Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3 precursors

Abstract

Foxp3(+)CD25(+)CD4(+) regulatory T cells are produced in the thymus (natural T regs) but can also differentiate from peripheral Foxp3(-)CD4(+) precursors (induced or adaptive T regs). We assessed antigen presenting cell (APC) requirements for the latter differentiation. With added transforming growth factor (TGF)-beta, both immature and mature populations of dendritic cells (DCs) induced antigen-specific Foxp3(+) T regs from Foxp3(-) precursors. Using endogenous TGF-beta, DCs from gut-associated mesenteric lymph nodes were capable of differentiating Foxp3(+)T regs. Spleen DCs were 100-fold more potent than DC-depleted APCs for the induction of T regs and required 10-fold lower doses of peptide antigen. Interleukin-2 (IL-2) was essential, but could be provided endogenously by T cells stimulated by DCs, but not other APCs. The required IL-2 was induced by DCs that expressed CD80/CD86 costimulatory molecules. The DC-induced Foxp3(+)T regs divided up to 6 times in 6 days and were comprised of CD62L and CD103 positive and negative forms. The induced Foxp3(+)T regs exerted suppression in vitro and blocked tumor immunity in vivo. These results indicate that DCs are specialized to differentiate functional peripheral Foxp3(+)T regs and help set the stage to use DCs to actively suppress the immune response in an antigen-specific manner.

Figure 1

BM-DCs plus TGF-β induce differentiation of Foxp3⁺T regs from DO11.10 RAG−/− Foxp3⁻CD25⁺CD4⁺ T cells. (A) Day-6 BM-DCs were stimulated with or without 50 ng/mL LPS for 16 hours. After washing, 6 × 10³ immature (without LPS) or mature DCs (with LPS) were cultured with Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11.10 RAG^−/− mice for 5 days with peptide in the presence or absence of TGF-β (2 ng/mL). Cells were stained with mAbs to CD4, KJ1.26 clonotype, and CD25-Abs. After fixation, cells were stained with anti-Foxp3 mAb. Plots were gated on CD4⁺ KJ1.26⁺ cells. (B) As in panel A, but the indicated numbers of DCs were cultured with Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11 RAG^−/− mice for 5 days with the indicated doses of peptide in the presence or absence of TGF-β (2 ng/mL). The absolute numbers of Foxp3⁺CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days are shown. (C) As in panel A, but absolute numbers of CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days are shown. (D) As in panel A, but freshly isolated CD25⁺CD4⁺T cells from DO11.10 RAG^+/+ mice (2 × 10⁴, > 90% Foxp3+) were cultured with immature or mature BM-DCs plus 0.1 μg/mL peptide in the presence or absence of TGF-β (2 ng/mL) or IL-2 (100 U/mL). (E) As in panel D, but freshly isolated CD25⁺CD4⁺T cells from DO11.10 RAG^+/+ mice were cultured with immature or mature BM-DCs (2 × 10⁴). Geometric mean fluorescence intensity (MFI) for Foxp3 within the circle is shown in the bottom of the plots. Data are representative from 2 to 3 independent experiments.

Figure 2

Foxp3⁺CD25⁺CD4⁺ T cells are induced from Foxp3⁻CD25⁻CD4⁺ T cells more effectively by spleen CD11c⁺ DCs than CD11c⁻ APCs, and DCs from mesenteric lymph nodes differentiate Foxp3⁺T reg in the absence of exogenous TGF-β. (A) Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11.10 RAG^−/− mice were cultured for 5 days with CD11c⁺ DCs (2 × 10⁴) or CD11c⁻ cells (2 × 10⁵) from spleen and indicated doses of OVA peptide in the presence or absence of TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were stained with mAbs to CD4, clonotype (KJ1.26), CD25, and Foxp3. Plots were gated on CD4⁺ KJ1.26⁺ cells. (B) As in panel A, but absolute numbers of Foxp3⁺CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days were shown. (C) As in panel A, but the absolute numbers of CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days were shown. (D) As in panel A, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured for 5 days at 0.03 μg/mL peptide with CD11c⁺ DCs (2 × 10⁴) from mesenteric or skin draining lymph nodes in the presence or absence of TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). (E) As in panel D, but anti–TGF-β mAb (10 μg/mL) or isotype control was added into the culture. Data are representative of 2 to 4 independent experiments.

Figure 3

Endogenous IL-2 from T cells stimulated by CD80/CD86^+/+ DCs is required for the differentiation of Foxp3⁺CD25⁺CD4⁺ T cells. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with spleen CD11c⁺ DCs with peptide plus TGF-β in the presence or absence of blocking anti–IL-2 Ab or control Abs (20 μg/mL). (B) Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with spleen CD11c⁺ DCs (2 × 10⁴) or CD11c⁻ APCs (2 × 10⁵) with indicated dose of peptide in the presence or absence of TGF-β. After day 5, culture supernatants were collected, and the concentration of IL-2 measured by Luminex. (C) CD4⁺ T cells from Foxp3-IRES-RFP knock-in OVA OTII CD4 transgenic mice (FIR-OTII) were stained with anti-CD4 and CD25 Abs, and Foxp3⁻CD25⁻CD4⁺ T cells were purified by flow cytometry (left). The purity of Foxp3⁻CD25⁻CD4⁺ T cells after sorting was higher than 99.5% (right; gated on CD4⁺ T cells. (D) The Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from FIR-OTII mice were cultured with spleen CD11c⁺ DCs (2 × 10⁴) for 5 days in the presence of peptide (0.03 μg/mL) with or without TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were gated on CD4⁺ T cells. The absolute numbers of Foxp3⁺CD4⁺T cells per culture from 4 different experiments with spleen DCs or immature BM-DCs are shown. P value is provided by Student t test. (E) As in panel D, but spleen CD11c⁺ DCs or immature BM-DCs were prepared from CD80/CD86^−/− or wild-type (WT) mice in the presence of peptide with or without TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were gated on CD4⁺ T cells. The absolute numbers of Foxp3⁺CD4⁺ T cells per culture from 4 different experiments with spleen DCs or immature BM-DCs are shown. P value is provided by Student t test. Data are representative of 2 to 4 independent experiments.

