Kinetics of Foxp3 induction and proliferation of DC-induced Foxp3⁺ cells. Naïve OTII CD4 T cells from nonreporter background were sorted as Vβ5^highCD25⁻, labeled with 5 μM CFSE, and cultured with splenic DC subsets in the presence of OVA_323–339 peptide (500 ng/ml), IL-2 (50 units/ml), and TGF-β (2 ng/ml). Foxp3 expression was analyzed at 48 h (A), 72 h (B), and 96 h (C). The CFSE dilution profiles for Foxp3⁺ (blue) and Foxp3⁻ (red) cells were overlaid. The percentage of Foxp3⁺ cells at each time point was plotted (D). Shown are representative results of two independent experiments.

Conversion is regulated by DC maturation status. (A) Conversion cultures with naïve OTII CD4 T cells (CD25⁻Foxp3GFP⁻) and splenic DC subsets were set up as before in the presence of TGF-β (2 ng/ml) and IL-2 (50 units/ml). Antibodies (αCD40 or αCD154, 5 ng/ml) or LPS (5 ng/ml) were added as indicated. (B and C) DC subsets were purified from CD40^−/− mice (B) or CD80/86^−/− mice (C) and used in the conversion assay. The percentage of Foxp3⁺ cells was analyzed on day 5 and plotted. All conditions were performed in duplicate wells and reported as means ± SEM. Shown are representative results of three independent experiments.

Multiple costimulatory and coinhibitory pathways regulate conversion in vitro. (A) Unfractionated splenic DCs were cultured with naive OTII CD4 T cells (CD25⁻Foxp3GFP⁻) in the presence of TGF-β (2 ng/ml). Antibodies (5 ng/ml) against PD-L1, PD-L2, CTLA-4, GITR, or control rat Ig were added in the beginning of the culture. (B) Conversion cultures were set up as in A but with the use of purified CD8α⁺ and CD8α⁻ DC subsets. (C) PD-L1 expression on DCs is required for the induction of Foxp3. WT or PD-L1^−/− splenic DC subsets were used as APCs in conversion cultures. The percentages of Foxp3⁺ cells were analyzed on day 5 and plotted. All conditions were performed in duplicate wells and reported as means ± SEM. Shown are representative results of three to five independent experiments.

Ex vivo splenic CD8α⁺ DCs induce Foxp3 expression more efficiently than CD8α⁻ DCs in the presence of TGF-β. Freshly isolated splenic DC subsets (CD8α⁺ CD11c^high and CD8α⁻ CD11c^high) were cocultured with naïve OTII CD4⁺ T cells (CD25⁻Foxp3GFP⁻) in the presence of OVA_323–339 peptide (500 ng/ml) and increasing amounts of TGF-β1. Foxp3GFP expression was analyzed on day 5 by flow cytometry. The total cell number was counted, and the number of Foxp3⁺ cells was calculated and plotted. (A) Representative FACS plots showing Foxp3GFP induction among OTII CD4⁺ T cells. (B–C) The percentage and absolute number of Foxp3GFP⁺ cells per well after 5 day culture. (D) CD8α⁻ DCs were mixed with CD8α⁺ DCs at the indicated ratios to stimulate OTII CD4 T cells in the presence of TGF-β (2 ng/ml). The total number of DCs per well was kept constant. Foxp3GFP expression was analyzed as above. All conditions were performed in duplicate wells and reported as means ± SEM. Shown are representative results of three independent experiments.

Tumor-induced conversion depends upon PD-L1 signaling. B16OVA tumor cells (200,000) were inoculated on the right flank of irradiated mice. Naïve OTII CD4⁺ T cells (1 × 10⁶) were adoptively transferred next day. Mice were analyzed when tumors reached ≥100 mm². Cells from tumor dLN, spleen, and tumor site were analyzed for Foxp3GFP expression among transferred OTII cells. Representative FACS plots for detecting OTII CD4⁺ T cells from tumor dLN and tumor tissues were illustrated in A. Percentages of conversion were summarized in B. The data were combined from four experiments. The average conversion efficiency (mean ± SEM) in tumor dLN was 8.09 ± 1.36% (n = 12); in spleen, 6.93 ± 0.96% (n = 12); and within tumor tissues, 68.65 ± 5.59% (n = 11). (C) Conversion of OTII CD4⁺ T cells in tumor dLN was inhibited by αCD40/LPS (0.64 ± 0.10%, n = 6) and αPD-L1 treatment (0.53 ± 0.15%, n = 9) but not by αPD-L2 treatment (6.37 ± 1.13%, n = 6). The conversion in the control tumor B16 group was 0.11 ± 0.03% (n = 4); in the no-treatment group, 4.90 ± 0.75% (n = 9); and in the control rat Ig-treated group, 4.55 ± 0.81% (n = 6). (D) The conversion in the spleen and TILs was blocked by αPD-L1 treatment. The conversion in the spleen (n = 5) was 4.74 ± 1.27% (notreat) and 0.23 ± 0.11% (αPD-L1-treated); and in TILs (n = 6), 66.67 ± 7.96% (notreat) and 1.12 ± 0.98% (αPD-L1 treated). (E) Conversion in the PD-L1^−/− mice was significantly reduced. The conversion in the tumor dLN was 5.123 ± 0.78% (WT, n = 10) and 0.2585 ± 0.083% (KO, n = 10); in the spleen, 6.01 ± 0.59% (WT, n = 10) and 0.74 ± 0.29% (KO, n = 10); and at the tumor site, 71.83 ± 7.551% (WT, n = 6) and 9.414 ± 6.905% (KO, n = 5). Unpaired Student's t tests was performed to obtain a P value. ***, P ≤ 0.001.