1. Volume 45, Issue 2, Pages 428-441 (August 2016)
Adipose-Resident Group 1 Innate Lymphoid Cells Promote Obesity-Associated Insulin Resistance 
Timothy E. O’Sullivan, Moritz Rapp, Xiying Fan, Orr-El Weizman, Priya Bhardwaj, Nicholas M. Adams, Thierry Walzer, Andrew J. Dannenberg, Joseph C. Sun 
Immunity 
Volume 45, Issue 2, Pages (August 2016)
DOI: /j.immuni
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2. Immunity 2016 45, 428-441DOI: (10.1016/j.immuni.2016.06.016)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Phenotypic and Functional Heterogeneity of Adipose Group 1 ILCs
(A) Representative plots show Eomes and DX5 expression on gated lin−NKp46+NK1.1+T-bet+ (group 1 ILC) cells in indicated peripheral organs of WT mice. (lin = TCRβ+CD3ε+CD19+TCRγδ+Ly6G+F4/80+ cells).
(B) Percentage of group 1 ILC subsets in bone marrow and peripheral organs, including VAT and SAT, is shown.
(C) Representative plots show Eomes and DX5 expression on gated group 1 ILCs in the adipose tissue of WT and Rag2−/− mice.
(D and E) Representative plots and histograms show indicated cell surface markers on subsets of group 1 ILCs in adipose tissue.
(F) Percentage of IFN-γ+ resting splenic NK cells or indicated adipose group 1 ILC populations after stimulation with IL-12 and IL-18. Data are representative of three independent experiments, with n = 5 per group. Samples were compared using an unpaired, two-tailed Student’s t test, and data are presented as the mean ± SEM (∗p < 0.05).
See also Figure S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Adipose Group 1 ILCs Have Differential Developmental Requirements for Nfil3 and T-bet
(A and B) (A) Representative plots and (B) absolute density of lin−NK1.1+ cells in the adipose tissue of WT, Nfil3−/−, and Rag2−/−Il2rg−/− mice.
(C and D) (C) Representative plots of indicated lymphocyte populations in the adipose tissue and (D) chimerism shown as percentage of indicated lymphocyte populations from the spleen, liver, and adipose tissue of WT (CD45.1) and Nfil3−/− (CD45.2) mixed bone marrow chimeric mice 8 weeks following reconstitution.
(E and F) (E) Representative plots and (F) absolute density of indicated group 1 ILC subsets in Rag2−/−Tbx21+/− or Rag2−/−Tbx21−/− mice. Data are representative of two independent experiments, with n = 4 mice per group. Samples were compared using an unpaired, two-tailed Student’s t test, and data are presented as the mean ± SEM. (∗p < 0.05, ∗∗p < 0.001, ∗∗∗∗p < ).
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5. Figure 3
Group 1 ILCs Consist of Immature and Phenotypically Stable Mature Subsets in the Adipose Tissue
(A) Schematic of the experiment. Briefly, total adipose tissue was harvested from Eomes-GFP reporter mice and either 2 × 104 DX5+Eomes+, DX5−Eomes+, or DX5−Eomes− group 1 ILCs were sorted (>95% purity) and adoptively transferred intravenously (i.v.) into Rag2−/−Il2rg−/− recipients.
(B) Representative plots show the percentage of each transferred group 1 ILC subset (NKp46+NK1.1+) recovered from the adipose tissue of recipient mice after 14 days.
(C) In a separate experiment, 2 × 105 DX5+Eomes+, 2 × 104 DX5-Eomes+, or 2 × 104 DX5−Eomes− group 1 ILCs were sorted from adipose tissue or bone marrow of Eomes GFP mice and adoptively transferred i.v. into Rag2−/−Il2rg−/− recipients. Representative plots show the expression of Eomes and DX5 of indicated adoptively transferred NKp46+NK1.1+ cells in the adipose tissue of recipient mice 14 days after transfer. Data are representative of three independent experiments, with n = 3 mice per group.
See also Figure S2.
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6. Figure 4 Adipose ILC1s Are Long-Term Tissue Resident
(A and B) (A) Schematic of the parabiosis experiment. Briefly, CD45.1+ and CD45.2+ mice were surgically connected for 30 or 130 days and (B) chimerism of T and mNK cells (shown as percentage of donor-derived populations) was analyzed in the spleens of parabionts.
(C and D) (C) Representative plots and (D) quantitation of indicated host and donor derived lymphocytes in the adipose tissue or small intestine. Data are representative of two independent experiments, with n = 3–4 (parabiotic pairs) per time point.
