CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells - PubMed (original) (raw)

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Sayuri Yamazaki et al. J Immunol. 2008.

Abstract

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.

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Figures

Fig. 1

Fig. 1. CD8+ spleen DCs differentiate Foxp3+ CD25+CD4+ T reg from Foxp3− CD25− CD4+ T cells in the absence of exogenous TGF-β in vitro

(A) Freshly isolated Foxp3−CD25−CD4+ T cells from DO11.10 RAG−/− mice (2×104) were cultured with the indicated doses of peptide and CD8− or CD8+ DCs (2×104) in the absence or presence of TGF-β (2 ng/ml). After 5 days of culture, cells were analyzed by FACS. Representative gates of CD4+ CD11c+ cells are shown on the left. (B) As in (A), but the number of Foxp3+ cells, the percentage of Foxp3+ T cells/CD4+ T cells, and the number of CD4+ T cells per culture in the presence or absence of TGF-β are shown. The number of Foxp3+ cells at 0 μg/ml peptide was < 102. A summary of 5–7 separate experiments. P values were provided by student-t test.

Fig. 2

Fig. 2. CD8+ DCs differentiate Foxp3+ CD25+ CD4+ T reg using endogenous TGF-β

(A) Freshly isolated Foxp3− CD25− CD4+ T cells from DO11.10 RAG−/− mice (2×104) were cultured with 0.03 μg/ml peptide with CD8+ DCs (2×104) in the presence of anti-TGF-β mAb or isotype matched control mAb (10 μg/ml). After 5 days of culture, cells were analyzed with FACS by gating on CD4+ CD11c− T cells. (B) As in (A), but the number of Foxp3+ cells, the percentage of Foxp3+ T cells/CD4+ T cells, and the number of CD4+ T cells per culture are shown. A summary of 3 separate experiments. P values were provided by student-t test. (C) As in (A), but cells were cultured in the presence of anti-IL-10 mAb or isotype control mAb (10 μg/ml). After 5 days of culture, cells were analyzed with FACS by gating on CD4+ CD11c− T cells. (D) As in (C), but the number of Foxp3+ cells, the percentage of Foxp3+ T cells/CD4+ T cells, and the number of CD4+ T cells per culture are shown. These data are representative of 3 experiments.

Fig. 3

Fig. 3. CD8+ DCs produce TGF-β

(A) Total RNA was isolated from splenic CD8+ DEC-205+ or CD8− DCIR2+ DCs. Affymetrix gene array analysis showing relative levels of mRNAs associated with the TGF-β and TGF-β receptor signaling pathways expressed by CD8− DCIR2+ (left panel) and CD8+ DEC-205+ DCs (right panel) purified from B6 mice. Each lane consists of three independent microarrays of mRNAs from different cell sorts. (B) CD8+ or CD8− DCs from naïve BALB/c mice or BALB/c mice that had been injected with poly IC 12 hours before were cultured for 15–40 hours in serum free medium and the concentrations of TGF-β in the supernatants were measured by ELISA. The concentration of TGF-β in the serum free medium was 0. A summary of 5 separate experiments. P value was provided by student-t test. (C) CD8+ or CD8− DCs from naïve BALB/c mice or BALB/c mice that had been injected with poly IC 12 hours before were purified by flow cytometry. Pre-sort (top) and post-sort (middle) plots are shown as gated on CD11c+ cells. The purified cells were further stained with CD86 (black line) and isotype control Abs (shaded gray line) (bottom). (D) The purified CD8+ or CD8−DCs (2×104) from naïve or poly IC injected mice as in (C) were cultured with freshly isolated Foxp3− CD25− CD4+ T cells from DO11.10 RAG−/− mice (2×104) in the presence of 0.03 μg/ml peptide. After 5 days of culture, cells were analyzed with FACS by gating on CD4+ CD11c− T cells. (E) As in (D), but the frequency of Foxp3+ T cells/CD4+ T cells per culture is also shown. These data are representative of 3 independent experiments with each experiment done in triplicate. P value was provided by student-t test.

Fig. 4

Fig. 4. Suppressive function of Foxp3+ CD25+ CD4+ T reginduced by CD8+ or CD8−DCs

(A) Foxp3− CD25− CD4+ T cells (2×104) from FIR-OTII mice were cultured with spleen CD8+ DCs (2×104) in the presence of peptide (0.03–0.1 μg/ml) without TGF-β. After 5–6 days, the induced Foxp3+ CD25+ CD4+ T reg and Foxp3− CD25− CD4+ T cells were purified by flow cytometry. Similarly, Foxp3− CD25− CD4+ T cells from FIR-OTII mice were also cultured with spleen CD8− DCs in the presence of peptide (0.03–0.1 μg/ml) with TGF-β (2 ng/ml), and the induced Foxp3+ CD25+ CD4+ T reg were purified by flow cytometery. The square in pre-sort indicates the gate for sorting. The purity of sorted cells is also shown (post-sort). (B) For the suppression assay, CD25− CD4+ responder T cells (1×104) from B6 mice were CFSE-labeled and stimulated with spleen APCs (5×104) and anti-CD3 mAb. To these, the suppressors (1×104) as above were added. After 3 days, CFSE-dilution was analyzed by flow cytometry. Dead cells were gated out by TOPRO-3 iodide. These data are representative of 3 independent experiments. (C) As in (B), but the number of live CFSE+ CD25−CD4+ responder cells per culture was shown. The indicated suppressors were added in the culture at the indicated ratio. This data is representative of 2 independent experiments.

