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Induced CD4+ forkhead box protein–positive T cells inhibit mast cell function and established contact hypersensitivity through TGF-β1  Wenru Su, MD, Huimin.

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Presentation on theme: "Induced CD4+ forkhead box protein–positive T cells inhibit mast cell function and established contact hypersensitivity through TGF-β1  Wenru Su, MD, Huimin."— Presentation transcript:

1 Induced CD4+ forkhead box protein–positive T cells inhibit mast cell function and established contact hypersensitivity through TGF-β1  Wenru Su, MD, Huimin Fan, MD, PhD, Maogen Chen, MD, Julie Wang, BS, David Brand, PhD, Xiaoshun He, MD, PhD, Valerie Quesniaux, PhD, Bernhard Ryffel, MD, PhD, Ling Zhu, PhD, Dan Liang, MD, PhD, Song Guo Zheng, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 130, Issue 2, Pages e7 (August 2012) DOI: /j.jaci Copyright © Terms and Conditions

2 Fig 1 iTreg cells inhibit proinflammatory cytokines released by MCs activated in an IgE-independent manner. BMMCs were cocultured with different CD4+ T cells at the indicated ratios for 24 hours. After stimulation with PMACI for 6 to 16 hours, TNF-α and IL-6 levels in the supernatants were determined by using ELISA (A and B), and the expression of TNF-α and IL-6 mRNA in BMMCs was determined by using real-time PCR (C-F). Values are presented as means ± SEMs of 3 replicates, and experiments were repeated at least 2 times with similar results. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

3 Fig 2 iTreg cells inhibit proinflammatory cytokines released by MCs by modulating NF-κB signaling. BMMCs were cocultured with iTreg cells at a 2:1 ratio for 24 hours. After stimulation with PMACI for 16 hours, MCs were isolated from cocultures by using negative selection through autoMACS. The expression of phosphorylated IκB-α (A) and NF-κB p65 (B) in BMMCs was determined by means of Western blotting. Values are presented as means ± SEMs of 3 replicates, and experiments were repeated at least 2 times with similar results. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

4 Fig 3 Membrane-bound TGF-β1 is essential for iTreg cell–mediated inhibition on MCs. BMMCs were cultured with or without iTreg cells for 24 hours either under direct cell-cell contact or in a transwell system (A); in the presence or absence of specific neutralizing antibodies for TGF-β1, OX40L, or isotype control IgGs or gap-junction inhibitor (B); or after pretreatment with TGF-β1 siRNA or control siRNA (CSiRNA; C) or SMAD3 inhibitor (D and E) after stimulation with PMACI. TNF-α and IL-6 levels were determined by using ELISA (Fig 3, A-E), and the SMAD3 expression in BMMCs was determined by means of Western blotting (F). Values are presented as means ± SEMs of 3 replicates, and experiments were repeated at least 2 times with similar results. **P < .01. CsiRNA, Blank control siRNA; Isotype, IgG isotype control for anti-OX40L or anti–TGF-β1. a-SMAD3, SMAD3 inhibitor (SIS3). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

5 Fig 4 Human iTreg cells suppress proinflammatory cytokine production by a human MC line. HMC-1 cells were cultured with or without different CD4+ T cells, followed by stimulation with PMACI for 6 to 16 hours. TNF-α (A) and IL-6 (B) levels were measured by means of ELISA, and NF-κBp65 (C), TNF-α (D), and IL-6 (E) expression in BMMCs was determined by using Western blotting and real-time PCR, respectively. Values are presented as means ± SEMs of 3 replicates, and experiments were repeated at least 2 times with similar results. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

6 Fig 5 iTreg cell treatment attenuates CHS. A, Experimental protocol. B, Ear thickness was measured at indicated time points after challenge in different experimental groups. C, Representative images of hematoxylin and eosin staining of ear samples in normal mice, mice with CHS, and mice with CHS after treatment with iTreg cells. D and E, Quantification of cellular components in ears of mice with CHS and draining lymph nodes. F, Gross draining lymph node weight. Each group includes 5 mice, and experiments were repeated once. The data are either representative or means ± SEMs of 2 separate experiments. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

