Nature Immunology: doi:10.1038/ni.2797

SupplementaryFigure1.InductionofmurineIL­17Aproducingneutrophils.(a)C57BL/6andRag2–/–Il2rg–/–micewereinjectedsubcutaneouslywithheat‐killed,swollenAspergillusfumigatusconidia.Threedaysafterinjection(primed),splenocyteswereincubatedwithanti‐CD3,NK1.1,orNIMP‐R14,permeabilizedandincubatedwithanti‐IL‐17A,andexaminedbyflowcytometry.AsapositivecontrolforIL‐17producingCD3+cells,C57BL/6miceweregivenasecondinjectionofconidia7dayslater,andsplenocyteswereexaminedonday10(immunized).Scatterplotsarerepresentativeoffourmicepergroupandtworepeatexperiments.(b)BonemarrowneutrophilswereisolatedfromnaïveC57BL/6micebydensitycentrifugation(Percoll)andincubatedwith:Ly6GantibodiesNIMPR14and1A8,+/‐CD11b,orwithantibodiestoCD4,CD3,B220,F4/80, γδ TCR,andNK1.1.Dataarerepresentativeofneutrophilsfromfourseparatemiceandtworepeatexperiments.(c)BonemarrowneutrophilswereisolatedfromIl17a–GFPmicethreedaysaftersubcutaneousinjectionofheat‐killed,swollenA.fumigatusconidia(Primed).CellswereincubatedwithNIMP‐R14,andco‐expressionwithGFP(Merge)wasdetectedbyquantitativeimageanalysisandflowcytometry.Representativecellsfrom4naïveand4primedmiceareshown.Thisexperimentwasrepeatedtwicewithsimilarresults.(d,e)NIMP‐R14+cellswereisolatedfrombonemarrow(d)orspleen(e)ofprimedandnaïveC57BL/6micebycellsortingusingaFACSAriaIII(BDBiosciences), yielding>98%purepopulationofNIMP‐R14+neutrophils.Il17ageneexpressioninpurifiedneutrophilpopulationswasexaminedbyquantitativePCR,andq‐PCRproductswerequalitativelyexaminedon1%agarosegel.(N:naïve).(a–e)representativedatafrom3repeatexperiments.

Nature Immunology: doi:10.1038/ni.2797

SupplementaryFigure2.Rorc,IL­6R,IL­23R,andIL­17Aexpressioninbonemarrowneutrophils.(a)TotalbonemarrowcellsfromnaïveRorc+/GFPmicewereincubatedwithPEconjugatedNIMP‐R14,andco‐localizationofGFP(Rorc)andPEwasdetectedbyquantitativeimageanalysisandflowcytometry.ImagesarerepresentativeofthreeRorc+/GFPmiceandtworepeatexperiments.(b)ConstitutiveRorcgeneexpressioninbonemarrowneutrophilsisolatedfromnaïveC57BL/6miceandincubated1hwithrecombinantmurineIL‐6(20µg/ml)andIL‐23(2µg/ml)(IL‐6+IL‐23).PCRproductswerequalitativelyanalyzedon1%agarosegelusingActb(β-actin)asaloadingcontrol.CyclingthresholdscoresareshowninSupplementaryTable1.(c)Co‐expressionofGFPandIL‐6andIL‐23receptorsinbonemarrowneutrophilsfromnaïveRorc+/GFPmice.(d)ConstitutiveIL‐23RandIL‐12Rβ1geneexpressioninneutrophilsisolatedfromnaïveC57BL/6 mice.(e,f)Il17ageneexpressioninbonemarrowneutrophilsfromC57BL/6miceincubated1hwithrecombinantmouse(rm)IL‐6and/orrmIL‐23atindicatedconcentrationsperml,andanalyzedbyquantitativePCR(e),or(f)incubated15,30and60minwith20µg/ml ofrmIL‐6and2µg rmIL‐23.(g)IL‐17proteinintotalcelllysatesofbonemarrowneutrophilsfromnaïveC57BL/6miceincubated3hwithrmIL‐6,and/orrmIL‐23atindicatedconcentrationsperml.Dataaremean+/‐SDfrom3wellspergroup.Alldatashownarerepresentativeoftwo(a–d)orthree(e­g)repeatexperiments.

