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Classical dendritic cells are required for dietary antigen–mediated induction of peripheral Treg cells and tolerance

Nature Immunology volume 17, pages 545–555 (2016)Cite this article

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Abstract

Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pTreg 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 pTreg 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 pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg 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 pTreg cells and their redundancy during the development of oral tolerance.

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Figure 1: Delayed-type hypersensitivity responses in mice depleted of APC subsets during antigen feeding.
Figure 2: Allergic airway responses in mice depleted of APC subsets during antigen feeding.
Figure 3: Contribution of macrophage-monocyte– and pre-DC–derived cells to the induction of pTreg cells in vivo.
Figure 4: Contribution of CX3CR1+ cells to APC pools and the induction of pTreg cells.
Figure 5: Characterization of mLN cDC subpopulations.
Figure 6: Analysis of steady-state cDC and lymphocyte populations in the mLNs and lamina propria of zDC(ΔIrf8) and Irf8fl/fl mice.
Figure 7: Induction of oral tolerance and pTreg cells in zDC(ΔIrf8) and Irf8fl/fl chimeras.
Figure 8: In vitro characterization of the pTreg cell–induction potential of cDC subpopulations in the mLNs.

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Acknowledgements

We thank M. Amoury and L. Cohn for thoughts for the beginning of the project; M. Nussenzweig (The Rockefeller University) for Csf1rLsL-DTR, zDCDTR and zDCCre knock-in mice and conceptual input; S. Mazmanian (California Institute of Technology) for germ-free C57BL/6 mice; B. Reis and V. Pedicord for assistance in mouse-dissection experiments, critical discussions and help in preparing the manuscript; K. Velinzon and N. Thomas for assistance in cell sorting; The Rockefeller University Genomics Center; A. Rogoz, T. Rendon, S. Gonzalez and the Rockefeller University Comparative Bioscience Center for animal care and genotyping; and the New York University histology core for lung histological staining. Supported by the Swiss National Foundation (D.E.), The US National Institutes of Health (1R56AI119062 to D.M.) and the Leona M. and Harry B. Helmsley Charitable Trust (D.E. and D.M.).

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Authors and Affiliations

  1. Laboratory of Mucosal Immunology, The Rockefeller University, New York, New York, USA

    Daria Esterházy, Mariya London, Veronica Jove & Daniel Mucida

  2. Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA

    Jakob Loschko & Thiago Y Oliveira

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  1. Daria Esterházy
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Contributions

D.E. designed the study, performed the experiments (unless stated otherwise below) and wrote the manuscript; J.L. generated the zDCCre mice and backcrossed them onto the Irf8fl/fl background, carried out their hematopoietic characterization (Fig. 7) and substantially contributed to the design of experiments; M.L. and V.J. contributed substantially to the establishment of oral-tolerance protocols and to the oral-tolerance experiments; T.Y.O. performed the bioinformatics analysis of the RNA-seq experiments; and D.M. initiated, designed and supervised the study, performed experiments and wrote the manuscript.

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Correspondence to Daniel Mucida.

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Integrated supplementary information

Supplementary Figure 1 Analysis of OVA uptake and clearance and APC depletion and replenishment in wild-type, MMDTR and zDCDTR chimeras.

(a) Lineage tree of major antigen presenting cell populations. Cells expressing zDC and depleted in zDCDTR mice are depicted in dark grey, cells expressing LysM and CSF1R and depleted in MMDTR mice are depicted in light grey. Macrophages can be monocyte– or yolk sack–derived and self-renewing (indicated by curved arrow). (b, c) Representative flow cytometry plots of small intestine lamina propria (LP), mLN (b), spleen, and blood (c) of WT, MMDTR and zDCDTR BMC mice 36h after DT injection. (d) Serum Flt3L levels in WT and zDCDTR BMC mice 24h post first DT injection. n=5, data representative of more than 20 experiments. (e) Coomassie-stained (top) and radiography of SDS-PAGE gel (bottom) loaded with indicated amount of 125I-labeled ovalbumin, showing purity and integrity of labeled protein. (f-h) Percentage of radioactivity recovered in the (f) urine and blood, (g) jejunum, inguinal lymph nodes, mLN and spleen and the (h) thyroid and liver of 8 weeks old C57BL/6 mice gavaged with 300,000 CPM 125I-OVA (4 μg) in 50 mg unlabeled OVA in 200 μl PBS over indicated time. n>2 per time-point. (i-m) Radioactivity in the (i) mLN, (j) spleen, (k) liver, (l) thyroid and (m) total blood of 16 weeks old WT, MMDTR and zDCDTR BMC mice 24 h after DT administration gavaged with 300,000 CPM 125I-OVA (4 μg) in 50 mg unlabeled OVA in 200 μl PBS and sacrificed 4 h later. n=3. (n) Analysis of total APC and subpopulation replenishment in the LP, Peyer’s patches (PP), mLN, spleen, and blood of WT, MMDTR and zDCDTR BMC mice 7 days post last DT injection. n=3. (o) Relative frequencies of TCRβ+CD4+, TCRβ+CD8+, and B220+ among CD45+ cells in WT, MMDTR and zDCDTR BMC mice 7 days post last DT injection. n=3. NS= not significant, *p<0.05, **p<0.01,***p<0.005.

