NMED-A65251A. Supplementary Figures.
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1 NMED-A65251A Supplementary Figures. Sup. Fig. 1. ILC3 cells are the main source of in obese mice a. We gated on T cells (upper panels) or T cells (lower panels), and examined production. b. CD IL-13 + cells were assessed by FACS (upper panels). CD IL- 17A + cells were further analyzed for IL-13 production (lower panels). c. HFD induce -, but not IL-22- or IFN- -producing CD innate lymphoid cells. Sup. Fig. 2. Mice from both Jackson Labs and Taconic develop HFD-induced AHR. To compare the effect of SFB in the development of ILC3 cells, mice from Taconic or Jackson lab fed a HFD for 12wks and AHR was measured. a. Graph represents the changes in lung resistance ( ). p<.1, and p<.5 ( chow vs HFD in mice from Jackson) and # p<.5 ( chow vs HFD in mice from Taconic) (Two-way ANOVA). b. ILC3 cells in mice from Jackson (upper panel) or Taconic (lower panel) were assessed. Total number of ILC3 cells was calculated (right panel). Sup. Fig. 3 IL-23 induces AHR in Rag2 -/- mice. a. FACS dot plots of Treg, M1/M2 macrophages in the lung (left panels) and adipose tissue (right panels) from chow or HFD fed mice. b. IL-1 production from M1 (CD11c +, upper panels) or M2 (CD26 +, lower panels) macrophages was analyzed by FACS. c.to induce AHR,.1 g of ril-6 or ril-23 were administered to Rag2 -/- mice intranasally for 3 consecutive days. Graph represents the changes in lung resistance ( ). p<.5 (ril-23), and not significant (IL-6) (Two-way ANOVA). d. Cells in BAL fluid were analyzed after measurement of AHR. p<.1, ril-6 or ril-23 treated mice were compared to saline treated mice (Student s t-test). e. ILC3 cells (CD45 + Lin - + cells) from ril-6, ril-23 or ril-1 treated mice were analyzed by flow cytomerty after PMA/Ionomycin stimulation. Sup. Fig. 4. IL-1 -induced ILCs express and ROR t, but not IL-17F. Nature Medicine: doi:1.138/nm
2 NMED-A65251A a., IL-17F, GM-CSF, ROR t and T-bet expression was analyzed in lung cells from Rag2 - /- mice. ril-1 treatment induces -producing ILCs, and most of + cells express ROR t. b. ril-1 induced CD45 + lineage - CCR6 + cells (ILC3 cells) from Rag2 -/- mice. Sup. Fig. 5 ril-1 restores AHR in Nlrp3 -/-, IL-6 -/-, and TCR -/- mice but not in Rorc -/- mice. Changes in lung resistance ( ) in Nlrp3 -/- mice (a), Il6 -/- mice, TCR -/- mice (d), and Rorc -/- mice (g), after ril-1 treatment. p<.5 (Nlrp3 -/- mice), ril-1 treated mice were compared to saline treated mice (Two way- ANOVA). Other comparisons not significant. b, e,h. Cells in BAL fluids from a, d, g. p<.1 (Nlrp3 -/- mice), ril-1 treated group was compared to saline treated group. (Student s t-test). Other comparisons not significant. ril-1 induced IL-17 + ILC3 cells in Nlrp3 -/- mice (c), Il6 -/- mice (f), TCR -/- mice (f), and Rorc -/- mice (i). Sup. Fig. 6. is required for the development of AHR. a. r induced minimal or no AHR. Graph represents the changes in lung resistance ( ) in treated WT mice. NS (Two way- ANOVA). b. Changes in lung resistance ( ) of r and + ril-1 treated Il17A -/- mice. p<.5, vs + ril-1 treated Il17A -/- mice (Two way- ANOVA). c. Inflammatory cells in BAL fluid of (b). p<.5, p<.1, and p<.1 (Student s t-test). d. Changes in lung resistance ( ) of anti-il-17 mab injected Rag1 -/- mice before ril-1 treatment (Two way- ANOVA). e. Inflammatory cells in BAL fluid of (d). p<.5, and p<.1 (Student s t-test). f. producing ILC3 cells assessed by intracellular staining. Sup. Fig. 7. Human ILC cells in the BAL fluid from patients with lung disease. a. Severe asthma (left), mild asthma (middle) or control (right panels). First, we gated on CD45 + cells (1 st rows), on lymphocytes (2 nd rows), then on Lin - cells (3 rd rows), before analyzing for IL-7R (CD127) expression (bottom rows). The numbers in the histograms in the 4 th rows represent the percent of Lin - cells that were CD127 +, and these numbers were then used to calculate the percent of BAL fluid lymphocytes that were Lin - IL-7R +, as shown in Supplemental Table 1, 9 th column. Nature Medicine: doi:1.138/nm