Nature Immunology: doi:10.1038/ni.2797

SupplementaryFigure3.ExpressionofIL­17A,receptorsforIL­6andIL­23,andexpressionofRorcandRORγtinhumanneutrophils.(a)Ahighlypurifiedpopulationofneutrophilswasisolatedfromperipheralbloodofhealthyvolunteersbygradient(Ficoll)centrifugation,andvisualizedbyWrights‐Giemsastaining.(b,c)Neutrophilswerestimulated1hwithrecombinanthuman(rh)IL‐6and/orrhIL‐23attheindicatedconcentrationsperml,andIl17ageneexpressionwasdetectedbyq‐PCR(b).IL‐17AproteinfromneutrophillysateswasmeasuredbyELISAafter3hincubationwith20µg/mlIL‐6and2µg/mlIL‐23(c).(d,e)Peripheralbloodneutrophilsfromahealthydonorwereeitherleftunstimulated(unstim)orwereincubated3hwithrhIL‐6(20µg/ml)andrhIL‐23(2µg/ml)(IL‐6+IL‐23),andreceptorexpressionwasexaminedbyflowcytometry(d)(numbersdenotepercentpositivecellsinIL‐6+IL‐23–stimulatedneutrophils).(e)IL‐23RandIL‐12Rβ1expressioninunstimulatedneutrophilsexaminedbyquantitativePCR.(f)Rorcexpressionineitherunstimulatedneutrophils(N)orafterincubationwithrhIL‐6andIL‐23(IL‐6+IL‐23).(g)RORγtproteinintotalcelllysatesandnuclearextractsofisolatedneutrophilsobtainedafter1hincubationwithsupernatantsfromeitherunstimulated(unstim)totalperipheralbloodmononuclearcellsorafter18hincubationwithAspergillushyphalextract(AspHE).a,b,d­f:Representativedatafromasingledonor;similardatawereobtainedfromaseconddonor(datanotshown).c:Mean+/‐SDofthreewellspergroup;datain(e­g)arefromtwodonorsandarerepresentativeofthreerepeatexperiments.

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SupplementaryFigureS4.IL­6+IL­23stimulatedIL­17RC,dectin­1anddectin­2inhumanneutrophils,andIL­23RinRorcGFP/GFPmurineneutrophils.(a)CellsurfaceIL‐17RAandIL‐17RCexpressionofperipheralbloodneutrophilsfromahealthyvolunteer(Donor2)after3hincubationwithmediaalone(unstim),ormediacontaining20µg/mlrhIL‐6,2µg/mlrhIL‐23,bothcytokines,orbothcytokinesplusAspergillushyphalextract(AspHE).(b)Clec7a(dectin‐1)andClec6a(dectin‐2)geneexpressioninrecombinanthumanIL‐6andIL‐23(IL‐6+IL‐23)stimulatedperipheralbloodneutrophilsfromtwohealthydonors.(c)NeutrophilsfromnaïveC57BL/6orRorcGFP/GFPmicewereisolatedbygradientcentrifugation,incubatedwithanti‐IL‐23R,andexaminedbyflowcytometry.ThehistogramshowsoverlapofcellsurfaceIL‐23RinneutrophilsfromC57BL/6andRorcGFP/GFPmice.Datain(a­c)arerepresentativeoftworepeatexperiments.

Nature Immunology: doi:10.1038/ni.2797

SupplementaryFigureS5.RepresentativeflowcytometryprofilesshowingROSactivitybybonemarrowneutrophils.Intracellularamine‐reactivefluoresceindiacetate(CFDA)wasusedasaROSindicatorandshowninrepresentativepopulationsofnaïveC57BL/6neutrophils,andrecombinantmouseIL‐6andIL‐23(IL‐6+IL‐23)–stimulatedbonemarrowneutrophilsisolatedfromC57BL/6,Il17a­/­andIl17rc­/­miceafterincubationwithrecombinantmouseIL‐17Aandanti‐IL‐17RC.Combinedmeanfluorescenceintensity(MFI)dataarerepresentativeoftworepeatexperimentswithfourmicepergroup.

