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940710.fig.003b
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Figure 3: Foxp3+ Treg cells in NOD transfer models. (a–c) Adoptive BDC2.5+ T cell transfer. (a) FACS purification of BDC2.5+Foxp3+ Treg cells (CD4+V 4+CD25+GFP+) from pooled spleen and LNs of NOD.Foxp × BDC2.5 mice after magnetic bead enrichment of CD25+ cells. Presort (top) and postsort (bottom) analyses of CD4/V 4 (left) and CD25/GFP (right) expression among gated lymphocytes are depicted. The gating scheme is illustrated by the line with arrowhead. Numbers in dot plots indicate the percentage of cells in the respective gate. (b) For diabetes induction, NOD.Rag1−/− recipient mice were injected with naïve BDC2.5+ T cells (5 × 105 cells/mouse), either alone (red circles, ) or coinjected with Foxp3+BDC2.5+ Treg cells (5 × 104 cells/mouse, blue circles, ) that had been FACS purified as shown in (a). See Figure 2(a) for details on the flow cytometric isolation of naïve BDC2.5+ T cells. (c) In addition to naïve BDC2.5+ T cells (5 × 105 cells/mouse), NOD.Rag1−/− recipient mice were coinjected with 1 × 105 BDC2.5+ T cells that exhibited retrovirus-mediated expression of either [Empty]-IRES-YFP (red circles, ) or [Foxp3]-IRES-YFP (blue circles, ). Retrovirus infections of initially naïve, TCR stimulated BDC2.5+ T cells were performed essentially as described previously [20]. (d) Adoptive transfer of polyclonal T cells. NOD.Rag1−/− recipient mice received splenocytes harvested from diabetic NOD donor mice (red circles, , average diabetes development at day ) or were coinjected with equivalent numbers of splenocytes from NOD donors that maintained normoglycemia until 26 weeks of age after treatment with recombinant anti-DEC-205 antibodies fused to whole proinsulin, beginning at 7 weeks of age (blue circles, , average diabetes development at day ) (adopted from [21]). Blood glucose concentrations of recipient mice in (b–d) were determined and plotted as described in the legend for Figure 1.