Figure 6: Nrp-1 acts as a key mediator of Foxp3⁺ Treg cell migration to B16/F10 melanomas.

(A,B) Measurement of B16/F10 TDLN and tumor-derived CD4⁺Foxp3⁺ Treg cells in IL10^−/− B16/F10 mice or WT B16/F10 mice after treatment with an anti-Nrp-1 polyclonal Ab by flow cytometry. TDLN and tumor tissue samples were processed as described in the Methods section. (C) Measurement of B16/F10 tumor-derived Nrp-1⁺ expression in gated CD4⁺Foxp3⁺ T cells from IL10^−/− B16/F10 mice or WT B16/F10 mice after treatment with the anti-Nrp-1 polyclonal Ab by flow cytometry using micro-dissected and digested tumor tissues. (D–F) Measurement of TGF-β, IL6 and VEGF expression in NP-40-processed B16/F10 tumor tissues (described in the Methods section) obtained from mice treated with anti-Nrp-1. The cytokines were measured using Luminex (Millipore Merck). (G) Blood vessels stained with an anti-CD31 antibody. The images were obtained using a microscope with a 10X objective. (H) Compared with the control group, the tumor tissues of mice treated with the anti-Nrp-1 antibody showed much fewer Ki-67-positive cells. Left panel, representative immunohistochemistry images obtained using a microscope with a 20X objective. Right panel, statistical analysis of the mean IOD for blood vessels and tumor cell proliferation performed using Image-Pro Plus software. Nonlinear regressions were performed using GraphPad Prism (San Diego, CA, USA). The data shown are from one representative experiment of two independent experiments with the same results. The results were evaluated using ANOVA for the determination of statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001 between experimental groups. The data are expressed as the mean values ± SEM from n = 4–6 mice.