Peripherally generated Foxp3⁺ iT reg cells lack Nrp-1 expression. (a) Cells from 1B3.Foxp3-GFP mice were sorted into T conv (CD4⁺CD25⁻Foxp3⁻) cells and stimulated in vitro with anti-CD28 plus anti-CD28 Abs or with irradiated spleen cells loaded with 1B3 cognate peptide in the absence (top) or presence (bottom) of TGF-β. After 5 d of incubation, live CD4⁺ cells were stained for Nrp-1 and Foxp3. One representative plot from triplicate wells is shown and is representative of three independent experiments. (b) Naive T cells (CD4⁺CD25⁻GITR⁻CD62L⁺) were transferred into RAG^−/− recipient mice. 2 wk after transfer, CD4⁺ T cells from LN were stained with Foxp3 and analyzed for Nrp-1 expression. Histogram overlay showing Nrp-1 expression on converted iT reg cells compared with T reg cells in LN of WT mouse is shown. Bottom right, graph shows Nrp-1 expression on iT reg cells or WT in LN from three individual mice and bar represents mean. Data are representative of two independent experiments. (c) Foxp3 staining in CD4⁺ from inguinal LN of DO11.RAG^−/− mice 2 wk after implantation of osmotic pumps delivering PBS or 0.1 µg or 1 µg of OVA peptide per day (n = 3 for each group). Representative plots showing Nrp-1 staining (filled histogram) or isotype control Ab (empty histograms) on gated Foxp3⁺ cells is shown with numbers depicting mean ± SD of Nrp-1^hi cells from three different mice. LN from untreated DO11.RAG^+/+ mice was used as a control. (d) Foxp3 staining in CD4⁺ cells from axillary LN (LN) or lamina propria of colon (LP) from 8–12-wk-old NOD mouse. Representative histograms showing Nrp-1 staining (solid) or isotype control Ab (dotted line) on gated Foxp3⁺ cells are shown. Numbers indicate percentage of Nrp-1^lo cells in the gate. Graph with percentage of Nrp-1^lo among Foxp3⁺ cells from five mice is shown on right, with bars representing means.