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. 2016 May;17(5):545-55.
doi: 10.1038/ni.3408. Epub 2016 Mar 28.

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance

Daria Esterházy  1 , Jakob Loschko  2 , Mariya London  1 , Veronica Jove  1 , Thiago Y Oliveira  2 , Daniel Mucida  1
Affiliations

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance

Daria Esterházy et al. Nat Immunol. 2016 May.

Abstract

Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.

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Figures

Figure 1
Figure 1. DTH responses in mice depleted of APC subsets during antigen feeding
(a) Flow cytometry contour plots of mLN macrophages and cDC subpopulations. (b) Experimental design of oral tolerance establishment towards CFA-mediated immunity. (c) Degree of ear swelling 48 h post second subcutaneous OVA challenge in wild-type, MMDTR, zDCDTR and CD11cDTR BMCs treated with DT during oral OVA (+) or PBS (−) exposure. (d) Serum anti-OVA IgG1 and (e) anti-OVA IgG2c levels in mice described in (c) 28 days post CFA immunization. Data represent pooled values (average±SEM) from two independent experiments, n=10 per group, ns= not significant, NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test).
Figure 2
Figure 2. Allergic airway responses in mice depleted of APC subsets during antigen feeding
(a) Experimental design of oral tolerance establishment towards Alum-mediated immunity. (b) Histological score for inflammatory infiltrate and (c) representative hematoxylineosin staining of lung tissue from naïve (upper row) and OVA-gavaged (lower row) wild-type, MMDTR and zDCDTR mice. Scale bar, 250 μm. (d) Representative flow cytometry contour plot for SiglecF+MHCII− gating (eosinophils, eos) in the BALF, and (e) analysis of total BAL eosinophil counts and (f) relative lung tissue eosinophil frequencies among CD45+ cells in wild-type, MMDTR and zDCDTR BMCs treated with DT during oral OVA (+) or PBS (−) exposure. (g) Serum total IgE in mice described in (b-e) 21 days post Alum immunization. Data represent pooled values (average±SEM) from two independent experiments, n=10 per group, NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test).
Figure 3
Figure 3. Contribution of macrophage-monocyte– and pre-DC–derived cells to pTreg cell induction in vivo
(a) Schematic representation of in vivo pTreg cell induction protocols. (b-f) Flow cytometry analysis of mLNs; (b) representative contour plots for Foxp3 and CD25 expression of TCRβ+CD4+CD45.1+ T cells; (c) Foxp3+, (d) CD45.1+, (e) CD25+ cell frequencies and (f) cell division index of adoptively transferred naïve CD45.1 OT-II cells in the mesenteric lymph nodes (mLN) of wild-type, MMDTR and zDCDTR BMCs, 48 h after first oral OVA gavage. n=4. (g) Flow cytometry analysis of CD45.1 OT-II Foxp3+ cell frequency in mLN of DT treated wild-type, MMDTR and zDCDTR BMCs, 7.5 days after first OVA gavage and 8 days post adoptive transfer of naïve CD45.1 OT-II cells. n=3. NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test). Data (average±SEM) are representative of three independent experiments.
Figure 4
Figure 4. Contribution of CX3CR1+ cells to APC pools and pTreg cell induction
(a) Relative frequencies of CD11c+, CD11c+CX3CR1− and CD11c+CX3CR1+ among CD45+ cells, and APC subpopulations among (b) CX3CR1− and CX3CR1+ (CD11c+) cells in the mLNs of wild-type, MMDTR and zDCDTR BMCs, 36 h after DT administration. n=3. (c) Pie charts of relative MM (light grey) and cDC (dark grey) contributions to APC subpopulations within CX3CR1+ versus CX3CR1− cell populations. n=4, ±SD. (d) Pie chart of flow cytometry analysis of APC subpopulations frequencies in the mLN within CD45+Lin−MHCII+CD11c+ cells in SPF versus GF mice and targeting of the populations in zDCDTR mice (dark grey). n=4, ±SD. (e) Foxp3+ cell frequencies and cell division index of adoptively transferred naïve CD45.1 OT-II cells in the mLN of SPF versus GF, CX3CR1GFP/+ versus zDCDTR (CX3CR1GFP/+) BMCs, 48 h after first oral OVA gavage. n=4 (f, g) Flow cytometry analysis of APC cell frequencies in the mLN of CX3CR1lsl-DTR control versus CX3CR1DTR BMCs, 36 h post DT administration. n=4. Numbers above CX3CR1 bars depict percentage reduction upon DT administration. (h-j) Foxp3+, CD25+ cell frequencies and cell division index of adoptively transferred naïve CD45.1 OT-II cells in the mLN of CX3CR1LsL-DTR control versus CX3CR1DTR BMCs, 48 h (h) or 7.5 days (i, j) after first oral OVA gavage. n=4, NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test). Data (average±SEM) are representative of two independent experiments.