7 Fig 6 iTreg cell treatment suppresses MCs in mice with CHS. iTreg cells were transferred to mice with CHS. A, Representative images. MCs were determined by using toluidine blue staining, and activated MCs were identified based on their irregular shape (red arrows) in ears of normal mice, mice with CHS, and iTreg cell–treated mice with CHS, as indicated. B and C, TNF-α and IL-6 mRNA expression determined based on real-time PCR. D, NF-κB expression determined by means of Western blotting. E and F, TGF-β1 mRNA (Fig 6, E) and protein (Fig 6, F) levels determined based on real-time PCR and ELISA. Each group includes 5 mice, and experiments were repeated once. Values are presented as means ± SEMs of 3 replicates. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

8 Fig 7 iTreg cell treatment attenuates CHS through TGF-β1. CHS was induced as above, and iTreg cells, either TGF-β1 or control knockdown iTreg cells, were adoptively transferred to mice with CHS. Ear thickness (A), frequency of MCs and numbers of irregular MCs (B), and TNF-α and IL-6 mRNA expression on ear tissues (C) were measured as above at the indicated time points after challenge. Each group includes 5 mice, and experiments were repeated once. Values are presented as means ± SEMs of 2 separate experiments or 3 replicates. **P < .01. ciTreg, iTreg cells treated with blank control siRNA; diTreg, iTreg cells treated with TGF-β1 siRNA. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

9 Fig E1 TGF-β–induced iTreg cells have a similar ability to express Foxp3 and develop the suppressive activity as nTreg cells. A, Splenic naive CD4+CD25−CD62L+CD44low cells from BALB/C mice were stimulated with anti-CD3/CD28–coated beads (1:5) in the presence of IL-2 (50 U/mL) and TGF-β (2 ng/mL) for 4 days. Splenic nTreg cells sorted from the thymus were stimulated with anti-CD3/CD28–coated beads (1:3) in the presence of IL-2 (300 U/mL) for 7 days. A, The cells were collected for intracellular Foxp3 staining. Scatter plots were gated on CD4 and are representative of 4 separate experiments. B, Carboxyfluorescein succinimidyl ester (CFSE)–labeled effector T cells stimulated with anti-CD3 in the presence of antigen-presenting cells were cultured at a 1:4 ratio with TGF-β–induced Treg or nTreg cells or without (Baseline) for 3 days. Representative CFSE plots of the T effector cells from 4 independent experiments are shown. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

10 Fig E2 iTreg cells modulate IL-6 production by MCs through TGF-β1 signal. BMMCs were cultured with or without iTreg cells for 24 hours under direct cell-cell contact or in a transwell system (A), in the presence or absence of anti–TGF-β1 and anti-OX40L antibodies or matched isytope control IgG and specific inhibitors for gap junction (B), or after pretreatment with TGF-β1 siRNA or control siRNA (CsiRNA; C). After stimulation with PMACI, IL-6 levels were determined by means of ELISA. Values are presented as means ± SEMs of 3 separate experiments. **P < .01. CsiRNA, Blank control siRNA; Isotype, IgG isotype control for anti-OX40L or anti–TGF-β. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

11 Fig E3 iTreg cells inhibit the degranulation of MCs activated in an IgE-dependent manner. Anti–DNP-IgE–presensitized BMMCs were challenged with DNP-BSA for 30 minutes in the presence or absence of equal amounts of Treg cells or T control cells. Degranulation was determined by measuring the activity of the MC granule–associated enzyme β-hexosaminidase. Values are presented as means ± SEMs of 3 separate experiments. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

12 Fig E4 Differential therapeutic effects of systemic administration of T-cell subsets. iTreg, nTreg, or T control cells were adoptively transferred into mice with CHS, as described in Fig 5, and ear thickness was measured at the time points as indicated after allergen challenge. *P < .05 and **P < .01 in contrast to T control cells transferred to mice with CHS by means of 1-way ANOVA. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

13 Fig E5 iTreg cell treatment inhibits the production of inflammatory cytokines in the ears of mice with CHS. iTreg cells were transferred to mice with CHS, as described in Fig 6. TNF-α (A), IL-6 (B), and IL-9 (C) production in the supernatants was determined by means of ELISA. Values are presented as means ± SEMs of 3 independent experiments. **P < .01. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

14 Fig E6 Knockdown of TGF-β1 in iTreg cells by siRNA significantly decreases their inhibitory capability on inflammatory cytokine production in mice with CHS. The experiment was conducted similarly to Fig 7. TNF-α and IL-6 production was determined by means of ELISA. Values are presented as means ± SEMs of 3 independent experiments. **P < 0. ciTreg, iTreg cells treated with blank control siRNA; diTreg, iTreg cells treated with TGF-β1 siRNA. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © Terms and Conditions

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