Supplementary Figure 1: Phenotype and function of VAT-resident NK cells.

(a) VAT leukocytes were stimulated in vitro with the indicated stimuli, and IFN-γ production was assessed in NK cells and macrophages (n = 3). (b) VAT leukocytes were stimulated in vitro with PMA and ionomycin, and IFN-γ production was assessed (n = 3). (c) Splenic and VAT-resident NK cells were isolated, and the relative number of cells expressing the indicated markers was determined by flow cytometry. Gated for CD3^–NK1.1⁺NCR1⁺ cells, except for the markers NK1.1 (CD3^–NCR1⁺) and NCR1 (CD3^–NK1.1⁺). Representative plots are shown of one of seven independent experiments, with three to five mice used per experiment. (d) Total splenocytes or VAT-derived leukocytes were stimulated with PMA and ionomycin, and the expression of CD107a on NK cells was determined by flow cytometry (n = 5). (e) The relative number of NK cells in total splenocytes or VAT-derived NK cells was determined and cells were mixed in the indicated ratio with CFSE-labeled target cells. After 5 h, effector cell–induced cell death (Topro3⁺) of target cells was determined by flow cytometry (n = 5). (f) Splenic and VAT-resident NK cells were stimulated in vitro with IL-15 or agonistic NCR1 antibodies, and expression of Ki67 was analyzed after 48 h. Gated for CD3^–NK1.1⁺ cells (n = 3). (g) Kinetics of the absolute number of γδ T cells in VAT after 4 weeks of NCD or HFD feeding (n = 5). (h) Kinetics of the absolute number of the indicated immune cell subsets in the spleens of mice after 12 weeks of NCD or HFD feeding (n = 5). (i) Relative number of regulatory T cells (CD3⁺CD4⁺FoxP3⁺) in VAT after 12 weeks of NCD or HFD feeding (n = 5). Shown are data from one of two to seven experiments with similar results (mean ± s.e.m., n = 4–5, *P < 0.05, **P < 0.01, ***P < 0.001). FMO, fluorescence minus one.