% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
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1 Supp. Figure 1. a spleen cells (x1 6 ) 16 % of live splenocytes % of live splenocytes b 1 1 c % of CD11c + splenocytes (closed shapes) % ROSA + (open shapes) % floxed STAT5 deletion pdc CD8 + CD4 + RFP +/ RFP +/ RFP +/- RFP +/ RFP +/ RFP +/ pdc CD8 + CD4 + Nature Immunology doi:1.138/ni.541
2 Supplemental Figure 1, STAT5 deficiency in splenic DCs has no effect on hematopoietic cell numbers. A, Total cellularity and percentages of hematopoietic cells in Cre - 5 fl/fl and mice. CD49b + CD3 - (NK cells), CD11b + F4/8 + (macrophages), Ly6c + Ly6g + (granulocytes) Representative of three independent experiments. B, Percentages of pdc (PCDA1 + CD11c + ), CD8 + DCs (PDCA1 - CD11c + CD11b - CD8 + MHCII + ), CD4 + DCs (PDCA1 - CD11c + CD11b + CD4 + MHCII + ) of total CD11c + splenocytes (closed shapes) and percentages of FLOX:STOP:RFP + DCs within each gated DC population. Representative of three independent experiments. C, Deletion efficiency of STAT5 flox locus in sorted splenic DCs. Graphed as percent of STAT5 flox allele deleted in the germline, in duplicate, representative of two experiments. Nature Immunology doi:1.138/ni.541
3 Supp. Figure. a epidermis dermis CD7 97% 94% R R1 R3 R4 CD11b R1: 3% R1: 1% R: 3.9% R: 3.% R3: 1% R3: 1% R4: 53% R4: 57% c e CD7 R3 R1/R CD11b R1/: 35% R1/R: 3% R3: 31% R3: 3% R4: 4% R4: 6% R4 % % d LN cells (x1 6 ) * f ROSA:RFP +/ 99.8% b CD8 7.4% 9.6% Nature Immunology doi:1.138/ni.541 g CD11b % of Max % of Max CD4 epidermis R 38% 4% R1 R R1/ R3 R4 R skin LN dermis R3 ROSA:RFP +/ R4 Background CD11c ROSA:RFP
4 Supplemental Figure, Normal skin DC development in Cre - 5 fl/fl and mice. A, MHCII + cells were analyzed for the expression of CD7 (langerin) and CD11b in the epidermis and dermis of Cre - 5 fl/fl and mice. Graphs and percentages are representative of three independent experiments. B, Fluorescent microscopy for MHCII in epidermal sheets. Scale bar is 1μm. C, Pooled axillary and inguinal skin dlns from Cre - 5 fl/fl and mice were analyzed as in (A). D, Total cell numbers from pooled axillary and inguinal LNs from Cre - 5 fl/fl and mice. E, FACs plots gated on CD11c + cells. The percentages of CD4 + and CD8 + DCs from pooled skin dlns of Cre - 5 fl/fl and mice, representative of three independent experiments. F, Percent of CD11c + DCs which are RFP + from pooled axillary and inguinal LNs from Cre - 5 fl/fl ROSA:RFP +/- and ROSA:RFP +/- mice. G, R expression on epidermal, dermal, and skin-draining LN DCs of Cre - 5 fl/fl and mice are analyzed as in (A) and (C). Histograms are representative of three independent experiments. *p>.1 Nature Immunology doi:1.138/ni.541
5 Supp. Figure 3. a mrna (fold) untreated FITC/DBP P =. GM-CSF IL-7 b mrna (fold) P =.5 untreated DNFB GM-CSF IL-7 Nature Immunology doi:1.138/ni.541
6 Supplemental Figure 3, Expression levels of STAT5 activating cytokines following CHS sensitization. A, WT mice were left untreated or sensitized on the ear with FITC/DBP for 4 hours, followed by qpcr for indicated STAT5 activating cytokines, normalized to HPRT and fold induction is relative to untreated ears. B, WT mice were left untreated or sensitized on the ear with DNFB for 4 hours, followed by qpcr for indicated STAT5 activating cytokines, normalized to HPRT and fold induction is relative to untreated ears. Minimum 3 mice per group, 3 independent experiments. Nature Immunology doi:1.138/ni.541
7 Supp. Figure 4. a d CCL17 mrna (fold) BAL cells (x1 6 ) PBS PBS OVA/ WT OVA/ P =.19 P <.1 PBS OVA/ IL-4 mrna (fold) b BAL diff count (% of total) P <.1 WT P =.88 PBS OVA/ PBS OVA/ PBS OVA/ PBS OVA/ PBS IFN-γ mrna (fold) Alveolar mϕ 14 Neutrophils Lymphocytes Eosinophils WT OVA/ c Total IgE (ng/ml) IL13 IL4 e WT PBS 8 1 WT OVA/ 4 5 P <.1 PBS 9 OVA/ Nature Immunology doi:1.138/ni.541
8 Supplemental Figure 4, Absence of STAT5 in DCs blocks -induced lung inflammation. A, Mice were treated with +OVA every other day for 7 treatments, followed by analysis 4 hrs after last treatment. BAL cell count (A) and diff count (B) were average of 4 mice per group. C, Intracellular staining of IL-4 and IL-13 following 4 hour restimulation. D, Expression levels of lung mrna, relative to WT PBS controls. E, Serum total IgE in mice following last treatment. Two independent experiments with 3-5 mice per group. Nature Immunology doi:1.138/ni.541
