Spleen VAT FMO. Nature Immunology: doi: /ni Ki67 DX5 CD27. CD11b 2B4 KLRG1 CD69 NKG2D CXCR3 CD44 NKG2A/C/E CD62L CD103 CD94 CD48.

Transcription

1 IFN (% of ells) CD7 Marophages Medium NCR LPS PMA/Ionomyin CD IFN (% of ells) T ells Marophages B ells DX CD9 KLRG NKGD B CDL CD CD CXCR CD9 CD NKGA/C/E NCR Ly9h Ly9G Ly9d Ly9i Ly9/i/h FMO CD7 CD9a CD7 TRAIL CD T Bet Eomes d g Cells CD7 (% of ) e Speifi killing (%) T ells h.... E/T ratio Cells ( ) f Ki7 Cells ( ) 7 % NKT ells Cells ( ) 7 % CD T ells Cells ( ) Ki7 (% of ells) CD T ells IL NKp i Cells ( ) Foxp CD T ells Nature Immunology: doi:./ni.

2 Supplementary Figure Phenotype and funtion of resident. (a) leukoytes were stimulated in vitro with the indiated stimuli, and IFNγ prodution was assessed in and marophages (n = ). () leukoytes were stimulated in vitro with PMA and ionomyin, and IFNγ prodution was assessed (n = ). () Spleni and resident were isolated, and the relative numer of ells expressing the indiated markers was determined y flow ytometry. Gated for CD NCR ells, exept for the markers (CD NCR ) and NCR (CD ). Representative plots are shown of one of seven independent experiments, with three to five mie used per experiment. (d) Total splenoytes or derived leukoytes were stimulated with PMA and ionomyin, and the expression of CD7a on was determined y flow ytometry (n = ). (e) The relative numer of in total splenoytes or derived was determined and ells were mixed in the indiated ratio with CFSElaeled target ells. After h, effetor ell indued ell death (Topro ) of target ells was determined y flow ytometry (n = ). (f) Spleni and resident were stimulated in vitro with IL or agonisti NCR antiodies, and expression of Ki7 was analyzed after h. Gated for CD ells (n = ). (g) Kinetis of the asolute numer of γδ T ells in after weeks of or feeding (n = ). (h) Kinetis of the asolute numer of the indiated immune ell susets in the spleens of mie after weeks of or feeding (n = ). (i) Relative numer of regulatory T ells (CD CD FoxP ) in after weeks of or feeding (n = ). Shown are data from one of two to seven experiments with similar results (mean ± s.e.m., n =, P., P <., P <.). FMO, fluoresene minus one. Nature Immunology: doi:./ni.

3 FSC A e IFN (% of ) FSC H M / M..... PI NKp M / M ratio FSC A % of CD T ells T Helper ells IFN IL7 IFN IL7 IFN IL7 f CD9 CD M / M CD..... F/ CD M / M ratio ( ) GR M CD g / % of M d GMI NKT ells ( ) / IFNg / Arginase M M GMI CD T ells ( ) ILR M M inos (% of Cells) Arg IL Rα inos / inos M M. %. % h. /a. Insulin p p7 β Atin p p7 β Atin /α / IFNg i Liver pp7 / Loading CD7CD CD7CD Liver CD7CD j Body Weight (g) k Fat Pad Weight (g)..... /Isotype /Isotype / /Isotype /Isotype / /Isotype /Isotype /. / Nature Immunology: doi:./ni.

4 Supplementary Figure resident are ativated upon feeding and promote M marophage formation. (a) Mie were fed an or an for weeks, after whih leukoytes were stimulated in vitro with PMA and ionomyin and ytokine prodution was assessed in and CD T H ells (n = ). () Mie were fed an or an and reeived PBS or α antiodies one every d. After weeks, numers of NK (CD NCR ), NKT (CD NCR ) and CD T ells (CD CD ) were quantified (n = ). () Gating strategy for adipose tissue marophages and. M marophages (M) are defined as CD9 CD F/ GR /Dim CD CD Dim. M marophages (M) are defined as CD9 CD F/ GR /Dim CD CD. are defined as CD NCR. (d) Marophage susets (as defined in ) were analyzed for the expression of M and M markers under homeostati onditions (arginase, ILRα) and after d of in vitro stimulation with LPS (inos, n = ). (e) Mie were fed an or an and reeived PBS or α antiodies one every d. After weeks, the ratio etween M and M marophages in was determined (n = ). (f,g) Mie were fed for weeks and injeted with PBS or neutralizing αifnγ antiodies one every other day (n = ). (f) Ratio etween M and M marophages in. (g) Phenotype of in. (h k) Mie were fed an or an for weeks and reeived depleting α or isotype ontrol antiodies every d. (h) Mie were fasted overnight and injeted i.p. with either PBS ( ) or. U/kg insulin (). Immunolot was used to quantify phosphorylated p7 (Thr9) in tissue samples of liver and. βatin was used as a loading ontrol. (i) Quantifiation shows pooled data from two independent experiments (n = ). (j) Body weight and (k) viseral fat pad weight of indiated groups after weeks of feeding (n = ). Shown are data from one of two to five experiments with similar results (mean s.e.m., n =, P., P <., P <.). Nature Immunology: doi:./ni.

