Figure 3.

Nrp1 is required for immune-cell suppression and modulation of conventional CD4+ T phenotype. Responder convT cells were sorted, CTV-labeled and cultured with Mitomycin-C treated–APCs plus soluble anti-CD3 antibody, alone or in the presence of sorted Treg cells. (A) Histograms showing proliferation of convT cells activated alone (gray) or at different Treg:convT ratios (1:1, 1:2, and 1:4) of wt (dark green), het (light green), or Nrp1KO (black) Treg cells. (B) Percentage of suppression activity was calculated as described in Materials and Methods. Representative data of at least three independent experiments. Squares represent mean ± SEM. (C) Representative dot plots showing Nrp1 expression on convT cells activated as in (A). (D) Pooled data depicting Nrp1 expression on CD45.1⁺convT cells cultured with Treg cells from the three indicated genotypes. Bars represent mean ± SEM. Data is representative of at least two independent experiments. (E) Pooled data depicting Nrp1 expression on CD45.2⁺ Treg cells before and after co-culture with convT cells at aforementioned different Treg:convT ratios. PS: post-sorting expression of Nrp1 on Treg cells. (F) Histograms showing proliferation of convT cells activated alone (gray) of at different Treg:convT ratios (1:2 and 1:4) of wt (dark green), het (light green), or Nrp1KO (black) Treg cells from transwell experiments (described in detail in Materials and Methods section and Supplemental Figure 3). (G) Percentage of suppression activity was calculated as described in Materials and Methods. Representative data of two independent experiments performed in triplicates. (H) Pooled data depicting Nrp1 expression on CD45.1⁺ convT cells cultured with wt or Nrp1KO Treg from transwell experiments. Bars represent mean ± SEM. Data is representative of two independent experiments performed in triplicates. For (B,D–E,H), Paired T-test; ^* < 0.05; ^** < 0.01 were used; ns, not significant. For (G) ^* < 0.05 for wt vs. Nrp1KO (both 1:2 and 1:4 ratios).