9 Supp. Figure 5. a. 1 FL-CD11b DC FL-CD4 DC FL-pDC CD8 b MFI (fold) GM-CSF * *** 3 CD86.5 MFI (fold) 1 OX4L I-A d 1.5 c MFI )fold) pdcs CD4 + DCs CD8 + DCs * * Time (h) CD8 CD86 Nature Immunology doi:1.138/ni.541
10 Supplemental Figure 5, activates specific DC subsets through upregulation of costimulatory molecules. A, Costimulatory molecule expression was determined by flow cytometry on FL-DC cultures over 96 hours following treatment with 5ng/ml or left untreated. B, Fold change in MFI of GM-CSF DCs following 48hr treatment with in triplicate. C, Fold change in MFI of the three splenic DC populations following treatment in triplicate. Representative of 3 independent experiments. Nature Immunology doi:1.138/ni.541
11 Supp. Figure 6. a IL-7 pjak1 total JAK1 pstat5 IP: α-jak1 + IL-6 + pjak total JAK WCL pstat3 IP: α-jak + WCL + b CCl17 (pg/ml) WT GFP-Cre - JAK fl/fl GFP-Cre + JAK fl/fl 1X X X total STAT5 5 mins total STAT3 5 mins 1 IL-7 IL Nature Immunology doi:1.138/ni.541
12 Supplemental Figure 6, activation of JAK proteins in FL-CD11b-DCs. A, CD11b-DCs were sorted from Flt3-L cultures and treated with 5ng/ml of indicated cytokines for 5 minutes. Whole cell lysates (WCL) were split into two, half were immunoprecipitated (IP) for indicated JAK, half were used as WCL control. Immunoblot analysis of indicated JAK and STAT phosphoand total-antibodies. Representative of 5 independent experiments. B, Jak fl/fl FL-CD11b-DCs were infected with GFP-Cre retrovirus. 7 hours post treatment, CCL17 ELISA was performed on WT, GFP + Jak fl/fl and GFP - Jak fl/fl CD11b-DCs. Representative of two independent experiments. Nature Immunology doi:1.138/ni.541
IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia
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Supplementary. presence of the. (c) mrna expression. Error. in naive or
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Supplementary Figure 1: TSLP receptor skin expression in dcssc. A: Healthy control (HC) skin with TSLP receptor expression in brown (10x
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Supplementary Figure S1. PTPN2 levels are not altered in proliferating CD8+ T cells. Lymph node (LN) CD8+ T cells from C57BL/6 mice were stained with
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a surface permeabilized
a surface permeabilized RAW 64.7 P388D1 J774 b CD11b + Ly-6G - Blood Monocytes WT Supplementary Figure 1. Cell surface expression on macrophages and DCs. (a) RAW64.7, P388D1, and J774 cells were subjected
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SUPPLEMENTARY INFORMATION
doi:1.138/nature1554 a TNF-α + in CD4 + cells [%] 1 GF SPF 6 b IL-1 + in CD4 + cells [%] 5 4 3 2 1 Supplementary Figure 1. Effect of microbiota on cytokine profiles of T cells in GALT. Frequencies of TNF-α
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Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs
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Supporting Information Table of Contents
Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting
Supplementary Fig. 1 No relative growth advantage of Foxp3 negative cells.
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SUPPLEMENTARY METHODS
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Supplemental Figure 1
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Supplemental Table I.
Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides,
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
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pplementary Figur Supplementary Figure 1. a.
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SUPPLEMENTARY INFORMATION
doi:10.1038/nature10134 Supplementary Figure 1. Anti-inflammatory activity of sfc. a, Autoantibody immune complexes crosslink activating Fc receptors, promoting activation of macrophages, and WWW.NATURE.COM/NATURE
pro-b large pre-b small pre-b CCCP (µm) Rag1 -/- ;33.C9HCki
a TMRM FI (Median) b TMRM FI (Median) c 20 15 10 5 0 8 6 4 2 0 pro-b large pre-b small pre-b 0 10 20 30 40 50 60 70 80 90 100 TMRM (nm) pro-b large pre-b small pre-b 0 1 2 4 8 16 32 64 128 256 CCCP (mm)
Supporting Information
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