5 GTT. Suutaneous Fat Pad Weight GTT Gluose (mm) % of d e f /Sham /etomy 7 Time (Minutes) SCID SCID ATM ( ) Fat Pad weight (g)..... Sham/ Sham/ etomy/ ATM M ATM ( ) Gluose (mm) M ATM WT WT SCID SCID 7 Time (minutes) Weight (g) Body Weight Gluose (AUC) g Weight (g) Fat Pad Weight..... WT SCID WT SCID CDKLRG CD KLRG CD KLRG SCID SCID SCID / SCID SCID SCID / /Isotype /Isotype /. /Isotype /Isotype / Supplementary Figure do not require the adaptive immune system to promote indued M marophage formation in. (a,) Mie underwent either sham operation or removal of adominal fat (etomy; n = ). (a) weeks after surgery, mie were sujeted to GTT. () Weight of suutaneous fat pads. () Wildtype and Prkd sid/sid (SCID) mie were fed an or an for weeks and sujeted to GTT. Shown are plasma gluose levels and AUC (n = ). (d,e) or fed SCID mie were injeted with α antiodies or PBS one every d. After weeks, (d) the phenotype of and (n = ) (e) the numer of total and M marophages in were determined (n = ). (f,g) SCID mie were fed an or an for weeks. Mie reeived depleting α or isotype ontrol antiodies every d. After weeks the (f) ody weight and (g) viseral fat pad weight of mie were determined (n = ). Shown are data from one of two experiments with similar results (mean ± s.e.m., n =, P., P.). AUC, area under the urve. Nature Immunology: doi:./ni.

6 Leukoytes CD ells (% of SVF) 7 (% of CD ) BrdU BAT SCfat Liver BAT SCfat No BrdU... BrdU (% of ) Supplementary Figure promotes proliferation of in. (a) Fration of leukoytes as a perentage of the stromal vasular fration (SVF) in spleen, rown adipose tissue (BAT), and suutaneous (SC) fat (n = ). () Fration of as a perentage of leukoytes in indiated organs under homeostati onditions (n = ). () Mie were fed for weeks and susequently injeted with mg BrdU. BrdU inorporation in was determined after h (n = ). Shown are pooled data from three independent experiments (a,) or one experiment (). Nature Immunology: doi:./ni.

7 mnkp Ig NCR Ig mnkgd Ig Ctr Ig T L n.d. IFN (% of ),,,, g/ml NCR Epidydimus IFN (% of ) Fat Capsule WT NCR / Neg. Ctr. NCR d inos (% of Cells) NCR/ IFN CD (% of Cells) NCR/ IFN e inos (% of Cells) M Only NCR NCR/ IFNg, g/ml NCR Supplementary Figure resident are ativated y stress ligands in adipose tissue upon feeding. (a) Wildtype mie were fed an or an for weeks. setions were stained with mncrimmunogloulin or mnkgdimmunogloulin fusion proteins and visualized with DAB. Irrelevant fusion proteins were used as negative ontrols. As a positive ontrol for NCRL staining, TL ells were inluded. Arrow shows fat apsule. Sale ar, μm. () Conentrationdependent IFNγ prodution in after in vitro NCR stimulation (n = ). () Spleni from Nr gfp/gfp mie were stimulated in vitro with plateound or NCR antiodies, and IFNγ prodution was assessed (n = ). (d) The SVF of was stimulated for h with agonisti NCR antiodies in the presene or asene of neutralizing IFNγ antiodies. After h, the fration of inos and CD marophages was determined (n = ). (e) were ativated in vitro for h with agonisti NCR antiodies in the presene or asene of neutralizing IFNγ antiodies. Susequently, supernatants of these ells were taken and used to stimulate marophages. After h, inos prodution in marophages was assessed (n = ). Shown are data from one of two to four experiments with similar results (mean ± s.e.m., n =, P., P <., P <.). n.d., not determined. Nature Immunology: doi:./ni.

