1. Volume 136, Issue 4, Pages 1308-1316.e3 (April 2009)
Smad7 Controls Resistance of Colitogenic T Cells to Regulatory T Cell-Mediated Suppression 
Massimo C. Fantini, Angelamaria Rizzo, Daniele Fina, Roberta Caruso, Massimiliano Sarra, Carmine Stolfi, Christoph Becker, Thomas T. MacDonald, Francesco Pallone, Markus F. Neurath, Giovanni Monteleone 
Gastroenterology 
Volume 136, Issue 4, Pages e3 (April 2009)
DOI: /j.gastro
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2. Figure 1
Smad7-dependent resistance of CD lamina propria CD4+ T cells to Tregs suppression. (A) LPMC from colons of CD patients and controls and CD4+CD25− cells from peripheral blood mononuclear cells (PBMC) of healthy donors (responder cells, R) were stained with CFSE and cultured in activating conditions in the presence of peripheral blood CD4+CD25+ Tregs (suppressor cells, S) at different ratios as indicated. After 5 days, proliferating CD4+ T cells were evaluated on the basis of CFSE dilution by flow cytometry gating on CD4+ cells. Numbers above the line in the histogram plots indicate the nonproliferating fraction of CD4+ T cells from 1 representative experiment. (B) Normalized percentage of CD4+ T cells proliferation in the presence or absence of Trges as depicted in A. Points represent the average percentage ± SD of responder cells' proliferation cultured in the absence of suppressor cells (R/S 1:0) (CD LPMC vs control LPMC, *P < .05). (C) LP CD4+ T cells isolated from CD LPMC were incubated in the presence of the Smad7 antisense (AS) or the control oligonucleotide (SS). After 5 days, Smad7 expression was evaluated by Western blotting. (D) CFSE-labeled LPMCs from CD patients (T responder cells) were perincubated for 24 hours with Smad7 antisense or with the control oligonucleotide. Pretreated cells or control (CTRL) cells from LP of uninflamed patients cells were then activated alone or in the presence of Tregs isolated from the peripheral blood of healthy controls at different ratios as indicated. After 5 days, CD4+ T-cell proliferation was evaluated by flow cytometry. Bars indicate average proliferation ± SD of responder (R) preincubated with the Smad7 AS oligonucleotide (dark gray) or control cells (black) relative to cells preincubated with the control (SS) oligonucleotide (light gray) in the presence of Tregs (S). *Indicates a statistically significant difference between cells incubated in the presence or absence of Tregs (P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
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3. Figure 2
(A) Representation of the CD2-SMAD7 construct. Smad7 expression in wild-type and Smad7tg mice was evaluated by real-time PCR (B) and Western blotting (C). A representative real-time PCR and Western blotting for Smad7 and β-actin in CD4+ T cells isolated from wild-type and Smad7tg mice are shown. (D) Representative Western blotting for phosphorylated Smad3 (p-Smad3) and total Smad3 in CD4+ T cells isolated from wild-type and Smad7tg mice and stimulated with TGF-β (1 ng/mL) for 15 and 30 minutes. (E) CFSE-labeled naive CD4+ T cells from wild-type and Smad7tg mice were activated with soluble anti-CD3 in the presence of APC and increasing doses of TGF-β1. After 3 days, proliferation was evaluated by flow cytometry. Bars indicate mean ± SD of 3 independent experiments. *P < .05, **P < .01.
Gastroenterology  , e3DOI: ( /j.gastro )
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4. Figure 3
Wild-type Tregs fail to suppress the proliferation and cytokine secretion in Smad7tg CD4+ T-cell cultures. (A) CFSE-labeled wild-type and Smad7tg naïve T cells (R) were activated by soluble anti-CD3 and syngenic APC alone or in the presence of different dilutions of wild-type Treg cells (S). Proliferation was evaluated by flow cytometry. One of 5 representative experiments is shown. Numbers above the bars indicate the percentage of nonproliferating cells. (B) Percent of proliferation of each dilution relative to R activated alone from 5 independent experiments was calculated and pooled together. Points indicate mean ± SD. *P < .01. (C) Supernatants of cells cultured as indicated in A in the presence or absence of Tregs (R/S 4:1) were analyzed for IFN-γ, IL-6, IL-2, and IL-17 by flow cytometry-based ELISA system. Bars indicate mean ± SD of 3 independent experiments. *P < .05.
