A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism

Abstract

Foxp3(+) regulatory T (T reg) cells play a key role in controlling immune pathological re actions. Many develop their regulatory activity in the thymus, but there is also evidence for development of Foxp3(+) T reg cells from naive precursors in the periphery. Recent studies have shown that transforming growth factor (TGF)-beta can promote T reg cell development in culture, but little is known about the cellular and molecular mechanisms that mediate this pathway under more physiological conditions. Here, we show that after antigen activation in the intestine, naive T cells acquire expression of Foxp3. Moreover, we identify a population of CD103(+) mesenteric lymph node dendritic cells (DCs) that induce the development of Foxp3(+) T reg cells. Importantly, promotion of T reg cell responses by CD103(+) DCs is dependent on TGF-beta and the dietary metabolite, retinoic acid (RA). These results newly identify RA as a cofactor in T reg cell generation, providing a mechanism via which functionally specialized gut-associated lymphoid tissue DCs can extend the repertoire of T reg cells focused on the intestine.

Figure 1.

Induction of Foxp3⁺ T cells after oral administration of antigen. DO11.10 SCID mice were given OVA or BSA in drinking water for 5 d. (A) Single cell suspensions of MLNs stained for CD4 and Foxp3 and analyzed by FACS. Numbers represent the proportion of Foxp3⁺ cells among the CD4⁺ population. (B) Percentage of Foxp3⁺ cells among the CD4⁺ population, and total number of Foxp3⁺ cells in OVA- and BSA-fed mice. Data are representative of two similar independent experiments.

Figure 2.

Induction of Foxp3⁺ T cells in the presence of MLN CD103⁺ DCs. (A) Single cell suspensions of spleen, MLNs, and colonic LP from BALB/c mice were prepared, and the proportion of CD103⁺ cells among the CD11c^high population was determined. (B) 0.25–1 × 10⁵ CD103⁺ or CD103⁻ MLN DCs were cultured with CFDA SE-labeled DO11.10 CD4⁺ T cells and 0.2 μg/ml OVA peptide. At day 7 of culture, T cells were stained for Foxp3 and CD4 and analyzed by FACS. The graph shows the percentage of Foxp3⁺ cells among CD4⁺ T cells in the presence of varying numbers of either DC subset. Data is representative of three independent experiments. (C) FACS plots showing the percentage of T cells expressing Foxp3 at the start of culture and after 7 d of culture with 10⁵ CD103⁺ or CD103⁻ MLN DCs. Plots are gated on CD4⁺ cells, and numbers represent the proportion of CD4⁺ cells in each quadrant. (D) CD4⁺CD25⁺ T cells from DO11.10 mice were isolated and cultured with 10⁵ CD103⁺ or CD103⁻ MLN DCs and 0.2 μg/ml OVA peptide. Before culture, and at day 6 of culture, T cells were stained for CD4, Foxp3, and clonotypic TCR (KJ-1.26). Plots are gated on KJ-1.26⁺CD4⁺ cells. Numbers represent the percentage of Foxp3⁺ cells among the KJ-1.26⁺CD4⁺ population. Data are representative of two independent experiments.

Figure 3.

Induction of Foxp3 is dependent on TGF-β. (A) CD4⁺ T cells from DO11.10 SCID mice were cultured with 5 × 10⁴ CD103⁺ or CD103⁻ MLN DCs, 0.2 μg/ml OVA peptide, and 50 μg/ml anti–TGF-β or isotype control. At day 6 of culture, T cells were stained for CD4 and Foxp3 and analyzed by FACS. Representative plots from two independent experiments are gated on CD4⁺ cells, and numbers represent the percentage of CD4⁺ cells in each quadrant. (B) CD103⁺ and CD103⁻ DCs were sorted from the MLNs of BALB/c mice. Tgfb2, ltbp3, and plat gene expression was assayed by quantitative PCR and normalized relative to expression of HPRT. Data shown are representative of two independent experiments. (C) CD4⁺ T cells from DO11.10 SCID mice were cultured with 10⁵ CD103⁺ or CD103⁻ MLN DCs, 0.2 ug/ml OVA peptide, and the indicated concentrations of rhTGF-β. At day 6 of culture, T cells were stained for CD4 and Foxp3 and analyzed by FACS. Representative plots from three similar experiments are gated on CD4⁺ cells, and numbers represent the percentage of Foxp3⁺ cells among CD4⁺ T cells.

Figure 4.

RA acts as a cofactor for Foxp3 induction. (A) CD103⁺ and CD103⁻ DCs were sorted from the MLNs of BALB/c mice. Aldh1a2 gene expression was assayed by quantitative PCR and normalized relative to expression of HPRT. Data shown are representative of two independent experiments. (B) CD4⁺ T cells from DO11.10 SCID mice were cultured with 5 × 10⁴ CD103⁺ or CD103⁻ MLN DCs, 0.2 μg/ml OVA peptide, and 2 ng/ml rhTGF-β. Some wells were additionally supplemented with 100 nM RA. T cells were stained for β7 integrin, CD4, and Foxp3 and analyzed by FACS. Plots are gated on CD4⁺ cells, and numbers represent the percentage of CD4⁺ cells in each quadrant. Data are representative of two independent experiments. (C) Graph depicts pooled data from the experiments described in B. Bars show the percentage of CD4⁺ cells expressing Foxp3. (D) Graph depicts absolute numbers of Foxp3⁺ T cells in culture under the indicated conditions.

Figure 5.

CD103⁻ DCs produce proinflammatory cytokines. CD103⁺ and CD103⁻ DCs were sorted from the MLN of BALB/c mice and cultured overnight in the presence of anti-CD40, an isotype control, or LPS. (A) Supernatants were harvested, and cytokine concentrations were analyzed by cytometric bead array. Graphs are representative of two independent experiments and depict cytokine concentrations in pg/ml. (B) IL-12p40 and IL-23p19 gene expression was assayed by quantitative PCR and normalized relative to expression of HPRT. Data are representative of two independent experiments. (C) CD103⁺ and CD103⁻ DCs were sorted from the MLN of BALB/c mice. Tlr2, tlr4, and tbx21 gene expression was assayed by quantitative PCR and normalized relative to expression of HPRT. Data are representative of two independent experiments.