Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice

Abstract

Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1-mutant mouse (Tim-1(Δmucin)) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1(Δmucin) mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1(Δmucin) mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1(Δmucin)lpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.

Fig. 1.

Generation and characterization of Tim-1^Δmucin mice. (A) The gene structures of the WT Tim-1 allele, the Tim-1^Δmucin targeting construct, and the targeted Tim-1 allele. Colored boxes represent coding sequences; Roman numerals represent exons. E, EcoR I sites. (B) (Left) CD19⁺ B cells isolated from WT and Tim-1^Δmucin mice were used to determine Tim-1 mRNA expression by RT-PCR. (Right) 293T cells were overexpressed with WT Tim-1 and Tim-1^Δmucin and then were used to determine Tim-1 protein expression by Western blot. (C and D) RT-PCR products were sequenced, and parts of cDNA and predicted encoding amino acid sequences of WT Tim-1 (C) and Tim-1^Δmucin (D) are shown. (E) Isolated WT and Tim-1^Δmucin B cells were examined for Tim-1 expression by flow cytometry. (F) Schematic representation of Tim-1 and Tim-1^Δmucin protein structures.

Fig. 2.

Immune phenotypes in Tim-1^Δmucin mice. (A) Spleen, lymph nodes, and thymi were analyzed for the expression of indicated markers by flow cytometry. Data shown are representative of n = 8–10 per group of 3- to 6-mo-old mice. (B) CD4⁺ T cells isolated from 3- to 6-mo-old mice were treated with anti-CD3 and anti-CD28. Cytokines in 48-h culture supernatants were measured by cytometric bead array. Proliferation was measured by [³H]-thymidine incorporation. (C) Splenocytes from 10- to 12-mo-old mice were examined for CD62L and CD44 expression on CD3⁺ cells by flow cytometry. (D and E) Splenocytes from 10- to 12-mo-old mice also were stained for IFN-γ and IL-17 (D) and IL-10 (E) expression after stimulation with phosphomolybdic acid (PMA)/ionomycin. (F) Splenocytes from 20-mo-old WT and Tim-1^Δmucin mice were analyzed for CD3, CD4, and CD8 expression by flow cytometry (gated on CD3⁺ cells). (G) Splenocytes from 10- to 12-mo-old mice were examined for CD80, CD86, and MHC class II expression on CD11c⁺ cells by flow cytometry. (H) DCs isolated from WT and Tim-1^Δmucin mice were cocultured with WT CD4⁺ T cells in the presence of anti-CD3. Cytokines in 48-h culture supernatants were measured by cytometric bead array. Proliferation was measured by [³H]-thymidine incorporation. *P < 0.05.

Fig. 3.

Foxp3⁺ Tregs are normal, but IL-10–producing Bregs are impaired in Tim-1^Δmucin mice. (A) Frequency of Foxp3/GFP⁺ Tregs in spleens of 4- to 6-mo-old WT Foxp3/GFP-KI and Tim-1^ΔmucinFoxp3/GFP-KI mice was analyzed by flow cytometry. (B) Effector T cells (Teff) and DCs from WT Foxp3/GFP-KI mice were cocultured with different ratios of Tregs in the presence of anti-CD3, and cell proliferation was measured by ³[H]-thymidine incorporation. (C) Splenocytes from 3-mo-old WT mice were stained with CD19, CD5, CD1d, and Tim-1 mAb, and Tim-1 expression on different CD19⁺ B-cell subsets was analyzed by flow cytometry. (D) Splenocytes from IL-10/Thy1.1 reporter mice were activated with LPS/PMA/ionomycin for 5 h, and Tim-1 expression and IL-10 production (Thy1.1⁺) in CD19⁺ B cells were analyzed by flow cytometry. (E) Splenocytes from WT and Tim-1^Δmucin mice were activated with LPS/PMA/ionomycin for 5 h, and IL-10 production in CD19⁺ B cells was analyzed by intracellular cytokine staining (Left and Middle). Isolated splenic CD19⁺ cells were activated with LPS for 24 h, and IL-10 production in culture supernatants was measured by cytokine bead array (Right). *P < 0.05.

Fig. 4.

Immune phenotypes in Tim-1^Δmucinlpr double-mutant mice. (A) Increased accumulation of abnormal CD3⁺B220⁺CD4⁻CD8⁻ cells in Tim-1^Δmucinlpr mice. CD3 and B220 expression in splenocytes of 14-wk-old mice was determined by flow cytometry. (B) Increased splenic and lymph node sizes in Tim-1^Δmucinlpr mice. (Left) Representative images of spleens and lymph nodes from 10- to 12-mo-old WT, Tim-1^Δmucin, lpr, and Tim-1^Δmucinlpr mice. (Right) Splenic and lymph node cells from 10- to 12-mo-old WT (n = 8), Tim-1^Δmucin (n = 12), lpr (n = 10), and Tim-1^Δmucinlpr (n = 15) mice were counted. (C–E) Cells from B were examined for indicated surface markers (C and D) or for IFN-γ and IL-17 production (E) by flow cytometry. Different immune cell populations then were enumerated based on the percentage. *P < 0.05.

Fig. 5.

Increased serum levels of Ig and anti-dsDNA autoantibodies in Tim-1^Δmucinlpr mice. (A) Levels of different Igs in sera from >10-mo-old WT (n = 8), Tim-1^Δmucin (n = 12), lpr (n = 10), and Tim-1^Δmucinlpr (n = 15) mice were determined by ELISA. (B) Heat map of lupus-associated autoantigen microarray. Serum IgG reactivity to 5 of 39 lupus-associated autoantigens is shown. Other lupus-associated autoantigens showed no significant difference among the groups of mice. (C) Levels of anti-dsDNA IgG in sera from A were determined by ELISA. *P < 0.05.