Gastroenterology  , e3DOI: ( /j.gastro )
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5. Figure 4
(A) RAG1−/− mice were transferred with Smad7tg and wild-type naïve T cells. The weight of each mouse was recorded daily. Each point represents the cumulative mean weight ± SD of 7 animals per group. *P < .05, **P < .01. (B) Representative endoscopic pictures of mice belonging to the 2 groups 4 weeks after the initial transfer. (C) Endoscopic score of colitis obtained from each animal. *P < .05. (D) Representative H&E staining of colon cross sections from the 2 groups at the end of the experiment. Original magnification, 40× and 100×. (E) Histologic score of colitis was obtained from each animal belonging to the 2 groups. *P < .05.
Gastroenterology  , e3DOI: ( /j.gastro )
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6. Figure 5
(A) IL-17A and IFN-γ intracellular staining of LPMC isolated from RAG1−/− mice reconstituted with either Smad7tg or wild-type naïve T cells. Dot plots were obtained gating on CD4+ cells. Percentages of the different populations are indicated in the plot quadrants. (B) Th1- and (C) Th17-related cytokines and transcription factors expression were evaluated by real-time PCR in freshly isolated LPMC isolated from the 2 groups of animals. Columns and error bars indicate mean ± SD of 5 different mice pooled together. P < .05. (D) Flow cytometry analysis of FoxP3 expression in LPMCs isolated from the 2 groups of animals was performed gating on the CD4+ population. Percentages of the different populations are indicated in the plot quadrants. Foxp3 transcripts were analyzed by real-time PCR in LPMC isolated from wild-type and Smad7tg mice (lower left panel; *P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
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7. Figure 6
Smad7tg CD4+ naïve T cells are resistant to Treg-mediated suppression in vivo. In the same experiment shown in Figure 4, 2 additional groups of RAG1−/−-deficient mice were reconstituted with either Smad7tg or wild-type naïve T cells (responder cells, R) together with an equal number of wild-type Treg cells (suppressor, S). (A) The weight of each mouse of the 2 groups was daily recorded. Each point represents the cumulative mean weight ± SD of 1 experiment in which 5 mice per group were analyzed (*P < .05; **P < .001). (B) Representative endoscopic pictures of mice belonging to the 2 groups of mice 6 weeks after the initial transfer. (C) Endoscopic score of colitis obtained from each animal (**P < .01). (D) Representative H&E staining of colon cross sections from the 2 groups at the end of the experiment; original magnification, 20× and 100×. (E) Histologic score of colitis was obtained from each animal belonging to the 2 groups (*P < .05). (F) Expression of Th1- and Th17-related cytokines (upper panel) and transcription factors (lower panel) were evaluated by real-time PCR in freshly isolated LPMCs isolated from the 2 different groups of mice. Bars indicate the average expression ± SD of 5 mice per group (*P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
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8. Supplementary Figure 1
(A) FoxP3 expression in CD4+CD25+ Tregs sorted from splenocytes of wild-type and Smad7tg mice. (B) CFSE-labeled wild-type naïve T cells (WtR) were activated by soluble anti-CD3 and syngenic APC alone or in the presence of different dilutions of either wild-type or Smad7tg Treg cells (S). Proliferation was evaluated by flow cytometry. Percent of proliferation of each dilution relative to R cells activated alone from 3 independent experiments was calculated and pooled together. Points indicate mean ± SD.
Gastroenterology  , e3DOI: ( /j.gastro )
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9. Supplementary Figure 2
(A) Representative H&E-staining of colon cross sections collected from 8-week-old Smad7tg and wild-type mice, original magnification, 20×. (B) IFN-γ and IL-17A intracellular staining of LPMC, cells from mesenteric lymph nodes, and splenocytes isolated from 8-week-old wild-type or SMAD7tg mice. Representative dot plots obtained gating on CD4+ cells are shown. Percentages of the different populations are indicated in the plot quadrants. Expression of Th1 (C) and Th17 (D) cytokines and lineage commitment transcription factors were evaluated by real-time PCR. Bars indicate average ± SD of 2 independent experiments (P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
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10. Supplementary Figure 3
Naïve T cells were sorted from splenocytes of Smad7tg and wild-type mice and activated with plate bound anti-CD3 and soluble anti-CD28 in the presence of Th0-, Th1-, or Th17-polarizing stimuli for 5 days. After 5 days, cells were stimulated for a further 5 hours with PMA and ionomycin in the presence of monensin before the staining with IL-17A (A) and IFN-γ (B) specific antibodies. Representative results of 3 independent experiments are shown. (C) Naïve CD4+ T cells from Smad7tg and wild-type mice were activated in vitro in the presence of TGF-β (5 ng/mL). After 5 days, cells were stained for FoxP3. Percentage of CD4+ cells expressing FoxP3 is indicated in the plot quadrants. (D) FoxP3 expression evaluated in LPMC of RAG1−/− mice reconstituted with wild-type or Smad7tg naïve T cells gating on the CD4+CD25bright population. Percentages of the different populations are indicated in the plot quadrants.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions