice (n = 8 per group) were scored daily for clinical signs of EAE. * P < 0.05.
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