Nature Immunology: doi:10.1038/ni.2797

Nature Immunology: doi:10.1038/ni.2797

SupplementaryFigureS6.AspergillusinfectedcorneasofprimedC57BL/6miceandCD18–/–mice.(a)NIMP‐R14+neutrophilsandintracellularIL‐17A(IL‐17Ab)inA.fumigatusinfectedcorneasofC57BL/6micethathadeitherbeenprimedbysubcutaneousinjectionofheat‐killedswollenconidia72hprior,orwerenaïvepriortocornealinfection.Micewereexamined24hafterinfection.(b)NIMP‐R14+cellsfrominfectedcorneas72hafterprimingbysubcutaneousinjectionofheatkilledswollenconidia.IL‐17+andIL‐17‐cellswereexaminedforpropidiumIodide(PI)uptake24hpost‐infection.(c)AbsenceofinfiltratingcellsincorneasofA.fumigatusinfectedCD18‐/–micenotgivendonorneutrophils.Flowcytometryoftotalcellspooledfromfourcorneas24hafterinfection.Cellswereincubatedwithanti‐CD11b,1A8,NIMP‐R14,F4/80,CD4,CD3, γδ TCR,NK1.1antibodiesoranti‐IL‐17A(intracellular).

Nature Immunology: doi:10.1038/ni.2797

SupplementaryTable1:Cyclingthreshold(ct)scores

UD:undetectable;>40cycles

Figure Neutrophilsample Gene CTscoreFig1g UnstimC57BL/6 Il17a UD Splnsupstim 27.04 Splnsupstim+αIL‐1β 27.58 Splnsupstim+αTGF‐β 26.96 Splnsupstim+αIL‐6 39.24 Splnsupstim+αIL‐23 39.63

Fig2e NaïveC57BL/6 Il17a UD IL‐6+IL‐23C57BL/6 28.56 NaïveRORc+/GFP UD IL‐6+IL‐23RORc+/GFP 28.38 NaïveRORcGFP/GFP UD IL‐6+IL‐23stimRORcGFP/GFP UD

Fig4b Unstimhuman Il17a UD PBMCsupstimhuman 28.06 PBMCsupstim+αIL‐1β 27.91 PBMCsupstim+αTGF‐β 28.59 PBMCsupstim+αIL‐6 39.90 PBMCsupstim+αIL‐23 39.33

Fig4c D1unstimhuman Il17a 39.07 D1IL‐6+IL‐23stimhuman 23.38 D2unstimhuman 38.83 D2IL‐6+IL‐23stimhuman 24.22

Fig5a NaïveC57BL/6 Il17ra 28.01 Naïve+AspHEC57BL/6 27.86 Naïve+rmIL‐17C57BL/6 28.47 Naïve+AspHE+rmIL‐17C57BL/6 28.18 IL‐6+IL‐23C57BL/6 28.63 IL‐6+IL‐23+AspHEC57BL/6 28.01 IL‐6+IL‐23+rmIL‐17C57BL/6 27.91 IL‐6+IL‐23+AspHE+rmIL‐17C57BL/6 28.34

Fig5a NaïveC57BL/6 Il17rc UD Naïve+AspHEC57BL/6 UD Naïve+rmIL‐17C57BL/6 UD Naïve+AspHE+rmIL‐17C57BL/6 UD IL‐6+IL‐23C57BL/6 29.13 IL‐6+IL‐23+AspHEC57BL/6 23.18

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Fig5a IL‐6+IL‐23+rmIL‐17C57BL/6 Il17rc 28.76 IL‐6+IL‐23+AspHE+rmIL‐17C57BL/6 23.44

Fig5b NaïveIl17a‐/‐ Il17ra 27.11 Naïve+AspHEIl17a‐/‐ 27.05 Naïve+rmIL‐17Il17a‐/‐ 26.96 Naïve+AspHE+rmIL‐17Il17a‐/‐ 27.34 IL‐6+IL‐23Il17a‐/‐ 26.88 IL‐6+IL‐23+AspHEIl17a‐/‐ 27.17 IL‐6+IL‐23+rmIL‐17Il17a‐/‐ 26.93 IL‐6+IL‐23+AspHE+rmIL‐17Il17a‐/‐ 27.06