Fig. 5

Fig. 5. T reg proliferate during differentiation by DC subsets

Foxp3− CD25−CD4+ T cells from DO11.10 RAG−/− mice (2×104) were CFSE labeled and cultured with CD8+ DCs (2×104) without TGF-β (2 ng/ml) or CD8− DCs (2×104) with TGF-β in the presence or absence of OVA peptide (0.03 μg/ml). At day 5, cells were stained with anti-Foxp3 mAb. Foxp3 expression and CFSE dilution are shown gated on live CD11c− CD4+ T cells. This data is representative of 3 independent experiments.

Fig. 6

Fig. 6. DC targeting of OVA via DEC-205 leads to the development of Foxp3+ CD25+ CD4+ T cells in vivo

(A) Thy1.2+ DO11.10 RAG−/− mice lack Foxp3+ CD25+ CD4+ T cells as shown in the plots. The Foxp3− CD25− CD4+ T cells were CFSE-labeled (2×106) and injected i.v. into Thy1.1+ BALB/c mice. The recipients were stimulated with indicated doses of DEC-OVA or 33D1-OVA i.p. one day later. After 3 or 10–14 days, spleen and lymph node Thy1.2+ cells were analyzed by FACS. (B) As in (A), but the frequencies of donor Thy1.2+ DO11.10 RAG−/−CD4+ T cells within gates at day 3 are shown in the top row. Foxp3, isotype control staining and CFSE dilution gated on Thy1.2+ transferred DO11.10 RAG−/−CD4+ T cells are shown in the middle and bottom rows. This data is representative of 2 independent experiments. (C) As in (A), but the frequencies of donor Thy1.2+ DO11.10 RAG−/− CD4+ T cells within gates at day 13 are shown in the top row. Foxp3, isotype control staining and CFSE dilution gated on the Thy1.2+ transferred DO11.10 RAG−/−CD4+ T cells are shown in the middle and bottom rows. (D) As in (A), but recipient mice were stimulated with PBS, 3 μg DEC-OVA or Iso-OVA i.p. one day later. After 13 days, a mixture of spleen and lymph nodes was analyzed by FACS. Foxp3 (left), isotype control staining (right) and CFSE dilution gated on the Thy1.2+ transferred DO11.10 RAG−/−CD4+ T cells are shown. Data are representative of 3 independent experiments. (E) Cell recoveries of Foxp3+ Thy1.2+ T cells from transferred DO11.10 RAG−/− CD4+ T cells at day10–14 are shown as percentages per Thy1.2+ T cells (left) and absolute numbers from each mouse (right). A summary of 5 separate experiments where each data point is a separate experiment. P value is from student t-test.

Fig. 7

Fig. 7. DC targeting of OVA via 33D1 better sustains naturally occurring Foxp3+ CD25+CD4+ T cells in vivo

(A) CD25+ CD4+ Foxp3+ T cells from Thy1.2+ DO11.10 transgenic mice were cultured with mature DCs, peptide and IL-2. After 7 days, the expanded T reg were > 90% CD25+Foxp3+. The expanded T reg (0.5~1×106) were CFSE-labeled and adoptively transferred into Thy1.1+ BALB/c mice. One day later, the mice were i.p. injected with 3 μg of 33D1-OVA, DEC-OVA Abs or PBS. Spleen and lymph node cells were mixed and analyzed by FACS at day 3 or 14. (B) Mixtures of spleen and lymph node cells at day 3 or 10 after injection of 33D1-OVA, DEC-OVA Abs or PBS were stained with anti-CD4 and anti-Thy1.2 (Top). Foxp3, isotype control staining and CFSE dilution gated on Thy1.2+ transferred DO11.10 T reg are shown in the middle and bottom rows. (C) As in (A), but freshly isolated CFSE-labeled Thy1.2 DO11.10 CD25+ CD4+ T reg were transferred into Thy1.1+ BALB/c mice. One day later, the mice were i.p. injected with 3 μg of 33D1-OVA, DEC-OVA Abs, Iso-OVA or PBS. Spleen and lymph node cells were mixed and analyzed FACS at day 3. Data are representative of 2 independent experiments (D) As in (A), but cell recovery of Thy1.2+ transferred DO11.10 T reg at day 10–14 is shown. A summary of 5 separate experiments. P value is from student t-test.

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