TGF-β signaling is required for the function of insulin-reactive T regulatory cells
Wei Du, F. Susan Wong, Ming O. Li, Jian Peng, Hao Qi, Richard A. Flavell, Robert Sherwin, Li Wen
Wei Du, F. Susan Wong, Ming O. Li, Jian Peng, Hao Qi, Richard A. Flavell, Robert Sherwin, Li Wen
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Research Article Autoimmunity

TGF-β signaling is required for the function of insulin-reactive T regulatory cells

Abstract

We have previously isolated insulin-reactive Tregs from diabetic NOD mice designated 2H6, from which TCR transgenic mice were generated. The T cells from these 2H6 transgenic mice recognize insulin but have suppressive properties in vitro. They protect NOD mice in vivo from spontaneous development of diabetes and adoptive transfer of disease caused by polyclonal diabetogenic spleen cells as well as the highly diabetogenic monoclonal BDC2.5 TCR transgenic T cells that recognize an islet granule antigen. Using cells from both NOD and BDC2.5 mice that express a dominant-negative TGF-β receptor type II (TGF-βDNRII), we show that 2H6 T cells protected from disease by producing TGF-β and that the ability of the target diabetogenic T cells to respond to TGF-β was crucial. We further demonstrate that TGF-β signaling in 2H6 cells was important for their protective properties, as 2H6 cells were unable to protect from adoptive transfer–induced diabetes if they were unable to respond to TGF-β. Thus, our data demonstrate that insulin-specific regulatory cells protect from diabetes by virtue of their production of TGF-β1 that acts in an autocrine manner to maintain their regulatory function and acts in a paracrine manner on the target cells.

Authors

Wei Du, F. Susan Wong, Ming O. Li, Jian Peng, Hao Qi, Richard A. Flavell, Robert Sherwin, Li Wen

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Figure 2

Insulin reactivity, cytokine profile, and CTLA-4 and FoxP3 expression of 2H6 TCR transgenic T cells.

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Insulin reactivity, cytokine profile, and CTLA-4 and FoxP3 expression of...
(A) Insulin reactivity of 2H6 TCR transgenic T cells. Splenocytes from 3 randomly selected 2H6 TCR transgenic mice (1 from line 22 and 2 from line 50) were used in the proliferation assay. Cells (105 per well) were cultured in Click’s medium for 72 hours (in triplicate). 3H-thymidine was added for the last 16–18 hours. (B) The reactivity to insulin B chain peptide 12–25 or 9–23 was also analyzed using the same protocol as in A. Results are illustrated as Δ cpm = proliferation with peptide – proliferation with medium. (C and D) TGF-β (C) and IFN-γ (D) production from cells of transgene-negative and -positive NOD mice. Splenocytes (105 cells per well) from both types of mice were cultured in Click’s medium in the presence or absence of anti-CD3 (1:100 dilution of 2C11 hybridoma supernatant) for 48 hours (in triplicate). The culture supernatants were collected. IFN-γ and TGF-β contents were measured with an IFN-γ ELISA kit (BD Biosciences) and a TGF-β ELISA kit (R&D Systems). (E) Expression of CTLA-4 and FoxP3 in transgene-positive and -negative NOD mice. Splenocytes (106) from both types of mice were stained for intracellular CTLA-4 and FoxP3 expression with or without TCR stimulation (anti-CD3). Monoclonal antibodies against CTLA-4 and FoxP3 were purchased from BD Biosciences and eBioscience, respectively. The staining was carried out according to the manufacturers’ protocols. The numbers in the top right quadrants represent the percentage of positive cells among the total cells analyzed.