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Insulin-like growth factor 2 enhances regulatory T-cell functions and suppresses food allergy in an experimental model  Gui Yang, MD, Xiao-Rui Geng, MD,

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Presentation on theme: "Insulin-like growth factor 2 enhances regulatory T-cell functions and suppresses food allergy in an experimental model  Gui Yang, MD, Xiao-Rui Geng, MD,"— Presentation transcript:

1 Insulin-like growth factor 2 enhances regulatory T-cell functions and suppresses food allergy in an experimental model  Gui Yang, MD, Xiao-Rui Geng, MD, Jiang-Ping Song, MD, PhD, Yingying Wu, PhD, Hao Yan, MS, Zhengke Zhan, MS, Litao Yang, PhD, Weiyi He, MS, Zhi-Qiang Liu, MD, PhD, Shuqi Qiu, MD, Zhigang Liu, MD, PhD, Ping-Chang Yang, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 133, Issue 6, Pages e5 (June 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Treg cells express IGF2R. A to D and I to L, Gating strategy (arrows). E-H and M-P, IGF2R+ T cells. The bars of Fig 1, H and P, show the summarized data of Fig 1, F and G (Fig 1, H) and Fig 1, N and O (Fig 1, P). Values are presented as means ± SDs. *P < .01 compared with the Treg cell group. Each experiment was repeated 3 times. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IGF2 facilitates Treg cell proliferation. The iTreg cells were generated in vitro and treated in culture, as denoted above each subpanel. The histograms indicate the frequency of Treg cell proliferation. The broken curves are the negative control. IGF2R gene knockdown results are presented in Fig E1. Experiments were repeated 3 times. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Adoptively transferred Treg cells in the intestine. A-C, Images show CFSE+ Treg cells (in green) in the small intestine. Treatment is denoted above each image. D, Bars indicate summarized CFSE+ cell counts. Values are presented as means ± SDs. *P < .01 compared with group A. #P < .01 compared with group B. Each group consists of 6 mice. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IGF2 promotes Treg cell suppressor function. A, Histograms indicate Teff cell proliferation. B, Summarized data of Fig 4, A. C, iTreg cells were cultured in the presence of anti-CD3/CD28 antibody (Ab), IGF2, or both. Bars indicate levels of TGF-β in culture medium. *P < .01 compared with saline group. #P < .01 compared with IGF2/Ab group. Experiments were repeated 3 times. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 IGF2 promotes Treg cell inhibitory effects on allergic inflammation. Sensitized mice were challenged with OVA, and treatment is shown on the x-axis. Data are presented as means ± SDs. *P < .01 compared with the saline group (A) or the naive group (B-F). #P < .01 compared with the saline group. $P < .01 compared with the iTreg and IGF2 groups. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 IGFR2 gene silencing results. Saline, Cells were treated with saline in the culture. shRNA, Cells were transduced with shRNA plasmid of IGFR2. cshRNA, Cells were treated with control shRNA with a sequence that did not target any genes. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 IGF1R levels in Treg and Teff cells. A or M and F or Q are the same panels as in Fig 1, C, and Fig 1, K. B and G, Bars indicate frequencies of IGF1R+ cells in the groups denoted on the x-axis. Data are summarized from C and D and H and I. Fig E2, C and D and H and I, Dot plots indicate frequencies of IGF1R+ cells in CD45RA+ or CD45RA− cells. L and P show IGF1R+ cells, IGF2R+ cells, or both among CD4+CD25− T cells. N and R, Bars indicate summarized data of Fig E2, L and P, respectively. E, J, K, and O are isotype IgG controls. Data from bars are presented as means ± SDs. The experiments were repeated 3 times. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 IGF2 promotes TCR activation–induced ERK phosphorylation in Treg cells. Treg cells were prepared as described in the text and cultured in the presence of anti-CD3/CD28 antibody (Ab), IGF2, or both for 72 hours. The treatment was denoted below the blots. BSA is used as an irrelevant protein and as a control reagent. Treg cell extracts were analyzed by using Western blotting. The immune blots show the status of ERK1/2 phosphorylation. Experiments were repeated 3 times. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 IGF2 promotes TCR-dependent Treg cell proliferation in vivo. OVA-specific iTreg cells were prepared in vitro, as described in the text; labeled with CFSE; and adoptively transferred into naive BALB/c mice. The mice were treated with either saline or OVA (50 μg per mouse in 0.3 mL of saline) and/or recombinant IGF2 (20 μg per mouse in 0.1 mL of saline administered intraperitoneally) daily from days 1 to 3. The mice were killed on day 4. LPMCs were isolated from each mouse and analyzed by means of flow cytometry. The CFSE+ cells were gated first and analyzed by using the CFSE dilution assay. A-E, Histograms show the frequency of CFSE+ cells. F, Bars show the proliferation rate of the CFSE+ cells. Each group consisted of 6 mice. Samples from individual mice were processed and analyzed separately. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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