Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1

Foxp3 from Dbc1^−/− mice is more stable than Foxp3 from WT mice, and Treg cells from Dbc1^−/− mice are more suppressive than Treg cells from WT mice, especially during inflammatory insult. (A) Treg cells isolated from thymi of C57BL/6 Foxp3-GFP mice were expanded for 4 d and were recultured in the presence of APCs with soluble anti-CD3 (1 μg/mL), anti-CD28 (1 μg/mL), anti–IL-4 (5 μg/mL), and anti–IFN-γ (5 μg/mL), with or without recombinant human (rh)-TGF-β (5 ng/mL), recombinant mouse (rm)-IL-6 (10 ng/mL), or rm-TNF-α (50 ng/mL). After 3 d these cells were harvested and stained for intracellular expression of Foxp3 and IL-17a. (B) The statistical percentages of Foxp3 expression among the different groups as indicated. (C) The statistical percentages of IL-17a expression among the different groups as indicated. “None” indicates no rm-TNF-α or rm-IL-6 treatment. (D–F) Suppressive activity of Treg cells from Dbc1^−/− and Dbc1^+/+ mice. Treg cells from Dbc1^−/− and Dbc1^+/+ mice without treatment (“None”) (D), pretreated with 50 ng/mL rm-TNF-α (E), or treated with 10 ng/mL rm-IL-6 and 5 ng/mL rh-TGF-β (F) were tested using an in vitro suppressive activity by indirectly measuring the proliferative rates of anti-CD3–activated T-responder cells (B6) labeled with CFSE. Cells were harvested on day 3 and stained with PE-conjugated anti-CD8 antibody. Samples were analyzed by flow cytometry and gated on the CD8⁺ population. The x axis shows the ratio of Treg cells to responder T cells. All data are representative of three independent experiments.

Dbc1^−/− mice develop less severe autoimmune disease symptoms during EAE induction. (A) EAE clinical scores for 8- to10-wk-old male Dbc1^+/+ and Dbc1^−/− mice (n = 7 in each group) were calculated on the indicated days after immunization with MOG35-55. (B) The statistical relationship between the percentage of Foxp3⁺ cells in the CD4⁺ population and EAE scores after EAE induction. (C) Representative flow cytometry data for IL-17a and IFN-γ expression in spleens and draining lymph nodes in each group. Mice were euthanized on day 33, and fresh cells taken from spleens were stained for Foxp3, IL-17a, and IFN-γ. (D) Representative microphotographs of spinal cord sections from mice in the Dbc1^+/+ (n = 4) or Dbc1^−/− (n = 1) group. (Original magnification: 200×.) Data are pooled from seven independent experiments. (E) EAE was induced in 8- to10-wk-old Dbc1^+/+ and Dbc1^−/− Foxp3-GFP mice (n = 4 mice in each group) following immunization with MOG35-55 as previously described. PC61, an anti-CD25 antibody, was injected 5 d before EAE induction. The curve shows the EAE clinical scores calculated in the different groups. (F and G) IL-17a and IFN-γ detected in E. The percentage of IL-17a⁺ cells in the CD4⁺ population in E (F) and the percentages of IL-17a⁺ and IFN-γ⁺ cells in E were analyzed by flow cytometry (G). (H) EAE was induced in B6 WT mice with MOG35-55. PC61 was injected to deplete Treg cells 7 d before EAE induction. Treg cells (2 × 10⁶ for each mouse) from Dbc1^+/+ or Dbc1^−/− mice were injected into these mice via the tail vein 6 d after MOG35-55 immunization. PBS was injected as a control. EAE clinical scores were calculated at the indicated days after immunization. (I) The percentage of IL-17a⁺ cells in the CD4⁺ population in spinal cord analyzed by flow cytometry in H. IL-17a expression was detected 18 d after EAE induction in the control group and 33 d after EAE induction in the Dbc1^+/+ and Dbc1^−/− groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Dbc1^−/− Treg cells function profoundly in preventing colitis. (A) The changes in weight in the different groups in the colitis model. Treg cells (5 × 10⁵) from the indicated mice were expanded for 4 d and then were cotransferred i.p. with 3 × 10⁵ syngeneic CD4⁺CD45RB^hi T cells into 8- to 10-wk-old Rag2^−/− mice (n = 6 mice in each group). Rag2^−/− mice receiving only CD4⁺CD45RB^hi T cells were used as a control. Body weights were calculated twice a week. (B) Colitis scores were calculated according to pathological anatomy and histological expression in the different groups. Each data point represents an individual mouse. (C) The percentages of cells expressing IL-17a in the CD4⁺ population. Fresh cells taken from spleens of mice in the Dbc1^−/−, Dbc1^+/+, and control groups were stimulated with phorbol12-myristate13-acetate (PMA) and Ionomycin for 1 h and with brefeldin A (BFA) for 4 h (5 h total), followed by staining for IL-17a. (D) Representative photomicrographs of colonic sections from control recipients (n = 11) or recipients that received Dbc1^+/+ (n = 7) or Dbc1^−/− (n = 3) Treg cells. Data from six independent experiments were pooled. (Original magnification: 200×.) (E) The changes in weight in the different groups in the colitis model. Syngeneic CD4⁺CD45RB^hi T cells (3 × 10⁵) from Dbc1^+/+ and Dbc1^−/− mice were transplanted i.p. into 8- to 10-wk-old Rag2^−/− mice (n = 4 mice in each group). Rag2^−/− mice injected with PBS were used as the control group. Body weights were calculated twice a week. (F) Colitis scores were calculated according to the pathological anatomy and histological expression in the different groups. Each data point represents an individual mouse. (G and H) Mesenteric lymph nodes were isolated and stimulated with PMA plus Ionomycin and BFA. Cells were stained for IFN-γ/IL-17a/CD4 and then were analyzed by flow cytometry. *P < 0.05; **P < 0.02; ***P < 0.01.