Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway by Kevin M. Elias, Arian Laurence, Todd S. Davidson, Geoffrey Stephens, Yuka Kanno, Ethan M. Shevach, and John J. O'Shea Blood Volume 111(3):1013-1020 February 1, 2008 ©2008 by American Society of Hematology

ATRA inhibits Th1 and Th2 polarization. ATRA inhibits Th1 and Th2 polarization. Polyclonal CD4+CD62L+ cells isolated by MACS from C57Bl6J mice were polarized for 5 days under neutral, Th1 (IL-12 and anti–IL-4), or Th2 (IL-4 and anti–IFN-γ) favoring culture conditions with or without 1 μM ATRA. (A) Cytokine production at day 5 by intracellular staining. Numbers on plots are percentages of total cells. (B) Cells were washed thoroughly after 5 days in culture, then resuspended at 0.5 × 106 cells/mL and restimulated overnight with plate-bound anti-CD3 and anti-CD28 in media without additional cytokines. Supernatants were collected and measured by ELISA; error bars represent standard deviations (n = 3). These experiments are representative of 3 independent experiments. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

ATRA inhibits Th17 polarization and promotes FoxP3 expression. ATRA inhibits Th17 polarization and promotes FoxP3 expression. (A) CD4+ T cells isolated by MACS beads from OTII TCR transgenic mice were incubated with isolated CD11c+ cells pulsed with 1 μg OVA peptide and cultured for 3 days under neutral or Th17-favoring (IL-6, TGF-β1, anti–IFN-γ, and anti–IL-4) conditions with or without 1 μM ATRA. The figure depicts intracellular staining for IL-17 production and FoxP3 expression, and it is representative of 2 independent experiments. Numbers on plots are percentages of total cells. (B,C) Polyclonal CD4+CD62L+CD25−CD44− cells isolated by flow cytometry were stimulated for 3 days with anti-CD3 and anti-CD28 under neutral or Th17-favoring conditions with or without ATRA. (B) Average IL-17 concentration in supernatants from 3 independent experiments as measured by ELISA. The P value was determined by a 2-tailed paired t test; a single asterisk denotes significance (P < .05). (C) FoxP3 expression normalized to β-actin levels and relative to Th-neutral conditions without ATRA; error bars represent standard deviation (n = 3); the data are representative of 2 independent experiments. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

Th17 cells express high levels of RARα, RARγ, and RORγt mRNA. Th17 cells express high levels of RARα, RARγ, and RORγt mRNA. (A,B) CD4+CD62L+ cells isolated by MACS were polyclonally stimulated for 3 days under Th-neutral, Th1, Th2, or Th17 conditions. Relative expression of RAR and RXR receptors was analyzed by quantitative RT-PCR. Expression is normalized to β-actin levels under Th-neutral conditions, and error bars represent standard deviation (n = 3). Detected RAR and RXR receptors are shown (RAR-β and RXR-γ were not present). (C) Naive CD4+ T cells were polyclonally stimulated for 3 days under Th-neutral or Th17 conditions in the presence or absence of 1 μM ATRA. Relative expression of RORγt mRNA was analyzed by quantitative RT-PCR. Expression is normalized to β-actin levels and relative to Th-neutral conditions; error bars represent standard deviation (n = 3). The inhibition of RORγt mRNA was confirmed in 3 independent experiments. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

RARα is necessary for TGF-β1 induction of FoxP3. RARα is necessary for TGF-β1 induction of FoxP3. (A) Naive CD4+ cells were polyclonally stimulated for 3 days under Th17 conditions or TGF-β1 alone for 3 days with TTNBP (pan-RAR agonist) or LE540 (pan-RAR antagonist). IL-17 and FoxP3 expression were measured on fixed cells by intracellular staining. Numbers on plots are percentages of total cells. (B) Naive CD4+ T cells were polyclonally stimulated for 3 days with TGF-β1 and 1 μM ATRA, 1 μM A7980 (selective RARγ agonist), or 1 μM AM580 (selective RARα agonist). FoxP3 expression was measured on fixed cells by intracellular staining. Data are representative of 2 independent experiments. Numbers on graphs are percentages of cells that are FoxP3.+ Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

ATRA enhances FoxP3 expression independent of Stat3. ATRA enhances FoxP3 expression independent of Stat3. Peripheral CD4+ cells from Stat3fl/flCD4-Cre mice or WT controls were polyclonally stimulated for 3 days under Th-neutral conditions, Th17 conditions, or with TGF-β1 alone in the presence or absence of 1 μM ATRA. IL-17 and FoxP3 expression was measured on fixed cells by intracellular staining. Data are representative of 2 independent experiments. Numbers on plots are percentages of total cells. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

ATRA enhances FoxP3 expression independent of Stat5. ATRA enhances FoxP3 expression independent of Stat5. CD8-depleted thymocytes from Stat5fl/flCD4-Cre mice were polyclonally stimulated for 3 days with media alone, IL-6, IL-2, and TGF-β1, or IL-2 and TGFβ-1 in the presence or absence of 1 μM ATRA. IL-17 and FoxP3 expression were measured on CD4+ gated fixed cells by intracellular staining. Data are representative of 2 independent experiments. Numbers on plots are percentages of total cells. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology

ATRA enhances FoxP3 expression independent of IL-2. ATRA enhances FoxP3 expression independent of IL-2. (A) Peripheral CD4 cells from Rag2−/− transgenic (OTII) mice were polyclonally stimulated for 3 days under Th17 conditions or TGF-β1 alone, in the presence of either IL-2 or anti–IL-2. All stimulations were performed with (lower panels) or without (upper panels) 1μM ATRA. IL-17 and FoxP3 expression were measured on fixed cells by intracellular staining. Data are representative of 2 independent experiments. Numbers on plots are percentages of total cells. (B) CD4+CD25− MACS-purified FoxP3-GFP cells were polyclonally stimulated for 3 days in the presence of TGF-β1, ATRA, and anti–IL-2. FoxP3-positive cells were isolated by flow cytometry and used to inhibit CD4+CD25− cells sorted by flow cytometry and stimulated with soluble anti-CD3 in the presence of irradiated Thy1.1− cells. Proliferation was measured by 3H-thymidine uptake and was compared with CD4+CD25+ (natural Treg) cells purified by flow cytometry. Error bars denote the standard error of the mean (n = 3), and significance was determined by an unpaired t test, and data are representative of 2 independent experiments. Kevin M. Elias et al. Blood 2008;111:1013-1020 ©2008 by American Society of Hematology