The combination of TGF-β– and CpG-activated BMDC induces IL-10–expressing CD4 T cells. (A) 10⁵ D10 BMDC that were either left unactivated or preactivated with the indicated agonists were used in combination with 1 µg/ml OVA peptide to stimulate 5 × 10⁵ naive CD4 T cells from 10BiT.Foxp3^gfp.OT-II mice, in the absence (top) or presence (bottom) of 5 ng/ml of recombinant human TGF-β1. On day 3, CD4 T cells were examined for expression of Thy1.1 and GFP. Numbers represent the percentage of CD4 T cells in the respective quadrants. (B) Naive CD4 T cells were stimulated as described in A with CpG-BMDC in the presence of TGF-β only or TGF-β plus anti–IFN-γ and anti–IL-4 (each at a final concentration of 10 µg/ml). On day 5, cells were restimulated for 5 h with PMA/ionomycin in the presence of brefeldin A, and viable CD4 T cells were analyzed for expression of Thy1.1, Foxp3, IL-17A, and IFN-γ. (C) Day-5 Thy1.1⁺ T cells (induced in the presence of CpG-BMDC and TGF-β) were MACS sorted and cultured for 24 h with Il10^−/− splenic DC in the absence (−) or presence (+) of 1 µg/ml OVA peptide. IL-10 protein in culture supernatants was quantitated by cytometric bead array. Mean cytokine concentrations ±SEM are shown. (ND, not detected). (D) 5 × 10⁵ CFSE-labeled OT-II CD4 T cells were stimulated with 1.0 µg/ml OVA peptide and 10⁵ IL-10–deficient splenic DC in the lower chamber of a transwell plate. In some cases, an equal number of DC or DC plus 5 × 10⁵ day-5 Thy1.1⁺ T cells (differentiated as in C) were similarly activated in the upper chamber. Where indicated, anti–IL-10R antibody was added to the lower chamber at a final concentration of 10 µg/ml. After 4 d, cells were harvested from the bottom chamber and the CFSE profile of the CD4 T cells was analyzed by FACS. Numbers in A and B indicate the percentages of CD4 T cells in the given quadrants. All data are representative of at least two experiments.

at-RA inhibits TGF-β–mediated induction of IL-10 in CD4 T cells. (A) Analysis of gut-homing receptor expression on CD4 T cells activated on CpG-BMDC in the presence of TGF-β. Shaded histograms, isotype control; open histograms, antibody to indicated marker. (B) Naive CD4 T cells were stimulated with CpG-BMDC and TGF-β only or TGF-β plus 100 nM at-RA for 3 d before being analyzed for expression of GFP and Thy1.1. (C) Naive CD4 T cells were stimulated on unactivated or preactivated BMDC alone or in the presence of TGF-β only, TGF-β plus 100 nM at-RA, or TGF-β plus 1 µM LE540. On day 5, cells were restimulated for 5 h with D10 BMDC and 1 µg/ml OVA peptide in the presence of brefeldin A and analyzed for expression of Thy1.1, GFP, and IL-17A. All FACS plots are gated on CD4 T cells and numbers represent the percentages of cells in the respective quadrants. (D) The polarized T cell populations were restimulated on day 5 with anti-CD3 and anti-CD28 antibodies for 24 h before the IL-10 content of culture supernatants was determined by cytometric bead array. Mean cytokine concentrations ± SEM are shown. *, P < 0.05. All data are representative of at least three experiments.

Mucosal DC-derived RA inhibits the induction of IL-10 in CD4 T cells. (A) 5 × 10⁵ naive 10BiT.Foxp3^gfp.OT-II CD4 T cells were stimulated with 1 µg/ml OVA peptide and 10⁵ CD11c⁺ DC from the spleen, MLN, sLP, or cLP in the absence (top) or presence (bottom) of 5 ng/ml TGF-β1. (B) CD4 T cells were stimulated with splenic or MLN DC as described in A in the presence of 100 µM TGF-β1 and RA or 1 µM LE540. CpG-act are DC that were preactivated for 16 h with CpG and then washed and cocultured with CD4 T cells in the presence of OVA peptide and TGF-β1. (C) CD11c⁺ MLN DC were FACS sorted into CD103⁻ and CD103⁺ fractions before being used in cocultures with T cells as in A. Data are representative of three to four independent experiments.

CpG-BMDC–mediated induction of IL-10 in CD4 T cells is Myd88 and ICOSL independent but IL-6 and IL-21 dependent. (A) Naive T cells were activated on CpG-BMDC from WT, Myd88^−/−, or Icosl^−/− mice in the presence of TGF-β and analyzed on day 3 for expression of Thy1.1 and GFP. (B) CD4 T cells were cultured with WT CpG-BMDC plus TGF-β in the presence of the additives indicated and analyzed on day 3 for expression of Thy1.1 and GFP. (C) D10 BMDC (left) were left unstimulated or stimulated for 24 h with the indicated TLR/NOD agonists. MACS purified CD4⁺Thy1.1⁺ cells (right), induced in the presence of CpG-BMDC plus TGF-β, were cultured for 24 h with Il10^−/− splenic DC in the absence or presence of OVA peptide. IL-6 was quantified by cytometric bead array and IL-21 by ELISA. Mean cytokine concentrations ±SEM are shown (ND, not detected; *, P < 0.05; ***, P < 0.001). All data are representative of at least three experiments.

RA inhibits transcription but not translation of IL-10. (A and B) CD4 T cells differentiated as described in Fig. 3 C were reactivated for 24 h with anti-CD3 and anti-CD28 antibodies, and IL-10 (A) and IL-21 (B) production was quantified by cytometric bead array. (C) On day 5 after activation, CD4 T cells differentiated in the presence of CpG-activated BMDC plus the additives indicated were harvested for analysis of Il10 mRNA by real-time PCR. Data are normalized to cells activated with CpG-activated BMDC and antigen only and are from one of two experiments with similar results. (D) CD4⁺Thy1.1⁺ cells were isolated from naive 10BiT mice as shown (top left) and left unstimulated or stimulated for 24 h with anti-CD3 and anti-CD28 antibodies only or with the addition of 100 nM at-RA or 1 µM LE540 before IL-10 protein in supernatants was quantified by cytometric bead array. Mean cytokine concentrations ±SEM are shown in A, B, and D, and all data are representative of three independent experiments. **, P < 0.005.

Reduced dietary vitamin A enhances and increased dietary vitamin A inhibits the induction of Il10 in vivo in the steady state. (A) Male 10BiT.Foxp3^gfp mice that were exposed to vitamin A–modified diets as described were killed at 12–14 wk of age. Single cell suspensions were prepared from the indicated tissues, and expression of GFP and Thy1.1 by CD4 T cells was determined by FACS. Plots are gated on CD4 T cells and numbers represent the percentage of CD4 T cells in quadrants 1, 2, and 4. (B) Graphical summary of data represented in A to denote the frequency of Il10-Thy1.1–expressing cells among Foxp3⁻ (bottom) and Foxp3⁺ (middle) cells in the respective tissue sites among the three diet groups. Graphs show mean frequencies ±SEM and all data are pooled from 8–12 mice per diet group. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Data are representative of three independent experiments.