Figure 5
Figure 5. Characterization of mLN cDC subpopulations
(a) Heat map of most differentially expressed genes of cDC subsets in the mLN determined by RNA-seq. (b) Arbitrary units of TGF-β synthesis and (c) retinoic acid synthesis gene cluster expression levels in mLN cDC subsets determined by RNA-seq. (a-c) n=3, representing biological replicates. (d) Flow cytometry analysis of mean fluorescence intensity (MFI) of FITC+ Aldefluor conversion product in indicated mLN cDC populations after 30 min of substrate addition, pre-incubated with or without RALDH inhibitor DEAB. n=4 (biological replicates). Data are representative of four independent experiments. (e) Heat map of expression levels of cDC subsets in the mLN determined by RNA-seq. n=3, as (a-c). Data shown as average±SEM; NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test).
Figure 6
Figure 6. Analysis of steady state cDC and lymphocyte populations in the mLN and lamina propria of zDC(ΔIrf8) and Irf8fl/fl mice
(a) Representative flow cytometry contour plots, (b) relative frequencies of CD11c+, CD11c+MHCIIhi (migratory) and CD11c+MHCIIint (resident) cDCs and total CD45+ cells in the mLN of Irf8fl/fl versus zDC(ΔIrf8) mice. (c-e) Representative flow cytometry contour plots (c) and relative frequencies among MHCII+CD11c+ cells (d) or CD11c+CD64− cells (e) of small intestine lamina propria (LP) CD103+, CD103+CD11b+ and CD103− DCs in 10 weeks old Irf8fl/fl versus zDC(ΔIrf8) mice. (f) mLN and small LP CD3+CD8+ and CD3+CD4+ cells, (f) mLN and small LP CD3+CD4+Foxp3+, CD3+CD4+CD62L+ and CD3+CD4+CD44+ cells in 10 weeks old Irf8fl/fl versus zDC(ΔIrf8) mice. n=4, NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test). Data (average±SEM) are representative of two independent experiments.
Figure 7
Figure 7. Assessment of oral tolerance and pTreg cell induction in zDC(ΔIrf8) and Irf8fl/fl BMCs
(a) Degree of ear swelling 48 h post subcutaneous OVA challenge in Irf8fl/fl versus zDC(ΔIrf8) BMCs given oral OVA (+) or PBS (−) before immunization. (b) Serum anti-OVA IgG1 and (c) anti-OVA IgG2c levels in mice described in (a), 28 days post CFA immunization. n=5 per group. (d-f) Flow cytometry analysis of (d) Foxp3+, (e) CD25+ cell frequencies and (f) cell division index of adoptively transferred naïve CD45.1 OT-II cells in the mLN of Irf8fl/fl versus zDC(ΔIrf8) BMCs, 48 h after first oral OVA gavage. n=4. (g) Foxp3+ cell frequencies of adoptively transferred naïve CD45.1 OT-II cells in the mLN of Irf8fl/fl versus zDC(ΔIrf8) BMCs, 7.5 days after first oral OVA gavage. n=4. Data (average±SEM) are representative of two independent experiments each (a-c, d-f, g-h). NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test).
Figure 8
Figure 8. In vitro characterization of pTreg cell induction potential of cDC subpopulations in the mLN
(a) Heat map of most differentially expressed genes in OT-II cells co-cultured with indicated mLN cDC subsets and OT-II peptide for 24 h determined by RNA-seq. n=3 (b) Treg and proliferation gene cluster expression levels of cDC subsets in the mLN determined by RNA-seq experiment shown in (a). n=3 (c) Flow cytometry analysis of Foxp3+ frequency and cell division index of sorted naïve CD45.1 OT-II cells co-cultured with indicated mLN cDC subsets, OT-II peptide, anti-IL-4 and anti-IFN-γ antibodies for 72 h. n=4-6 (n=4 for resident and n=6 for migratory DC co-culture) from three independent DC donor pools. Data is representative of three independent experiments. (d) Flow cytometry analysis of Foxp3+ frequency of sorted naïve CD45.1 OT-II cells co-cultured with indicated mLN cDC subsets obtained from mice that were fed an OVA-rich diet for 48 h, anti-IL-4 and anti-IFN-γ antibodies for 72 h. n=4-6 from three independent DC donor pools. Data shown as average±SEM; NS= not significant, *P<0.05, **P<0.01, ***P<0.005 (two-tailed t-test).
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