Supplementary Figure 2 mLN cell counts and long-term pTreg cell induction in wild-type, MMDTR and zDCDTR chimeras.

(a) Total CD4+TCRβ+ cell numbers in the in the mLNs of WT, MMDTR and zDCDTR BMC mice, 60h post first DT treatment. (b) Percentage of CD45.1+ cells among CD4+TCRβ+ cells in the mLNs upon 2 doses of DT treatment without OVA gavage, analyzed 48h after first DT dose. (c) Percentage of CD45.1+ cells among CD4+TCRβ+ cells in the mLNs of WT, MMDTR and zDCDTR BMC mice, 7.5 days post first OVA gavage (see Fig. 3.a). (d) Cell division index and (e) total OT-II cell number in the mLNs of WT versus zDCDTR BMC mice, 7.5 days after first OVA gavage and 8 days post adoptive transfer of naïve OT-II cells. Each symbol indicates an individual mouse (d, e). n=4 per group, data (average±SEM) are representative of two independent experiments; NS= not significant, *p<0.05, **p<0.01,***p<0.005.

Supplementary Figure 3 Analysis of APC subset contributions to mLN APCs.

(a) Relative frequencies of APC subpopulations in the mLNs of eight weeks old GF versus SPF C57BL/6 mice. n=3. (b) Relative frequencies of APC subpopulations among CD45+ cells in the mLNs of GF versus SPF WT and zDCDTR BMC mice, 24h after DT administration. n=3. (c) Total CD45+ and total CD4+TCRβ+ cell counts in the mLNs of GF versus SPF WT and zDCDTR BMC mice, 24h after DT administration. n=3. (d) Percentage of CD45.1+ cells among total CD4+TCRβ+ cells in the mLNs of GF versus SPF WT and zDCDTR BMC mice, 24h after DT administration. n=3. (e) Total CD45+ and total CD4+TCRβ+ cell counts in the mLNs of CX3CR1LsL-DTR control (CTRL) and CX3CR1DTRBMC mice, 5 days after last DT administration. n=5 (f) Percentage of CD45.1+ cells among total CD4+TCRβ+ cells in the mLNs of CTRL and CX3CR1DTR BMC mice, 24h or 5 days after last DT administration. n=5. NS= not significant, *p<0.05, **p<0.01,***p<0.005.

Supplementary Figure 4 cDC and lymphocyte frequencies in the mLNs draining the proximal-to-distal gastrointestinal tract.

(a) Dissection pictures of murine mLNs draining the duodenum (D), jejunum (J), ileum (I) and colon (C), indicated in white. Celiac (Ce) and pancreatic (P(d), adjacent to D, and P(j), adjacent to J) lymph nodes, the portal vein and the thoracic duct are indicated in black. Relative frequencies of (b) CD45+CD11c+, (c) CD11c+MHCIIint, (d) MHCIIintCD8α+CD11b−CD103+, (e) MHCIIintCD8α−CD11b+CD103−, (f) CD11c+MHCIIhi, (g) MHCIIhiCD11b−CD103+, (h) MHCIIhiCD11b+CD103+, (i) TCRβ−CD19+, (j) TCRβ+CD8+, (k) TCRβ+CD4+, (l) TCRβ+CD4+Foxp3+NRP+and (m) TCRβ+CD4+Foxp3+NRP− cells in the duodenum (D), jejunum (J), ileum (I) and colon (C) draining mLNs from 8 weeks old female C57BL/6 mice. Each symbol indicates an individual mouse (b-m). n=4. NS= not significant, *p<0.05, **p<0.01,***p<0.005. Data shown as average±SEM. (n) PCA of RNA-seq data from cDCs in the mLNs. Each circle represents average of triplicates per indicated cell population. (o-q) Heat map of (o) TGF-β synthesis, (p) retinoic acid synthesis and (q) cytokine gene cluster levels of DC subsets in the mLNs determined by RNA-seq. n=3.

Supplementary Figure 5 cDC subset depletion in the spleen of zDC(ΔIrf8) mice.

(a) Representative flow cytometry contour plots and (b) relative frequencies of CD8α+ and DEC205+ DCs and (c) total CD45+ counts in the spleen of Irf8fl/fl versus zDC(ΔIrf8) mice. n=4, data representative of more than six independent experiments. Each symbol indicates an individual mouse (b, c). NS= not significant, *p<0.05, **p<0.01,***p<0.005. Data shown as average±SEM.

Supplementary Figure 6 OT-II cell differentiation by IRF8 and IRF4-dependent cDCs from the mLNs.

PCA of RNA-seq data from OT-II cells co-cultured with indicated mLN cDC population. Each circle represents average of triplicates per cell population.

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Esterházy, D., Loschko, J., London, M. et al. Classical dendritic cells are required for dietary antigen–mediated induction of peripheral Treg cells and tolerance. Nat Immunol 17, 545–555 (2016). https://doi.org/10.1038/ni.3408

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