Nrp-1 is differentially expressed on natural and iT reg cells. (a) Expression plots showing the relative intensity of gene expression in iT reg cells (1B3.RAG^−/−) and nT reg cells (1B3). The y axis compares the expression profiles of T reg cells from 1B3 mice, whereas the x axis compares the expression profiles of iT reg cells from 1B3.RAG^−/− mice (mean values are shown from n = 4 each group). Selected genes are marked and genes up-regulated specifically in nT reg cells are highlighted in the box. (b) CD4⁺ T cells from LNs (pooled) of 1B3.RAG^−/− or WT-NOD (4–5 wk) mice were stained with Nrp-1, PD-1, CD73, Foxp3, and Helios. (c) CD4⁺ cells from LN (pooled) of NOD, or B6 WT mice were stained with Foxp3 and Nrp-1. Foxp3⁺ T cells were gated into Nrp-1^hi and Nrp-1^lo subsets and analyzed for Helios expression (histograms). Graphs (bottom) compare percentages of Nrp-1– and Helios-positive cells among Foxp3⁺ T reg cells from five individual mice (NOD) with bars representing means. (d) Relative mRNA expression levels of Foxp3, Eos, Tnfrsf9, and Lypd1 in Foxp3.GFP⁺Nrp-1^hi (Nrp-1^hi) or Foxp3.GFP⁺Nrp-1^lo (Nrp-1^lo) or CD4⁺Foxp3.GFP⁻ (T conv) cells (mean ± SD from four replicates). (e) Foxp3⁺ cells in CD4 single-positive thymocytes of a 5-wk-old NOD mouse were separated into CD24^hiQa-2^lo and CD24^loQa-2^hi and analyzed for Nrp-1 staining (representative histogram is shown; dotted line is isotype control). Percentages of Nrp-1^hi cells in both Foxp3⁺ subsets are shown with each dot representing an individual mouse and bars representing means.