Nrp-1 expression is not significantly altered by activation. (a) Cells from 1B3.Foxp3-GFP mice were sorted into T conv (CD4⁺Foxp3⁻) or T reg cells (separated into Nrp-1^lo, Nrp-1^hi, or total) and activated in vitro with irradiated spleen cells loaded with 1B3 cognate peptide for 72 h. Resting state cells are shown on the top. After activation, CD4 cells were stained with Foxp3 (middle) or with CD69 and Nrp-1 (bottom). Live cells gated on CD4⁺ are shown from one of three replicates. Data are representative of three independent experiments. (b) 2.5 × 10⁵ T reg cells (Nrp-1^hi or Nrp-1^lo) from NOD.Thy1.1 mice were adoptively transferred into a WT Thy1.2 congenic host. Histograms show relative Nrp-1 expression in Nrp-1^hi or Nrp-l^lo T reg cells before (top) and 1 wk after transfer (bottom). Numbers in the bottom depict mean ± SD of Nrp^hi cells (among Foxp3⁺) from three different mice. Data are representative of two independent experiments. (c) 1.5 × 10⁵ Foxp3⁺Nrp-1^hi or Foxp3⁺Nrp-1^lo T reg cells were sorted from a WT.Thy1.1⁺ mouse and transferred into RAGKO mice with similar number of congenically marked T conv (CD4⁺Foxp3⁻) cells. 1 wk after transfer, Thy1.1⁺CD4⁺Foxp3⁺ T cells from spleen and LN were analyzed for Nrp-1 expression. Top histograms show Nrp-1 staining before transfer, and a representative plot with Nrp-1 expression after transfers with Nrp-1^hi or Nrp-1^lo T reg cells is shown. Bottom graph depicts mean + SD with data from four mice showing Nrp-1 staining on Foxp3⁺ cells in T reg compartment (Thy1.1). Data are representative of two independent experiments. (d) Methylation status of CpG motifs of the TSDR of the Foxp3 locus in different T reg cell subsets. Foxp3⁺Nrp-1^hi or Foxp3⁺Nrp-1^lo T reg cells or Foxp3⁻ T conv cells were isolated from NOD.Foxp3-GFP mouse. Numbers in the pie chart indicate mean ± SD of percent demethylation in the population from six replicates.