Immature Dendritic Cell Therapy Confers Durable Immune Modulation in an Antigen-Dependent and Antigen-Independent Manner in Nonobese Diabetic Mice

Figure 6

Assessment of Treg spontaneous proliferation and function induced by DC therapy. (a) Assessment of in vitro spontaneous proliferation of Tregs following DC therapy. CFSE-labeled spleen cells from female NOD mice from different groups were cultured in serum-free media without stimulation and allowed to proliferate for 72–84 h. Cells stained with CD4 and Foxp and analyzed by flow cytometry. The proliferating Foxp3+ cells were analyzed by gating on total CD4+ cells. Data shown is representative of 3 experiments from mice aging from 13–41 weeks old. (b) For suppressor T cell assay. Female 9-week-old NOD mice were treated with 3 weekly injections of DC, then Treg function was assessed at 13 weeks of age. CD4+CD25+ Tregs were purified and cocultured with CD4+CD25+ T cell-depleted spleen cells at ratios of 0 : 1, 1 : 2, and 1 : 4 and stimulated with anti-CD3 antibodies (0.05 μg/ml). Proliferation was assessed by 3H-thymidine incorporation. The suppression rate = (proliferation (cpm) without CD4+CD25+ T cells − proliferation (cpm) with CD4+CD25+ T cells)/proliferation (cpm) without CD4+CD25+ T cells.

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