See also Figure S3.
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7. Figure 5
Robust Proliferation and Persistent Accumulation of Group 1 ILCs in the Subcutaneous Adipose Tissue during HFD Feeding
WT mice were fed either a control LFD (10%) or a HFD (60%), and peripheral tissues were harvested and analyzed for group 1 ILCs at each time point following administration of diet.
(A) The absolute density (total cell number per mg of tissue) of each indicated group 1 ILC in the VAT or SAT of HFD fed mice was calculated, and absolute HFD cell density was normalized to LFD controls and presented as relative cell density.
(B) WT female mice were ovariectomized and, 1 week later, fed either a LFD or HFD for 10 weeks. Graph shows the relative cell density of indicated group 1 ILCs in the adipose tissue at week 10 after diet administration.
(C) Representative plots show Ki67 intracellular staining of lin−NKp46+NK1.1+ cells in the spleen or SAT of LFD or HFD mice on week 4 following diet administration.
(D) Absolute density of Ki67+ group 1 ILCs in the SAT of LFD or HFD mice 4 weeks after diet administration.
(E) Absolute density of BrdU+ group 1 ILCs in the SAT of LFD or HFD mice 4 weeks after diet administration. Data are representative of two independent experiments, with n = 3–4 per cohort. Samples were compared using an unpaired, two-tailed Student’s t test, and data are presented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < ).
See also Figure S4.
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8. Figure 6
IFN-γ Production by Group 1 ILCs Polarizes Proinflammatory Macrophages in the Adipose Tissue and Contributes to Obesity-Associated Insulin Resistance
IFN-γ YFP reporter (GREAT) or WT mice were fed either a HFD or control LFD for 3 weeks, and peripheral organs were harvested.
(A and B) (A) Representative histogram and (B) quantitation of YFP fluorescence in indicated group 1 ILCs from LFD or HFD cohorts.
(C) Quantitation of intracellular IFN-γ staining of splenic NK cells and indicated adipose group 1 ILC subsets from LFD or HFD mice after stimulation with IL-12 and IL-18.
(D and E) Briefly, WT or Ifng−/− mice were fed a HFD for 3 weeks, and 2 × 104 indicated group 1 ILCs were sort purified from adipose tissue of each group and adoptively transferred into separate Rag2−/−Il2rg−/− mice that had previously received HFD for 2 weeks. Recipient mice were fed a HFD for an additional 2 weeks.
(D) Schematic of experiment.
(E) The absolute density (cells/mg) of M1 macrophages (lin−F4/80+CD11b+CD11c+) was analyzed in the SAT of recipient mice compared to control LFD or untreated controls.
(F–J) 2 × 104 iNK or ILC1 were sort purified from the adipose tissue of WT mice fed a HFD for 4 weeks and adoptively transferred into Rag2−/−Il2rg−/− recipients starting on week 2 of HFD. iNK or ILC1 were adoptively transferred into recipients every 2 weeks for 6 weeks. Mice were analyzed on week 12 of HFD. (F) Schematic of experiment. (G) Absolute density of M1 macrophages in the SAT, (H) fasting plasma insulin concentration, (I) glucose tolerance test, and (J) insulin tolerance test of each indicated cohort are shown. Data are representative of two independent experiments, with n = 3–5 per cohort. Samples were compared using an unpaired, two-tailed Student’s t test, and data are presented as the mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < ).
See also Figures S5 and S6.
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9. Figure 7
Production of IL-12 in Adipose Tissues following HFD Drives Group 1 ILC Proliferation, Accumulation, and Proinflammatory Macrophage Polarization
WT mice were either fed a control LFD or HFD for 1, 2, 3, or 4 weeks, and adipose tissue was harvested.
(A) Graphs show IL-12p35 and IL-12p40 mRNA levels in SAT and VAT for each cohort.
(B–D) WT mice were fed a LFD; or WT, Il12rb2−/−, and Stat4−/− mice were fed a HFD for 4 weeks. (B) Absolute density of group 1 ILCs in the SAT (C) percentage of Ki67+ cells and (D) absolute density of M1 macrophages in the SAT of each cohort are shown. Data are representative of two independent experiments, with n = 3–5 per cohort. Samples were compared using an unpaired, two-tailed Student’s t test, and data are presented as the mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < ).
See also Figure S7.
Immunity  , DOI: ( /j.immuni )
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