© 2016 Nature America, Inc. All rights reserved. N.A.B. N.A.B. ( USA. 1 T cell–mediated inflammation, which directs their maturation into into maturation their directs which inflammation, cell–mediated T mucosa intestinal the to home constitutively progenitors These progenitors. and mice cell–deficient T in absent cells, mast Mucosal ments. system immune Fc receptors recognition xenobiotic and parasites multicellular bacteria, to resistance host mediating in function cell’s mast sentinel the to central is mediators of array This cytokines and chemokines prostaglandins, leukotrienes, matory by of is the generation pro-inflam accompanied activation mast-cell glycosaminoglycans heparin with core proteoglycan a to bound proteases serine mast-cell-specific of range broad a TNF,and serotonin histamine, including mediators, formed pre- containing tissue-resident granules secretory with packed densely cells sentinel distinct morphologically are cells Mast immunity. urochordates as far as compendium. ImmGen the of power the using system immune the of context broader the within them placed and lations 2 popu these with type associated profiles -expression the we assessed with infection associated helminthic against defense host and traditionally inflammation granulocytes are pipelines data-analysis and protocols cell-isolation standardized rigorously through system immune mouse the characterize that patterns gene-expression the determine of to seek who biologists computational and consortium immunologists a is Project (ImmGen) Genome Immunologic The underscores their differential capacities to detect signals environmental and influence the inflammatory milieu. with other circulating granulocytes than with mast cells. The derivation of mast-cell and signatures transcriptional than previously appreciated. We observed minimal homology between mast cells and basophils, which shared more overlap from independently all other profiled cells, and that mast cells demonstrated considerably greater across heterogeneity tissues tissues and basophils isolated from spleen and blood. We found that mast cells were distinct, clustering transcriptionally characterize the function of these cells, we assessed the profiles transcriptional of mast cells isolated from peripheral connective E immunoglobulin (IgE) and have been linked to host defense and diseases. Todiverse immune-system-mediated better Mast cells are ancient evolutionarily sentinel cells. Like basophils, mast cells express the receptor high-affinity for Daniel F Dwyer a unique identity within the Expression profiling of constitutive mast cells reveals nature nature Received 18 August 2015; accepted 23 March 2016; published online 2 May 2016; Division Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, USA. Massachusetts, γ at el ae on i to an eihrl ise compart tissue peripheral main two in found are cells Mast least at back date that cells ancient evolutionarily are cells Mast receptors, which allows them to respond to targets of the adaptive 3 1 Full Full list of members and affiliations appears at the end of the paper. 2 , arise from bone marrow (BM)-derived agranular mast-cell mast-cell agranular (BM)-derived marrow bone from arise , [email protected] 1 3 and are further recruited to the intestine 2 . 7– 1 , 9 2 9 . Mast cells can be activated through pattern- through activated be can cells Mast . 3 , , Nora A Barrett or tissue damage tissue or , 1 4

. Tissue-resident mast cells and circulating circulating and cells mast Tissue-resident . , which predates the emergence of adaptive adaptive of emergence the predates which , ) ) or K.F.A. ( aDV A NCE ONLINE PUBLIC ONLINE NCE [email protected] 5 10 . Granule release following following release Granule . 1 , 1 , 1 2 and express Fc express and , 4 , , K Frank Austen 1 4 and lung A TION

1 ε 5 R1 and and R1 during 2 . Here Here . 4 ). ). These These authors jointly supervised this work. should Correspondence be addressed to 5 , 6 - - - 1 .

, 2 , 4 ported previous findings, confirming important roles for mast mast for roles important confirming findings, previous ported or mast-cell-associated of cells mast deletion Cre- recombinase-mediated with mice in and proteases deficient, cell mast-cell-specific lacking mast mice are in which c-Kit, receptor stem-cell-factor the encoding gene the in mutations with strains mouse in studied been related. closely be to and mediators mast produced, have cells and believed basophils been cell progenitor a from replenished lived, with a half-life of several days in the periphery short- are basophils circulating cells, mast connective-tissue Unlike and cytokines, including interleukin 4 (IL-4) and IL-13 (refs. (refs. IL-13 and 4 (IL-4) interleukin including cytokines, and leukotrienes of generation rapid by the accompanied degranulation, Fc express cells, mast inflammation allergic during tissue peripheral infiltrate profile protease and a restricted granules histamine containing cytoplasmic smaller fewer, and nuclei multi-lobular with unknown. is tissues different across heterogeneity mast-cell of degree full the microenvironment, tissue the by regulated is and mast-cell expression of proteases replace ment by than BM-derived precursor cells. rather While studies have indicated self-renewal that or longevity through maintained is compartment mast-cell connective-tissue the that suggests which sites mucosal to recruitment their to relative tissues connective in adult mice have of shown mast engraftment poor donor-derived cells progenitors derived from the fetal liver tissues nective con most in present constitutively are cells mast connective-tissue cells mast mucosal granulated doi:10.1038/ni.344 & The Genome Immunological Project Consortium The contribution of mast cells to inflammation and immunity has immunity and to inflammation cells of mast contribution The Compared with mast cells, basophils are smaller circulating cells cells circulating smaller are basophils cells, mast with Compared 2 , 2 9 I sm css nwr eei apoce hv sup have approaches genetic newer cases, some In . 1 7 and are seeded during embryogenesis by circulating by circulating embryogenesis during and are seeded 5 ε R1. Signaling through Fc through Signaling R1. 2 Harvard Harvard Medical School, Boston, Massachusetts, 1 6 16 . In contrast to mucosal mast cells, cells, mast mucosal to contrast In . , 2 2 0 8 and receptors . Due to their Fc their to Due . 1 8 . . BM-transfer experiments in ε e c r u o s e r R1 induces basophil basophil R1 induces 2 2 1 7 is heterogeneous , and are actively ε 22 R1 expression expression R1 , 2 3 2 . Basophils . Basophils 4 and, like like and, 25