Fig5b NaïveIl17a‐/‐ Il17rc UD Naïve+AspHEIl17a‐/‐ UD Naïve+rmIL‐17Il17a‐/‐ UD Naïve+AspHE+rmIL‐17Il17a‐/‐ UD IL‐6+IL‐23Il17a‐/‐ 28.13 IL‐6+IL‐23+AspHEIl17a‐/‐ 23.98 IL‐6+IL‐23+rmIL‐17Il17a‐/‐ 28.74 IL‐6+IL‐23+AspHE+rmIL‐17Il17a‐/‐ 24.09

Fig5c Unstimhuman Il17ra 26.67 Unstimhuman+AspHE 26.53 Unstimhuman+rhIL‐17 26.89 Unstimhuman+AspHE+rhIL‐17 27.01 IL‐6+IL‐23human 26.42 IL‐6+IL‐23+AspHEhuman 27.07 IL‐6+IL‐23+rhIL‐17human 26.91 IL‐6+IL‐23+AspHE+rhIL‐17human 26.53

Fig5c Unstimhuman Il17rc UD Unstimhuman+AspHE UD Unstimhuman+rhIL‐17 UD Unstimhuman+AspHE+rhIL‐17 UD IL‐6+IL‐23human 29.23 IL‐6+IL‐23+AspHEhuman 26.18 IL‐6+IL‐23+rhIL‐17human 29.38 IL‐6+IL‐23+AspHE+rhIL‐17human 26.71

Fig5e NaïveC57BL/6 Clec4n UD IL‐6+IL‐23C57BL/6 28.31 NaïveC57BL/6 Clec7a 27.76 IL‐6+IL‐23C57BL/6 25.15

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Fig5f Unstimhuman Clec6a UD IL‐6+IL‐23human 28.94 Unstimhuman Clec7a 29.39 IL‐6+IL‐23human 27.14

Fig5g NaïveC57BL/6 Il17rc 39.98 IL‐6+IL‐23C57BL/6 27.96 IL‐6+IL‐23+AspHEC57BL/6 23.73 NaïveClec7a‐/‐ 39.73 IL‐6+IL‐23Clec7a‐/‐ 28.13 IL‐6+IL‐23+AspHEClec7a‐/‐ 24.19 NaïveClec4n‐/‐ 39.81 IL‐6+IL‐23Clec4n‐/‐ 27.86 IL‐6+IL‐23+AspHEClec4n‐/‐ 27.92

Fig5h UnstimRORc+/GFP Il17rc UD IL‐6+IL‐23RORc+/GFP 27.73 UnstimRORcGFP/GFP UD IL‐6+IL‐23RORcGFP/GFP 27.57

Fig5h UnstimRORc+/GFP Clec4n UD IL‐6+IL‐23RORc+/GFP 28.63 UnstimRORcGFP/GFP UD IL‐6+IL‐23RORcGFP/GFP 28.71

Fig6a NaïveC57BL/6 Il17rc UD IL‐6+IL‐23C57BL/6 27.83 NaïveIl17rc‐/‐ UD IL‐6+IL‐23Il17rc‐/‐ UD NaïveIl17a‐/‐ UD IL‐6+IL‐23Il17a‐/‐ 27.79 NaïveCybb‐/‐ UD IL‐6+IL‐23Cybb‐/‐ 27.53

Fig6a NaïveC57BL/6 Il17a UD IL‐6+IL‐23C57BL/6 28.79 NaïveIl17rc‐/‐ UD IL‐6+IL‐23Il17rc‐/‐ 28.83 NaïveIl17a‐/‐ UD IL‐6+IL‐23Il17a‐/‐ UD NaïveCybb‐/‐ UD IL‐6+IL‐23Cybb‐/‐ 29.21

FigS1d Naïvebonemarrowneutrophils Il17a 39.77 Primedbonemarrowneutrophils 27.03

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FigS1e Naïvespleenneutrophils Il17a 39.07 Primedspleenneutrophils 27.94

