Fig. 3.

Coexpression of Nrp1 and TGF-β components analyzed by flow cytometry. (A) Approximately 3% of mouse splenic T cells coexpressed LAP and Nrp1 (percent-positive cells are indicated on the histograms). (B) Splenic T cells were incubated with Nrp1-Fc, washed, and incubated with LAP-TGF-β1. This approximately tripled the number Nrp1⁺LAP⁺ cells. (C and D) Splenic T cells were stained in four colors with antibodies against CD4, CD25, Nrp1, and LAP (C) or forkhead box P3 (FoxP3; D). The two-dimensional plots were gated on CD4⁺CD25⁺ cells. The number of Nrp1^–FoxP3⁺ T cells is circled in D and shows that Nrp1⁺ cells generally express more FoxP3. (E) Tr-marker expression in CD4⁺Nrp1⁺ cells (percent±sem) in CD-1 mice. Similar results were obtained in C57BL/6 mice (not shown). (F) When Nrp1⁺ cells were incubated with LAP, the number of LAP⁺ cells increased from 40% to 95%, and this was blocked by an anti-Nrp1 mAb. Nrp1^– T cells only minimally captured LAP. (G) Sorted CD4⁺Nrp1^– T cells incubated with Nrp1-Fc and LAP-TGF-β1 capture LAP-TGF-β1 (LAP staining shown), and this was blocked by an anti-Nrp1 mAb. Silver peak, Isotype control; gray line/cross hatched peak, anti-LAP staining; solid black line, incubation with Nrp1-Fc and LAP-TGF-β1; dotted black line, incubation with Nrp1-Fc, then anti-Nrp1 mAb, and then LAP-TGF-β1. (H) T cells were incubated (or not) with Nrp1-Fc and then with active TGF-β1. Silver line, Isotype control; gray line/cross hatched peak, TGF-β1 staining; solid black line, T cell incubated with TGF-β1; dotted black line, T cells incubated with Nrp1-Fc and then TGF-β1. (A–H) The results are representative of two or more independent experiments. Cells were washed between treatments with Nrp1-Fc and TGF-β1 components.