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© 2016 Nature America, Inc. All rights reserved. clustered separately from all other lymphoid and myeloid cells analyzed sorted showed populations that expression the five variable mast-cell cells. NKT and cells (NK) killer CD4 neutrophils, cells, dendritic splenic and cells; B-1a and macrophages eosinophils; peritoneal blood including immunocytes, for obtained database ImmGen the in results with compared and microarray by assessed 1 Table ( samples parallel with assessed was purity final and ( sorting of rounds multiple through purity ( basophils and cells ing, which indicated that the cells isolated were mast morphologically ( for of ( the mast isolation cells CD45 of 0.1% they comprised where blood, peripheral and spleen the from CD49b integrin Fc of co-expression of basis the on sorted were Basophils CD45 of 10% and 0.05% the stomach) ( to proximal submucosa the in reside they (where esophagus the and tissue) serosal and submucosa the in reside they (where trachea the layer), muscular the in reside they (where tongue the dermis), the in reside they (where ear cavity, the peritoneal the from (CD117) c-Kit Fc of co-expression of basis the on sorted were cells Mast immunocytes among distinct transcriptionally are cells Mast RESULTS inflammation. tissue of absence the in periphery in turnover the mast-cell connective-tissue unappreciated previously for evidence found and expression gene in heterogeneity substantial we identified studied, populations Among the mast-cell populations. and and found cells a basophils shared signature by small the two cell of signatures mast Wecells. transcriptional the unique demonstrated eosinophils and shared unexpectedly few to distinct transcriptsclosest with mast transcriptionally were basophils that Wefound analyzed. from all other lymphoid independently and populations myeloid-cell clustering cells mast with distinct, was transcriptome mast-cell that the showed data Our blood). peripheral and spleen (the locations two from basophils isolated and cavity) peritoneal the and trachea distinct anatomical locations (the skin, the tongue, the esophagus, the Cre. of expression mast-cell-specific with strains novel of tion genera the for loci candidate provide and disruption Cre-mediated for targets identify functions, mast-cell clarify might cells other immune in expression their to relative cells mast in expressed inantly these strains. Thus, defining the and pathways uniquely or dom basophils in detected strains, been have Kitty Hello and Cre-Master mast-cell-deficient the of tion A3, to used Cre direct expression for as carboxypeptidase the genera such proteins, mast-cell-associated some Additionally, strains. these in of mast subsets deletion cell or differential compartment mast-cell of influence the protocols, in differences reflect might findings Such discrepant to allergens. contact sitization sen in cells for mast role a pro-inflammatory by establishing strains protease 5 has contradicted early findings obtained with c-Kit-mutant sensitivity resistance to animal arthritis crystal-induced anaphylaxis systemic and local IgE-dependent in cells e c r u o s e r  ( Supplementary Fig. 2b Supplementary Fig. 1 Fig.

Hierarchical clustering using the top 15% of genes with the most most the with genes of 15% top the using clustering Hierarchical Here we isolated mast constitutive cells from connective-tissue five c ). RNA extracted from sorted mast cells and basophils was was basophils and cells mast sorted from extracted RNA ). ). Lymphoid cells and myeloid cells clustered independently, independently, clustered cells myeloid and cells Lymphoid ). 3 3 , the Cre-mediated deletion of cells expressing cell mast expressing cells of deletion Cre-mediated the , Supplementary Fig. 1 Supplementary + T cells, CD8 cells, T Fig. 1 Fig. ) was validated through histochemical stain ) histochemical through was validated + 3 + 0 3 cells ( cells b sniiain o od allergen food to sensitization , 2 el i ec cmatet ( compartment each in cells ). Cells underwent enrichment to high high to enrichment underwent Cells ). . . In other models, such as contact hyper Supplementary Fig. 2a Supplementary + 3 T cells, cells, T 4 Fig. 1 Fig. , which are reduced in number in in number in reduced are which , ). ). Mast cells constituted between a γ ). The gating strategy used used strategy gating The ). δ Kit T cells, B-2 cells, natural natural cells, B-2 cells, T Supplementary Fig. 3 Fig. Supplementary mutation beyond the the beyond mutation Supplementary Supplementary ) and basophils ) and basophils 2 9 , uric-acid- , ε ε i. 1 Fig. R1 R1 3 α α 1 and and and and and and a ). ). ), ), ------