FigS2B NaiveC57BL/6 Rorc 27.62 IL‐6+IL‐23C57BL/6 28.30

FigS2d NaiveC57BL/6 Il23R 28.36 NaiveC57BL/6 Il12Rβ1 27.81

FigS2e NaiveC57BL/6 Il17a UD 20µgIL‐6stimC57BL/6 UD 20µgIL‐23stimC57BL/6 UD 10µgIL‐6/2µgIL‐23stimC57BL/6 UD 20µgIL‐6/2µgIL‐23stimC57BL/6 26.83 20µgIL‐6/1µgIL‐23stimC57BL/6 27.18 20µgIL‐6/500ngIL‐23stimC57BL/6 28.83 20µgIL‐6/250ngIL‐23stimC57BL/6 29.66 20µgIL‐6/125ngIL‐23stimC57BL/6 32.37 20µgIL‐6/62ngIL‐23stimC57BL/6 38.66 20µgIL‐6/31ngIL‐23stimC57BL/6 UD

FigS2f NaïveC57BL/615min Il17a UD IL‐6+IL‐23C57BL/615min UD NaïveC57BL/630min UD IL‐6+IL‐23C57BL/630min 33.19 NaïveC57BL/61h UD IL‐6+IL‐23C57BL/61h 26.13

FigS3b Unstimhuman Il17a UD 20µgIL‐6stimhuman UD 20µgIL‐23stimhuman UD 10µgIL‐6/1µgIL‐23stimhuman UD 20µgIL‐6/2µgIL‐23stimhuman 27.01 20µgIL‐6/1µgIL‐23stimhuman 28.98 20µgIL‐6/330ngIL‐23stimhuman 29.33 20µgIL‐6/110ngIL‐23human 33.13

FigS3e Unstimhuman Il23R 26.54 Unstimhuman Il12Rβ1 25.93

Nature Immunology: doi:10.1038/ni.2797

FigS3f Unstimhuman Rorc 28.10 IL‐6+IL‐23human 28.51

FigS4b D2unstimhuman Clec7a 29.81 D2IL‐6+IL‐23stimhuman 27.13 D3unstimhuman 29.11 D3IL‐6+IL‐23human 27.54

FigS4b D2unstimhuman Clec6a UD D2IL‐6+IL‐23human 28.33 D3unstimhuman 39.89 D3IL‐6+IL‐23human 28.16

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SupplementaryTable2:

Antibodiesforflowcytomtery

Clone IgGControl Distributor

Anti‐mouseNIMP‐R14 NIMP‐R14 RatIgG2b in‐house,AbcamAnti‐mouseLy6G 1A8 Rat IgG2 κ BioLegendAnti‐mouseIL‐23R 753317 RatIgG1 R&DAnti‐mouseF4/80 BM8 RatIgG2a κ eBioscienceAnti‐mouseCD3 145‐2C11 HamsterIgG eBioscienceAnti‐mouseCD4 GK1.5 RatIgG2b κ eBioscienceAnti‐mouseB220 RA3‐6B2 RatIgG2a κ eBioscienceAnti‐mouseNK1.1 PK136 MouseIgG2a κ eBioscienceAnti‐mouse γδ TCR GL3 HamsterIgG eBioscienceAnti‐mousegp130 KGP130 RatIgG2a κ eBioscienceAnti‐mouseIL‐6R 255821 RatIgG2a R&DAnti‐mousedectin‐1 218820 RatIgG2a R&DAnti‐mousedectin‐2 Goatpolyclonal

FAB1525GoatIgG R&D

Anti-mouse IL-17RC GoatpolyclonalFAB2270

GoatIgG R&D

Anti‐mouse IL-12Rβ1 GoatpolyclonalFAB1998

GoatIgG R&D

Anti‐mouseCD11b M1/70 RatIgG2b κ eBioscienceAnti‐mouseIL‐17A eBio17B7 RatIgG2a κ eBioscienceAnti‐mouse/humanRORγt

AFKJS‐9 RatIgG2a eBioscience

Anti‐humanIL‐17A eBio64DEC17 MouseIgG1 κ eBioscienceAnti‐humanIL‐6R B‐R6 MouseIgG1 eBioscienceAnti‐humanIL‐17RA 424LTS MouseIgG1 κ eBioscienceAnti‐humandectin‐2 545943 MouseIgG1 R&DAnti‐humanIL‐23R 218213 MouseIgG2b R&DAnti‐humandectin‐1 259931 MouseIgG2b R&DAnti‐humanIL‐17RC 309822 MouseIgG2b R&DAnti‐humangp130 28126 MouseIgG1 R&DAnti‐humanIL‐12Rb1 69310 MouseIgG1 R&D