8 Body Weight Fat Pad Weight M ATM ( ) Weight (gram) Weight (gram).... d / NCR tnfrsfa NCR /α /NCR. NCR Insulin pakt pgskα p p7 perk/ Pan ERK/ pakt pgskα p p7 Liver perk/ Pan ERK/ pakt pgskα p p7 p ERK/ pan ERK/ pakt / / /NCR pgsk / / /NCR pp7 / / /NCR perk/ / / /NCR panerk/ / / /NCR Liver pakt / pgsk / pp7 / perk/ /..... panerk/ / / /NCR / /NCR / /NCR. / /NCR / /NCR Nature Immunology: doi:./ni.

9 Supplementary Figure NCR defiieny or NK ell depletion redues indued insulin resistane ut does not affet weight gain. (a ) Mie were fed an or an and reeived PBS or α antiodies one every d (n = ). (a) After weeks, the numer of M marophages was determined in. (,) After weeks, () total ody weight and () viseral adipose fat pad weight were determined. (d) Mie were fasted overnight and injeted i.p. with either PBS ( ) or. U/kg insulin (). Immunolot was used to quantify pakt (Ser7), pgskα (Ser/9), pp7 (Thr9), perk/ (Thr/Tyr) and total ERK/ in tissue samples of liver and. was used as a loading ontrol (n = ). Shown are data from one of two experiments with similar results (mean ± s.e.m., n =, P., P <., P <.). Nature Immunology: doi:./ni.

10 . IFNγ / Donor ells.. IFNγ / WT NK IFNγ / NCR NK Gluose (mm) IFNg / IFNg / e i IFNγ ( ) KLRG (%) Plate ound αncr Plate ound αncr Solule αncr.. /WT NK IFN ells (% of Control) CD9 (GMI) IFN /NCRIg /NCR.. g/ml NCR j M ATM ( ) f Nr / Nr / BW BW Nr 7 Time (Minutes) WT/WT NK Iso. A. mnr /NCRIg /NCR CDL (GMI) Iso. A. mnr k Donor Reipient Ratio M / M g.... CCL B D PD. WT/WT NK mnr mnr /NCRIg /NCR TEC9 INS l mnr Weight (gram) mnr Isotype Nr Ig Only Nr Ig A d M (% of total ) /PBS /CtrIg /NCRIg h IFN ells (% of Control) /WT NK IFN.7 g/ml NCRIg NCR PMA/Iono. Supplementary Figure 7 Nature Immunology: doi:./ni.

11 Therapeuti loking of NCR prevents M marophage formation. (a) fed Ifng / mie (CD.) reeived PBS, wildtype (CD. ) or NCR ( ) every d. Shown are representative plots of the experiment shown in Figure 7a,. Donor ells were identified ased on their expression of CD. or. () Indiated groups were fed an or an for weeks and sujeted to GTT (n = ). (,d) fed Ifng / mie reeived PBS or wildtype ( ) one every d and were ompared with PBSinjeted, fed animals. After weeks, the phenotypes of () donor and reipient and (d) marophages in were analyzed (n = ). (e) were stimulated in vitro with plateound αncr, in the presene or asene of the same lone of αncr antiodies solule in the medium. (f) Wildtype, NCR, BW ells and BW ells transgenially expressing NCRζ were stained with the antiodies mncr and mncr to demonstrate the speifiity of these reagents for NCR. (g) Six ell lines that are known to express NCR ligands were stained with Nrimmunogloulin alone or in omination with NCRspeifi antiodies. NCRspeifi antiodies loked inding of NCRimmunogloulin to target ell lines, indiating that this reagent shows target inding that is idential to that of NCR. (h) were stimulated with PMA and ionomyin or agonisti Nr antiodies in the presene or asene (ontrol) of Nrimmunogloulin. NCRimmunogloulin is ale to redue NK ell ativation upon NCR stimulation, again demonstrating the speifiity of NCR immunogloulin fusion protein for NCR ligands. (i k) Mie were fed an or an for weeks. Animals reeived PBS or NCR ligand loking NCRimmunogloulin fusion proteins twie per week. Nr / mie are genetially defiient for NCR. M marophage and NK ell numers were analyzed after weeks (n = ). (i) NK ell numers and (j) M marophage numers in. (k) M/M ratio in showed that NCR defiieny or its loking prevented the asolute (Fig. 7e) and relative inrease of M ells in. (l) Fat pad weight of mie after weeks of feeding (n = ). Shown are data from one of one to two experiments with similar results (mean ± s.e.m., n =, P., P <., P <.). Nature Immunology: doi:./ni.