FIGURE 5.

B cell depletion reduces BDC2.5 T_reg number and function. (A) Foxp3⁺ T_reg cells from PBMC were analyzed by flow cytometry following treatment of hCD20-BDC2.5NOD mice with 2H7 or mIgG. The percentage of BDC2.5 T_reg cells decreased in 2H7-treated mice (n = 6–8 mice/group; p < 0.05). (B) Percentages of T_reg in BDC2.5 T cells in different lymphoid organs were variable. Representative dot plots show the percentage of BDC2.5 T_reg (Foxp3⁺CD25^high+low) cells. The bar charts show the pooled data from at least six mice per group (mean ± SEM). (C) BDC2.5 T_regs in islets from 2H7- or mIgG-treated hCD20-BDC2.5NOD mice. After islet isolation, the islet cells were dispersed, and infiltrating cells were stained with the corresponding mAbs and analyzed by flow cytometry. There were fewer infiltrating T_reg cells in islets after B cell depletion. Dot plots are shown after gating on CD4⁺ cells. At least two mice were pooled for islet isolation. Experiments were performed three times and summarized in the bar graphs on the right. (D) Cross-suppression assay. Foxp3⁺ BDC2.5 T_reg cells were sorted from 2H7- or mIgG-treated hCD20-BDC2.5-FirNOD mice and used as T_reg (Foxp3⁺) and T_eff cells (Fir⁻CD25⁻CD4⁺) were also sorted from 2H7- or mIgG-treated hCD20-BDC2.5-FirNOD mice. As indicated in (D), the cross-suppression assay was performed by coculturing T_eff cells from 2H7-treated mice with T_reg cells from mouse IgG-treated mice or T_eff cells from IgG-treated mice with T_reg cells from 2H7-treated mice. Cocultures of 2H7-treated T_eff and T_reg cells or IgG-treated T_eff and T_reg cells were used as controls. A cell ratio of 2:1 (T_eff/T_reg) was used in the cocultures. Proliferation is shown as stimulation index. T_reg cells from 2H7-treated mice do not suppress T_eff cell proliferation as effectively as T_reg cells from mIgG-treated mice. The average [³H]thymidine incorporation in the absence of mimotope = 74.65 ± 13.48 cpm. Similar results were obtained from three independent experiments.