Antibodiesforcytokineneutralization

Clone IgGControl Distributor

Anti‐mouseIL‐23p19 AF1619 GoatIgG R&DAnti‐humanIL‐23p19 AF1716 GoatIgG R&DAnti‐mouseIL‐6 MP5‐20F3 RatIgG1 R&DAnti‐humanIL‐6 6708 MouseIgG1 R&DAnti‐mouse IL-1β 30311 RatIgG1 R&DAnti‐human IL-1β 8516 MouseIgG1 R&DAntibodiesforcytokineneutralization

Clone IgGControl Distributor

Anti‐human TGF-β1 9016 MouseIgG1 R&DAnti‐humanTGF-β1 AB246 Goat

polyclonalIgGR&D

Nature Immunology: doi:10.1038/ni.2797

AntibodiesforWesternblotanalysis

Clone IgGControl Distributor

Anti‐mouse/human RORγt

AFKJS‐9 RatIgG2a eBioscience

Anti‐β-actin 8H10D10 MouseIgG2b CellSignalingAnti‐TATAboxbindingprotein

1TBP18 MouseIgG1 Abcam

SecondaryAntibodiesWesternblotanalysis

Clone IgGControl Distributor

Goatanti‐ratIgG2aHRP PA184709 RatIgG2a eBioscienceGoatanti‐mouseIgG2bHRP

SC2062 RatIgG2a eBioscience

Goatanti‐mouseIgG1HRP

SC2060 MouseIgG1 Abcam

Nature Immunology: doi:10.1038/ni.2797

SupplementaryTable3:

Gene GenBankID 5’­Forwardsequence­3’ 3’­Reversesequence­5’mIl7a NM_010552 TCAGCGTGTCCAAACACTGAG CGCCAAGGGAGTTAAAGACTThl17a NM_002190 TCCCACGAAATCCAGGATGC GGATGTTCAGGTTGACCATCACmClec7a NM_020008 GACTTCAGCACTCAAGACATCC TTGTGTCGCCAAAATGCTAGGhClec7a NM_197950 ACAATGCTGGCAACTGGGCTCT AGAGCCATGGTACCTCAGTCTGmClec4n NM_020001 AAGCGGAGCAGAATTTCATCA CCATTTGCCATTACCTTGTGGAhClec6a NM_001007033 TCAGTGAAGGGACAAAGGTGCC CTCCCATCTCAACACAGTTCTGCmIl17rc NM_178942 TTCTGCGGTATTTGACTGTTTCG GTCCCGGACTTCAAGACCChIl17rc NM_032732 CTGCCCTTGTGCAGTTTGG CAGATTCGTACCTCACTCCCTAmIl17ra NM_008359 CGGAGAATTAGTCCCTGTGTTG GAACAGTCACTTCATACTCCTGGhIl17ra NM_014339 GCTTCACCCTGTGGAACGAAT TATGTGGTGCATGTGCTCAAAmIl23R NM_144548 AACAACAGCTCGGATTTGGTAT ATGACCAGGACATTCAGCAGThIl23R NM_144701 CAGGTCACTATTCAATGGGATGC GCAGTTCTTAATTGCTGCTTGGhIl12Rβ1 NM_005535 TAGGGACCTGAGATGCTATCG CCCGGAGCTAAGGCAACACmIl12Rβ1 NM_008353 GCTGCTGCGTTGAGAAGACA CACAGGACGTGAGAAACATTGThRorc NM_005060 GTGGGGACAAGTCGTCTGG AGTGCTGGCATCGGTTTCGmRorc NM_011281 TCCACTACGGGGTTATCACCT AGTAGGCCACATTACACTGCTGAPDH NM_002046 GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAAActb(β‐actin) NM_001033319 AGAAACGCTGGCCTTAACCC GCTGCGAACTCCTGTGGAA

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