1. 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):
February 1, 2008
©2008 by American Society of Hematology

2. 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:
©2008 by American Society of Hematology

3. 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:
©2008 by American Society of Hematology

4. 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:
©2008 by American Society of Hematology

5. 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:
©2008 by American Society of Hematology

6. 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:
©2008 by American Society of Hematology

7. 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:
©2008 by American Society of Hematology

8. 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:
©2008 by American Society of Hematology