3 NMED-A65251A b. The top row of each pair of rows represents unstimulated cells, and the bottom row of each pair represents cells activated with PMA and ionomycin. The numbers in the upper left hand quadrants represent the percent of the Lin - CD127 + cells that were +. These numbers (in the PMA and ionomycin stimulated populations) were then used to calculate the percent of BAL fluid lymphocytes that were Lin - IL-17 +, as shown Supplemental Table 1, right most column. Nature Medicine: doi:1.138/nm
4 Sup. Fig. 1 a TCR b Chow HFD Chow HFD TCR IL-13 TCR 1.88 Chow 2.77 HFD HFD TCR IL-13 c 25K 2K 15K 1K K IL IFN Nature Medicine: doi:1.138/nm.3423
5 arl(% of baseline) Sup. Fig. 2 4 JAX Chow JAX HFD TAC Chow 3 TAC HFD # 2 # b chow HFD JAX TAC ILC3 cells(% of lymphocyte) NS chow HFD chow HFD JAX TAC Nature Medicine: doi:1.138/nm.3423
6 Sup. Fig.3 a Treg Foxp3 Macrophage CD Lung C how H FD CD M2 M2 AM M CD11c AM M Adipose tissue C how H FD M2 M2 M1 M b M1 IL-1β 1 M IL-1β Chow HFD CD11c CD26 c (%of baseline) ril-23 ril n.s d Cells in BALF (X1 4 ) n.s ril-23 ril-6 n.s n.s n.s n.s Macs Neu Eos Lymph e ril-6 ril ril-1α Nature Medicine: doi:1.138/nm.3423
7 Sup. Fig. 4 a 25K ril-1β b ril-1β 2K 15K 1K K FCS IL-17F cells CCR GM-CSF T-bet RoRγT Nature Medicine: doi:1.138/nm.3423
8 Sup. Fig. 5 a b c (% of baseline) Nlrp3 ril-1β 4 ril-1β Nlrp3 1 Nlrp3 ril-1β 5 Nlrp Cells in BALF(X1 4 ) 2 1 Macs Neu Eos Lymph Nlrp d e WT ril-1β f (% of baseline) WT ril-1β 4 WT Rorc ril-1β Rorc 3 2 WT ril-1β δtcr ril-1β Il6 ril-1β WT δtcr Il6 NS Macs Neu Eos Lymph g h WT ril-1β i (% of baseline) Cells in BALF(X1 4 ) Cells in BALF(X1 4 ) TCR ril-1β Il6 ril-1β WT δtcr Il6 WT Rorc ril-1β Rorc Macs Neu Eos Lymp RoRγT Rorc WT δtcr 4.46 ril-1β.51 Il Nature Medicine: doi:1.138/nm.3423
9 Sup. Fig.6 a (% of baseline) (% of baseline) d WT r WT NS Rag1 ril-1β Rag1 anti- Ab+rIL-1β Rag n.s (% of baseline) e Cells in BALF(X1 4 ) b Il17 Il17 ril-1β Il17 ril-1β+r Macs Neu Eos Lymp c 75 Cells in BALF(X1 4 ) f 5 25 Rag1 ril-1β Rag1 anti- Ab+rIL-1β Rag Macs Neu Eos Lymph Rag1 RoRγT Il17 Il17 ril-1β Il17 ril-1β+r ril-1β anti- Ab+rIL-1β Nature Medicine: doi:1.138/nm.3423
10 Sup. Fig. 7 a Severe Asthma Mild Asthma Control 25K 2K 15K 1K 5K CD45 25K P1 P2 P P4 P5 P6 P7 P8 P9 25K 25K K 2K 15K 1K 5K CD45 25K K 1K 5K CD45 25K P K 2K 2K 15K 15K 15K 1K 1K 1K 5K FSC K 1K 15K 2K 25K K FSC K 1K 15K 2K 25K K K 1K 15K 2K 25K FSC K 25K 25K 2K 2K 2K 15K 1K 5K K 1K 5K K K K CD CD CD127 b (-) Severe Asthma Mild Asthma Control P1 P2 P3 P5 P7 P8 P (-) (-) P1 P+I P+I P+I IL IL IL Nature Medicine: doi:1.138/nm.3423
11 Supplemental Table 1.Ten Consecutive Subjects with Pulmonary Disease Tested for ILC3-like Cells Patient Asthma severity 1 Sex Age (years) Race Co-illness FEV1 (L/sec) (%) BMI Lin - IL-7R + (% of lymphocyte) Lin - IL-17 + (% of lymphocyte) P1 Severe F 42 Asian Depression 1.52(62%) P2 Severe M 21 African None 4.5(9%) P3 Severe F 5 American None 2.33(77%) P4 Severe F 39 Hispanic P5 Severe M 52 White Obesity with gastric bypass Allergic rhinitis,ge reflux 1.5(48%) n/a 1.63(49%) P6 Mild M 29 Hispanic none 4.68(118%) P7 Mild M 26 White Sinusitis, Exsmoker 4.7(13%) n/a P7 Mild F 64 White Chronic rhinitis 3.26 (119%) P8 No asthma F 42 White Ulcerative colitis 3.42(12%) P1 No asthma F 57 White Celiac d, diabetes 2.5(92%) Based on NHLBI Guidelines In last 2 columns, LIN - IL-7R + or LIN - IL-17 + populations werecalculated as a percentage of total lymphocytes(the lymphocyte gate was considered as 1%). The gating strategies are provided in Supplemental Figures 7. GE= gastro-esophageal. Nature Medicine: doi:1.138/nm.3423
