Dendritic cells with TGF-beta1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells

Abstract

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from naïve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-beta1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4(+)CD25(+)Foxp3(+) T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.

Fig. 1.

Splenic DCs efficiently induce Foxp3 expression from naïve CD4⁺CD25⁻ T cells. (A) Cell surface expression of CD40, CD86, and MHC class II (I-A^g7) of freshly isolated NOD splenic CD11c⁺ DCs. (B) Foxp3 expression by precultured sorted CD4⁺CD25⁻CD62L⁺ BDC2.5 T cells and induction in T cells after culture with or without 2 ng/ml TGF-β1 on day 6 of culture. Expression of CD62L by precultured CD4⁺CD25⁻ BDC2.5 T cells is also shown. (C) Time-course of induction of CD4⁺CD25⁺Foxp3⁺ BDC2.5 T cells from naïve CD4⁺CD25⁻Foxp3⁻ BDC2.5 T cells in the presence of 2 ng/ml TGF-β1. (Upper) Total number of Foxp3⁺ T cells per well. (Lower) Percentages of Foxp3⁺ T cells determined by intracellular staining on days 2, 3, 4, 5, 6, and 10 of DC-T cultures. The isotype control for day 3 is shown. (D) Quantification of Foxp3 mRNA by real-time RT-PCR. Samples were prepared from enriched CD25⁺ fractions of the resulting T cells from cocultures with or without 2 ng/ml TGF-β1 or freshly isolated CD4⁺CD25⁺ and CD4⁺CD25⁻ BDC2.5 T cells. Values were standardized by 18s RNA and expressed as fold of increase compared with precultured freshly isolated CD4⁺CD25⁻ cells. (E) Dose-response of TGF-β1 determined on day 6 of DC-T cocultures at indicated concentrations of TGF-β1. The isotype control for the 0.01 ng/ml dose is shown. All results are representative of two to four separate experiments.

Fig. 2.

Characterization of CD4⁺CD25⁺Foxp3⁺ BDC T cells induced by DCs and TGF-β1. (A) Intracellular expressions of IFN-γ, IL-10, and IL-17 on day 6 of DC-T coculture. The addition of 20 ng/ml IL-6 induced IL-17 expression. (B) Expression of CD62L, CTLA-4, and GITR on day 6 of DC-T coculture. Cells cultured in the absence of TGF-β1 were also shown for comparison. Shaded histogram, isotype control; blue line, +TGF-β1; red dotted line, −TGF-β1. (C) BDC clonotype expression. (Left) Freshly isolated BDC2.5 CD4⁺ T cells. (Right) T cells on day 6 after culture. Shaded histogram, isotype control; blue line, T cells cultured with DCs and TGF-β1; green line, T cells cultured with anti-CD3, anti-CD28, and TGF-β1. All results are representative of two to four separate experiments.

Fig. 3.

CD4⁺CD25⁺Foxp3⁺ BDC T cells induced by DCs and TGF-β1 suppress BDC-specific T cell proliferation and cytokine production. After 6 days of culture with (A) or without (B) TGF-β1, CD11c⁺ cells were depleted and CD25⁺ cells were enriched. FACS plots constructed after the enrichment are shown (Left). Proliferation assays were set up with CD4⁺CD25⁻ cells from BDC2.5 mice [responders (R)], APCs from NOD mice, and BDC peptide (100 ng/ml). Increasing numbers of the induced CD4⁺CD25⁺ cells [suppressors (S)] were added at ratios indicated. Proliferation was assessed by [³H]thymidine uptake (Center), and IFN-γ was measured from culture supernatants (Right) as described in Materials and Methods.

Fig. 4.

CD4⁺CD25⁺Foxp3⁺ BDC T cells induced by DCs and TGF-β1 block the development of diabetes in NOD.scid recipients. NOD.scid mice were injected i.v. with 10⁷ spleen cells from diabetic NOD females with either nothing (red triangles) or the indicated numbers of CD4⁺CD25⁺ BDC T cells from cultures with splenic DCs in the presence (+TGF-β1 BDC T, blue circles) or absence (−TGF-β1 BDC T, green squares) of TGF-β1. P < 0.0001, control vs. 3 × 10⁵ +TGF-β1 BDC T cells; P < 0.0001, control vs. 3 × 10⁴ +TGF-β1 BDC T cells.

Fig. 5.

CD4⁺CD25⁺Foxp3⁺ BDC T cells induced by DCs and TGF-β1 protect syngeneic islet grafts from ongoing autoimmune destruction in spontaneously diabetic NOD recipients. A total of 500 NOD islets were transplanted into kidney subcapsular space either alone (red) or with 3 × 10⁵ +TGF-β1 BDC T cells (blue) or −TGF-β1 BDC T cells (green). Day 0 indicates the day of islet transplantation. (A) Blood glucose levels post transplantation. Each line represents one mouse. (B) Summary of graft survival after transplantation for individual mice is shown in (A). P < 0.0001 for comparison of graft survival among all three groups; P = 0.0069, −TGF-β1 BDC T vs. islets only; P = 0.0001, +TGF-β1 BDC T vs. islets only. Data represent the combination of three separate transplant experiments with cells from three separate cultures.