TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes - PubMed (original) (raw)

TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes

Yufeng Peng et al. Proc Natl Acad Sci U S A. 2004.

Abstract

CD4+CD25+ regulatory T cells are essential in the protection from organ-specific autoimmune diseases. In the pancreas, they inhibit actions of autoreactive T cells and thereby prevent diabetes progression. The signals that control the generation, the maintenance, or the expansion of regulatory T cell pool in vivo remain poorly understood. Here we show that a transient pulse of transforming growth factor beta (TGF-beta) in the islets during the priming phase of diabetes is sufficient to inhibit disease onset by promoting the expansion of intraislet CD4+CD25+ T cell pool. Approximately 40-50% of intraislet CD4+ T cells expressed the CD25 marker and exhibited characteristics of regulatory T cells including small size, high level of intracellular CTLA-4, expression of Foxp3, and transfer of protection against diabetes. Results from in vivo incorporation of BrdUrd revealed that the generation of a high frequency of regulatory T cells in the islets is due to in situ expansion upon TGF-beta expression. Thus, these findings demonstrate a previously uncharacterized mechanism by which TGF-beta inhibits autoimmune diseases via regulation of the size of the CD4+CD25+ regulatory T cell pool in vivo.

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Figures

Fig. 1.

Fig. 1.

Regulated TGF-β expression in TTA/TGF-β NOD mice. (A) TTA regulatory system. Under the control of a rat insulin promoter (RIP), tetracycline-controlled transactivator (TTA) is expressed specifically in insulin-producing cells. In the presence of tetracycline or its analogue doxycycline (Dox), TTA (+Dox) is unable to bind the TRE and TGF-β transcription is inactive, whereas in the absence of doxycycline (–Dox) TTA binds TRE and induces TGF-β expression via the cytomegalovirus minimal promoter (P min CMV). DNA fragments RIP-TTA and TRE-TGF-β were coinjected to generate double transgenic mice in NOD background. (B) Regulated TGF-β transcription. NOD Transgenic mice were fed with normal or doxycycline-supplemented food. Total RNA was isolated from pancreatic tissue. The transcripts of TGF-β (Upper) and TTA (Lower) were amplified by using RT-PCR. Lanes 1 and 3, constitutively on for 5 weeks since birth; lanes 2 and 4, constitutively on for 5 weeks followed by 1-week turn-off; lane 5, transgene negative control; lane 6, constitutively off for 2 months since birth; lanes 7, 8, and 10, constitutively off for 2 month since birth followed by a turn-on for 4 days (lanes 7 and 10) or 1 week (lane 8); lane 9, spleen RNA controls. Each lane represents a different mouse. (C) Regulated TGF-β protein expression. Pancreatic tissues were isolated from line 4 (a_–_c) and line 45 (d), then fixed and stained for TGF-β protein (red, counterstained by hematoxylin/eosin). (a) Constitutively off for 8 weeks since birth. (b) Constitutively on for 5 weeks since birth. (c) Constitutively on for 5 weeks since birth followed by 1 week turn-off. (d) Constitutively on for 8 weeks since birth.

Fig. 2.

Fig. 2.

Transient expression of TGF-β inhibits diabetes. (A) Summary of the different groups (1–5) of transgenic mice with turning on and off period time of the expression of TGF-β. Development of diabetes was monitored in line 4 (B) and line 45 (C) transgenic mice during 60 weeks. Filled circle, transgene negative; open circle, TGF-β turned on and off (groups 2–5); filled triangle, TGF-β constitutively turned off (group 1).

Fig. 3.

Fig. 3.

TGF-β expression does not affect intraislet antigen-presenting function or T cell differentiation profile. CFSE-labeled purified naïve BDC2.5 T cells were injected into 4-week-old transgenic mice (line 45) in which TGF-β was either turned off (A) or on (B) from birth to 4 weeks of age. Four days posttransfer, pancreatic lymph node cells were collected and lymphocytes were analyzed by FACS. Dot plots represent CFSE intensity on gated CD4+ T cells. The results are a representative of eight experiments. Similar results were obtained from line 4. Intraislet T cells were pooled from five transgenic mice (line 45) and stimulated with phorbol 12-myristate 13-acetate plus ionomycin for 6 h. Contour plots represent the distribution of IL-4-versus IFN-γ-expressing cells among gated CD4+ T cells isolated from mice with TGF-β constitutively off for 10 weeks since birth (C), with TGF-β off for 8 weeks since birth and turned on for the next 5 weeks (D), with TGF-β on for 8 weeks since birth (E), or with TGF-β on for 16 weeks since birth (F).

Fig. 4.

Fig. 4.

TGF-β induces high frequency of intraislet CD4+CD25+ regulatory T cells Intraislet T cells were pooled from five transgenic mice (line 45) and then stained for the expression of CD4 and CD25 markers. Contour plots represents results from mice with TGF-β constitutively off for 10 weeks since birth (A), TGF-β off for 8 weeks since birth and turned on for the next 5 weeks (B), TGF-β on for 8 weeks since birth (C), or TGF-β on for 16 weeks since birth (D). Gated CD4+CD25+ T cells from pancreatic lymph nodes (E) or islets (F) isolated from transgenic line 45 with TGF-β on for 8 weeks since birth were compared for intracellular expression of CTLA-4 as well as cell size (FSC) and cell granulosity (SSC). (G) Intraislet CD4+ T cells were purified from transgenic line 45 with TGF-β on for 8 weeks since birth (Tg pos) or from transgenic negative mice (Tg neg), and levels of mRNA of Foxp3 expression were determined by real-time quantitative PCR (filled bars). Splenic CD4+CD25+ and CD4+CD25– T cells from NOD mice were used as controls (open bars). (H) Intraislet T cells were purified from transgenic line 45 with TGF-β on for 8 weeks since birth, and 105 cells were transferred to NOD-SCID recipient mice together with 20 × 106 splenic cells from diabetic NOD mice (open bars). As control, 20 × 106 splenic cells from diabetic NOD mice were transferred alone to recipient mice (filled bars). Diabetes development was monitored for 10 weeks posttransfer.

Fig. 5.

Fig. 5.

TGF-β induces proliferation of intraislet CD4+CD25+ T cells. Control NOD mice (B) and transgenic line 45 with TGF-β on for 8 weeks since birth (A) were injected for 4 consecutive days with 1 mg of BrdUrd. Lymphocytes were isolated from islets, mesenteric lymph nodes, spleen, and thymus, and stained with anti-CD4 and anti-CD25 and anti-BrdUrd antibodies. Contour plots show the distribution of BrdUrd versus CD25 expression on gated CD4+ T cells. The results are representative of four pooled mice.

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