and zDC chimeras. - Nature Research...Macrophages can be monocyte– or yolk sack–derived and self-renewing (indicated by curved arrow). (b, c) Representative flow cytometry plots

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 g) in 50 mg unlabeled OVA in 200 l 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 g) in 50 mg unlabeled OVA in 200 l 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.

Nature Immunology: doi:10.1038/ni.3408


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 (averageSEM) are representative of two independent experiments; NS= not significant, *p<0.05, **p<0.01,***p<0.005.



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 BMCmice, 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.



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) MHCIIintCD8�+CD11b–CD103+, (e) MHCIIintCD8�–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 averageSEM. (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.



cDC subset depletion in the spleen of zDC(Irf8) mice.

(a) Representative flow cytometry contour plots and (b) relative frequencies of CD8+ and DEC205+ DCs and (c) total CD45+ counts in the spleen of Irf8fl/fl versus zDC(Irf8) 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 averageSEM.



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 percell population.


anti-‐B220 1:200 48-‐0452-‐82 eBioscience anti-‐B220 1:200 553086 BD Pharmigen anti-‐CD3e 1:200 48-‐0031-‐82 eBioscience anti-‐CD4 1:200 83-‐0042-‐42 eBioscience anti-‐CD4 1:200 BE0003-‐1 BioXCell anti-‐CD8a 1:200 BE0004-‐1 BioXCell anti-‐CD8a 1:200 56-‐0081-‐82 eBioscience anti-‐CD8a 1:200 553029 BD Pharmigen anti-‐CD8a 1:200 100744 Biolegend anti-‐CD11b 1:200 47-‐0112-‐82 eBioscience anti-‐CD11b 1:200 BE007 BioXCell anti-‐CD11b 1:200 553309 BD Pharmigen anti-‐CD11b 1:200 101236 Biolegend anti-‐CD25 1:200 17-‐0251-‐82 eBioscience anti-‐CD25 1:200 553070 BD Pharmigen anti-‐CD11c 1:200 25-‐0114-‐82 eBioscience anti-‐CD11c 1:200 17-‐0114-‐82 eBioscience anti-‐CD11c 1:200 56-‐0114-‐82 eBioscience anti-‐CD11c 1:200 553800 BD Pharmigen anti-‐CD11c 1:200 553800 Biolegend anti-‐CD19 1:200 BE0150 BioXCell anti-‐CD44 1:200 56-‐0441-‐85 eBioscience anti-‐CD45 1:200 25-‐0451-‐82 eBioscience anti-‐CD45.1 1:200 25-‐0453-‐82 eBioscience anti-‐CD45.2 1:200 560693 BD Pharmigen anti-‐CD62L 1:200 560516 BD Pharmigen anti-‐CD64 1:200 139306 Biolegend anti-‐CD69 1:200 45-‐0691-‐82 eBioscience anti-‐CD103 1:100 557495 BD Pharmigen anti-‐CD115 1:200 46-‐1152-‐82 eBioscience anti-‐Foxp3 1:100 12-‐5773-‐82 eBioscience anti-‐I-‐A/I-‐E (MHCII) 1:4000 6-‐5321-‐82 eBioscience anti-‐I-‐A/I-‐E (MHCII) 1:4000 56-‐5321-‐82 eBioscience anti-‐IL4 1:200 554432 BD Pharmigen anti-‐IL5 1:200 554395 BD Pharmigen anti-‐IL17A 1:200 56022 BD Pharmigen anti-‐IFN-‐g 1:200 25-‐7311-‐82 eBioscience anti-‐IgE 1:200 RMGE00 Invitrogen

anti-‐IgE 1 to 10000 13-‐5992-‐82 eBioscience

anti-‐IgG1 1:50000 A90-‐105B Bethyl anti-‐IgG2c 1:25000 A90-‐136B anti-‐Ly6C 1:200 560595 BD Pharmigen anti-‐Ly6G 1:200 48-‐5931-‐82 eBioscience anti-‐Neuropilin 1:200 BAF566 RandD Systems anti-‐NK1.1 1:200 562921 BD Pharmigen


anti-‐NK1.1 1:200 553163 BD Pharmigen

anti-‐OVA IgG1 1:800-‐1:100000 520501 Biolegend

anti-‐SiglecF 1:200 552126 BD Pharmigen anti-‐TCRβ 1:200 109220 Biolegend anti-‐TCRβ 1:200 BE00102 BioXCell anti-‐TER119 1:200 553672 Biolegend anti-‐Va2 1:200 48-‐5812-‐82 eBioscience

IgE isotype control 1:256-‐1:100000 554118 BD Pharmigen

Supplementary Table 1. List of antibodies, dilutions used, catalogue number and vendor.


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and zDC chimeras. - Nature Research...Macrophages can be monocyte– or yolk sack–derived and self-renewing (indicated by curved arrow). (b, c) Representative flow cytometry plots