1. Volume 141, Issue 5, Pages 1802-1812 (November 2011)
Intestinal Dendritic Cells Specialize to Activate Transforming Growth Factor-β and Induce Foxp3+ Regulatory T Cells via Integrin αvβ8 
John J. Worthington, Beata I. Czajkowska, Andrew C. Melton, Mark A. Travis 
Gastroenterology 
Volume 141, Issue 5, Pages (November 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
RA is not essential for the enhanced ability of CD103+ intestinal DCs to induce Tregs. (A–E) A total of 5 × 104 CD4+ Foxp3GFP− T cells were incubated with 2.5 × 103 CD103+/− DCs in the presence of anti-CD3 antibody and/or RA, RA inhibitors, and/or anti–TGF-β antibody, and the induction of iTregs (CD4+ Foxp3GFP+) was assessed by flow cytometry after 5 days. (A) CD103+/− DC subsets from mLN or spleen in the absence of exogenous TGF-β. (B) CD103+/− DCs from mLN in the presence of 2 ng/mL TGF-β and 100 nmol/L RA or 1 μmol/L of RA inhibitors (LE-135, LE-450). (C) CD103+/− DCs from mLN in the absence of exogenous active TGF-β and/or 1 μmol/L RA inhibitors and/or 40 μg/mL anti–TGF-β antibody. (D) CD103+/− DCs from mLN in the presence of 100 nmol/L RA and/or 40 μg/mL anti–TGF-β antibody. Data are representative of at least 3 independent experiments. *P < .05, **P < .01. (E) Representative flow cytometry plots from C and D analyzing effects of RA inhibitors and RA on CD4+ T-cell expression of Foxp3GFP and α4β7.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
CD103+ intestinal DCs have an increased ability to activate latent TGF-β. (A) Latent TGF-β production by CD103+/− mLN DCs was assessed by qPCR and levels normalized to the housekeeping gene HPRT. Data represent 3 independent experiments. (B) Production of active TGF-β by CD103+/− DCs from mLN was assessed by culturing DCs with an active TGF-β reporter cell line.12 Data represent 3 independent experiments. **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
CD103+ intestinal DCs express elevated levels of the TGFβ-activating integrin αvβ8 and activate more TGF-β in an integrin αvβ8-dependent manner. RNA from (A) total DCs or (B) splenic/mLN CD103+/− DCs was analyzed for integrin β8 expression by qPCR. β8 levels were normalized to the housekeeping gene HPRT and presented relative to levels in total spleen DCs (for A) or spleen CD103-ve DCs (for B). Data shown are representative of 2 and 4 independent experiments, respectively. (C) TGF-β activation by control or αvβ8−/− CD103+/− mLN DCs was detected by coculture with an active TGF-β reporter cell line.12 Data represent 5 independent experiments. (D) RNA from control or αvβ8−/− CD103+/− DCs was analyzed for TGF-β expression by qPCR. Data shown represent 2 independent experiments. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
Integrin αvβ8-mediated TGF-β activation is critical for the enhanced ability of CD103+ intestinal DCs to induce Foxp3+ Tregs. A total of 5 × 104 CD4+ Foxp3GFP− T cells were cultured with 2.5 × 103 control or αvβ8−/− CD103+/− DC subsets in the presence of anti-CD3 antibody plus (A) 40 μg/mL mIgG or anti–TGF-β antibody or (B) 2 ng/mL exogenous active TGF-β. Foxp3+ Treg induction was assessed by flow cytometry after 5 days. Representative flow cytometry plots and data are shown from 3 or 5 independent experiments, respectively. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
Elevated expression of integrin αvβ8 by small and large intestinal lamina propria CD103+ DCs is required for enhanced induction of Foxp3+ Tregs. (A) Integrin β8 expression in small/large intestinal lamina propria CD103+/− DCs was assessed by qPCR, normalizing levels to the housekeeping gene HPRT. Data are presented as relative to small intestinal CD103-ve DC levels and represent 3 independent experiments. (B and C) A total of 5 × 104 CD4+ Foxp3GFP− T cells were cultured with 2.5 × 103 control or αvβ8−/− CD103+/− DC subsets in the presence of anti-CD3 antibody plus (B) mIgG or anti–TGF-β antibody or (C) exogenous active TGF-β. Foxp3+ Treg induction was assessed by flow cytometry after 5 days. Representative flow cytometry plots and data are shown from 2 to 6 independent experiments. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Figure 6
Integrin αvβ8-mediated TGF-β activation is critical for the enhanced ability of intestinal DCs to induce Foxp3+ Tregs in vivo after oral administration of antigen. A total of 2 × 106 carboxyfluorescein succinimidyl ester–labeled CD4+ T cells from OTII/Rag−/− mice were injected intravenously into control or Itgb8 (CD11c-Cre) mice. Recipient mice were fed ovalbumin in drinking water and mLN analyzed for CD4+ Vα2+ Foxp3+ cells after 6 days. (A) Representative flow cytometry plots and pooled data showing iTreg in mLN of recipient mice (n = 3). (B) Total number of transferred cells in mLN of recipient mice. (C) Proportion of CD103+ DCs in the mLN, small intestine lamina propria (SILP), Peyer's patches (PP), and large intestine lamina propria (LILP) of control and Itgb8 (CD11c-Cre) mice. Data from 8 independent experiments. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions