Figure 6.

Nrp-1 Enhances T Cell Sensitivity to Antigen and Contributes to Treg Cell Suppression

(A–C) Ectopic expression of Nrp-1 in CD4⁺CD25⁻ cells increases sensitivity to antigen ex vivo. Th::Nrp-1 or Th::control cells from DO11.10xWT mice were cocultured with iDCs (iDC only) or with ova-loaded iDCs (1–100 μg/ml; 12 hr). (A) CD69 expression on Th::Nrp-1 (red) and Th::control cells (black) after 24 hr of culture (representative example of two independent experiments). T cells cultured in the absence (no iDC) or presence of 50 ng/ml PMA stimulation (PMA) are shown as controls. (B) Th::Nrp-1 (dark bars) and Th::control cells (light bars) cotransduced with a retroviral vector carrying a NF-κB-driven GFP reporter gene are shown. GFP fluorescence was measured by flow cytometry after 24 hr of culture with the antigen-loaded iDCs. Error bars represent the standard error of the mean (SEM) from three independent experiments. (C) Relative proliferation of Th::Nrp-1 and Th::control cells after 56 hr of culture with untreated iDCs or ova-loaded iDCs (iDC-ova) (10 μg/ml; 12 hr), normalized to proliferation measured with untreated iDCs (pooled data from two independent experiments; n = 3), is shown.

(D) Relative in vivo expansion of Th::Nrp-1 or Th::control cells from DO11.10xSCID mice in the spleens of nonimmunized mice or mice immunized with ova-loaded iDCs (100 μg/ml; 12 hr) (iDC-ova). The transduced cells were identified on the basis of the coexpression of GFP (two independent experiments, n = 6 for each group; p = 0.0359, unpaired t test). Error bars represent the SEM.

(E and F) Anti-Nrp-1 treatment interferes with suppressive function of Treg cells. CD4⁺CD25⁻ (Th) cells were cocultured with CD4⁺CD25⁺ (Treg) cells (both prepared from DO11.10 mice) and either untreated iDCs or ova-loaded iDCs, in the presence or absence of anti-Nrp-1 or isotype control. Proliferation was determined by ³H thymidine incorporation. (E) Effect of anti-Nrp-1 treatment at different concentrations of antigen. CD4⁺CD25⁻ cells were cocultured with ova-loaded iDCs (indicated concentrations; 12 hr), in the presence or absence of CD4⁺CD25⁺ cells, with or without anti-Nrp-1 or isotype control (10 μg/ml) (pooled data from three independent experiments performed in duplicates; p = 0.009, unpaired t test). Error bars represent the SEM. (F) Dose-dependent effect of anti-Nrp-1 treatment. CD4⁺CD25⁻ cells were cocultured with (dark bars) or without (light bars) CD4⁺CD25⁺ cells, in the presence of the indicated amounts of anti-Nrp-1 or isotype control (n = 4).