Figure 6

Regulatory T cells expanded with the combined optimized treatment show superior suppressive function, both in vitro and in vivo; and exhibit enhanced secretion of the anti-inflammatory cytokine IL-10. Analysis of spleen is shown. As in Fig. 3, NOD.Foxp3^EGFP females received either the combined optimized treatment, the combined treatment, A^g7/2.5 mi tetramer only, IL-2:mAb complexes only or were left untreated. Spleens were isolated at day 8 of treatment and used for the subsequent experiments. (A) In vitro suppression of CD4⁺Foxp3⁻ T cell proliferation by sorted CD4⁺Foxp3⁺ Treg cells isolated from mice treated as mentioned above. Proliferation was measured by flow cytometry via CellTrace Violet dye dilution after 3 days of co-culture at the indicated population ratio in the presence of anti-CD3 antibody as stimulus. Student t test (95% confidence) was applied for the calculation of statistical differences between naive and treated groups. The results of one of two independent experiments are shown (n = 3 per point). (B) Isolated splenocytes from treated (as referred) NOD.Foxp3^EGFP females were seeded into 96-well culture plates without stimulation or with the addition of 2.5 mi peptide or anti-CD3 antibody. Culture supernatants were collected at day 3 and assessed by ELISA for the presence of IL-10 and IFN-γ. The ratio between IL-10 secretion and IFN-γ production is shown for each condition. Data represent a compilation of three independent experiments. Statistical analysis (Student t test, 95% confidence): *, vs. naive; +, vs. only tetramer treatment; ♦, vs. IL-2:mAb complexes treatment; ●, vs. combined treatment. (C) Total splenocytes harvested from treated NOD.Foxp3^EGFP females were transferred along with splenocytes from naive BDC-2.5 transgenic females to young NOD.SCID recipients (spleens from treated NOD.Foxp3^EGFP female mice were extracted on day 8 after treatment as shown in Fig. 3C for individuals receiving tetramer only and the combined optimized treatment or on the day following the last dose of IL-2:mAb complexes for individuals receiving IL-2:mAb complexes only). Diabetes onset was determined by blood glucose measurements (animals with two consecutive measures above 200 mg/dl were considered diabetic). Percent of diabetes-free mice is depicted. Mantel-Cox test was applied to evaluate statistical differences between groups (n = 6-15/group).