Figure 4

DC-induced, differentiating, peripheral Foxp3⁺CD25⁺CD4⁺ T cells undergo substantial proliferative activity. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice (2 × 10⁴) were CFSE labeled and cultured with CD11c⁺ DCs (2 × 10⁴) from spleen with or without OVA peptide (0.03 μg/mL) in the presence or absence of TGF-β. At day 3 or day 6, cells were stained with anti-CD4 and KJ1.26 clonotype Abs. After fixation, cells were stained with anti-Foxp3 or isotype control mAb. Foxp3 expression and CFSE dilution were shown gated on CFSE⁺CD4⁺KJ1.26 clonotype⁺ T cells. indicates proliferating cells expressed Foxp3. (B) As in panel A, but CFSE-labeled DO11.10 RAG^−/− Foxp3⁻CD25⁻CD4⁺ T cells were cultured with spleen CD11c⁺ DCs (2 × 10⁴) or DC-depleted spleen cells (CD11c⁻ APC, 2 × 10⁵) at the indicated dose of peptide in the presence of TGF-β (2 ng/mL). Foxp3 expression and CFSE dilution were analyzed at day 6. Data are representative of 2 independent experiments.

Figure 5

DC-induced Foxp3⁺T regs have a comparable phenotype to natural T regs. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with CD11c⁺ spleen DCs and 0.03 μg/mL peptide in the presence of TGF-β. These cultured cells (top) or freshly isolated spleen cells from DO11.10 RAG^+/+ mice (bottom) were stained with mAbs to CD4, clonotype (KJ1.26), and the indicated Abs or isotype controls. Plots were gated on CD4⁺ KJ1.26⁺ cells for the induced T regs (top) and on CD4⁺ cells for freshly isolated spleen cells (bottom). (B) Freshly isolated spleen cells from DO11.10 RAG^+/+ mice (non–T regs, natural T regs) or the cultured cells as in panel A but with low peptide (0.03 μg/mL) or high peptide (0.3 μg/mL) were stained with CD4, clonotype (KJ1.26), CD62L, CD103, and Foxp3 Abs. Plots for non-T regs were gated on Foxp3⁻CD25⁻CD4⁺ T cells, and plots for natural T regs were gated on Foxp3⁺CD25⁺CD4⁺ T cells. Plots were gated on Foxp3⁺CD4⁺KJ1.26⁺ cells for cultured cells. Data are representative of 2 to 4 independent experiments.

Figure 6

DC-induced Foxp3⁺ T cells are suppressive in vitro. (A) Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured for 7 days with CD11c⁺ spleen DCs (2 × 10⁴), peptide (0.03 μg/mL), and TGF-β (2 ng/mL). Cells were stained with anti-CD4, CD25, and Foxp3 Abs. (B) As in panel A, but live cells were stained with anti-CD4 and CD25 Abs for sorting. The square indicates the gate for sorting. (C) Sorted cells from (B) were fixed and further stained with anti-Foxp3 Ab. The purity of sorted natural CD25⁺CD4⁺ T regs was also shown. (D) CD25⁻CD4⁺ responder T cells (2 × 10⁴) from DO11.10 mice were CFSE-labeled and stimulated with spleen APCs (10⁵) and anti-CD3 mAb. To these, the induced Foxp3⁺ T regs purified as in panel C were added in graded numbers. After 3 days, CFSE dilution was analyzed with flow cytometry. Dead cells were gated out by TOPRO-3 iodide. Data are representative of 2 independent experiments.

Figure 7

DC-induced Foxp3⁺ T cells suppress OVA specific immunity in vivo. (A) BALB/c mice were sublethally irradiated (4.5 Gy). After 6 hours, they were injected intradermally on back with OVA-expressing A20 tumor (4 × 10⁶). (B) As in panel A, but irradiated recipients were injected intradermally with OVA-expressing A20 tumor and were also injected intravenously with freshly isolated CD25⁻CD4⁺ T cells (2 × 10⁶) from DO11.10 RAG^−/− mice. (C) As in panel B, but irradiated recipients were injected intradermally with OVA-expressing A20 tumor and were also injected intravenously with freshly isolated CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice along with the TGF-β cultured Foxp3⁺ T regs (2 × 10⁶). The TGF-β cultured Foxp3⁺ T regs were induced by DCs and TGF-β from RAG^−/− DO11.10 mice and purified as in Figure 6A and supplemental Figure 9. (D) As in panel C, but instead of the TGF-cultured Foxp3⁺ T regs, TGF-cultured Foxp3⁻ T cells (2 × 10⁶) were injected. The TGF-cultured Foxp3⁻ T cells were from the same culture with panel C and purified as in Figure 7A-C. (E-H) The same experiment with panels A-D was repeated.