Supplementary Figure 1. IL-12 serum levels and frequency of subsets in FL patients. (A) IL-12
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Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured
Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured under Th0, Th1, Th2, Th17, and Treg conditions. mrna
ILC1 and ILC3 isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the ILC1, ILC2 and
Supplementary Methods and isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the, ILC2 and subsets. For this purpose we performed intracellular flow cytometry
Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
Supplementary. presence of the. (c) mrna expression. Error. in naive or
Figure 1. (a) Naive CD4 + T cells were activated in the presence of the indicated cytokines for 3 days. Enpp2 mrna expression was measured by qrt-pcrhr, infected with (b, c) Naive CD4 + T cells were activated
Supplementary Table 1. Oligonucleotide primers used in quantitative and qualitative PCRs of this study.
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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
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pro-b large pre-b small pre-b CCCP (µm) Rag1 -/- ;33.C9HCki
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CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 +
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Gene+c fate mapping. x loxp. Foxp3 3 UTR ROSA26 RFP IRES GFP CRE. STOP loxp. Stable Foxp3 expression. Foxp3 expression in new Treg.
1 Introduc+on (CD4 + CD25 + Foxp3 + )are indispensable for immune homeostasis. Muta+ons in Foxp3 gene leads to fatal autoimmune disorder. Condi+onal dele+on of Foxp3 reprograms cells into pathogenic Th
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for six pairs of mice. (b) Representative FACS analysis of absolute number of T cells (CD4 + and
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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,
Eosinophils! 40! 30! 20! 10! 0! NS!
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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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Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.
![Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.](/thumbs/90/103696086.jpg "Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.")
SUPPLEMENTAL DATA Supplemental Table 1: Demographics and characteristics of study participants Lean (n=15) Obese (n=12) Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± 9.5 44.8 ± 9.1 White, n
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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
% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
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Supplementary Figure 1. Ex vivo IFNγ production by Tregs. Nature Medicine doi: /nm % CD127. Empty SSC 98.79% CD25 CD45RA.
SSC CD25 1.8% CD127 Empty 98.79% FSC CD45RA CD45RA Foxp3 %IFNγ + cells 4 3 2 1 + IL-12 P =.3 IFNγ p=.9 %IL-4+ cells 3 2 1 IL-4 P =.4 c %IL-1 + cells IFNγ 4 3 2 1 Control Foxp3 IL-1 P =.41.64 4.76 MS 2.96
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of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
Supplementary online material Supplementary figure legends Supplementary Figure 1 Exposure to T reg cells causes loss of T resp cells in co-cultures. T resp cells were stimulated with CD3+CD28 alone or
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Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
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and follicular helper T cells is Egr2-dependent. (a) Diagrammatic representation of the
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<10. IL-1β IL-6 TNF + _ TGF-β + IL-23
3 ns 25 ns 2 IL-17 (pg/ml) 15 1 ns ns 5 IL-1β IL-6 TNF
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