inflammatory product D prostaglandin inflammatory taglandin D The last category included similar to each other and were closest to eosinophils ( eosinophils to closest were and other each to similar neu from ( furthest and trophils eosinophils and basophils to closest were ( different most the were peritoneum and skin the from subsets mast-cell the and similar, most the were tongue and esophagus trachea, the from subsets mast-cell the cells, ( expression variable most the with transcripts ( measurements distance the other cell populations analyzed were quantified through Euclidean and myeloid other ( cells lymphoid from distantly and other each with closely group ing tissues different from cells mast with profiled, populations cell other the from cells mast of distinction the highlighted further mast cells showed enrichment ( which for transcripts the with overlap little had that genes of cluster distinct associ a of a transcripts expression smaller had high Basophils types. cell other of other ated with many of expression expression low high and genes both of set on based was immunocytes and ( cells neutrophils dendritic and macrophages containing second a (eosinophils, and populations) basophil both granulocytes containing one into divided group further was cluster myeloid the and expected, as biosynthesis, biosynthesis, ‘polysaccharide ’ and ‘’ ( contained transcripts encoding important for heparin-sulfatemetabolism’, ‘sulfur which included signature mast-cell the in ment than in neutrophils ( mast cells in expression G; moregene was of higher this fivefold than showed enrichment for the expression of component C2 of the classical C3 convertase ( which encodes a metalloprotease, and Plau included transcripts for many canonical mast-cell proteases, as well as encoding products in other functional categories ( the category of ‘serine proteases’, relative to the abundance showedof transcriptsthe signature most significantmast-cell enrichment the for that genes encodingrevealed products system in pathway-classification other cells analyzed ( sion was twofold or greater in mast cells relative to their expressionWe in all next identified a transcriptional cells signaturemast tissue-resident of of signature 128 Transcriptional genes whose expres immunocytes. other from that them distinguished program gene a expressed populations mast-cell sue-resident ( eosinophils blood in expression their to transcripts, relative 869 of expression higher had basophils, basophils blood blood in whereas expression their to relative transcripts, 503 of expression had higher eosinophils a of or level twofold higher. Blood blood basophils revealed differential expression of 1,372 transcripts at and eosinophils blood of comparison pairwise contrast, In ferences. skin in mast cells ( expression their to relative transcripts, 1,135 of expression higher had basophils blood whereas basophils, blood in expression to relative their of transcripts, 1,428 expression had higher mast cells Skin greater. or twofold of level arbitrary an at transcripts 2,563 of dermal mast cells and blood basophils revealed differential expression ( and neutrophils basophils between to and the distance was mast similar cells basophils between distance The transcriptional relationships among mast cells, basophils and basophils cells, mast among relationships transcriptional The , which encodes a -type , Fig. 2 Fig. aDV Fig. Fig. 2 2 synthase, a factor important for synthesis of the mast-cell A a Fig. 1 Fig. NCE ONLINE PUBLIC ONLINE NCE b ). Basophils from the blood and spleen were very very were spleen and blood the from Basophils ). ); ); this further underscored their transcriptional dif Fig. 3 Fig. Fig. 3 Fig. 1 Fig. d ). b Fig. 2 Fig. a Hpgds ). Additional pathways that showed enrich c ). ). Functional analysis with the PANTHER ). The distinction of mast cells among among cells mast of distinction The ). Fig. 1 a , , which encodes hematopoietic pros ), calculated using the top 15% of of 15% top the using calculated ), Fig. Fig. 2 A c ). Principal-component analysis C2 TION Fig. 2 Fig. 2 Ctsg ( , which encodes complement Table a

). Pairwise comparison of comparison ). Pairwise , which encodes a nature immunology nature ). Mast cells as a whole whole a as cells Mast ). Table Fig. 1 Fig. 1 ). Table Fig. 2 Fig. 1 c ). ). Mast cells also ). Among mast mast Among ). 1 Fig. 2 Fig. c ). This group ). Thus, tis Thus, ). Adamts9 Table a ). The The ). 1 ). ). ------,

© 2016 Nature America, Inc. All rights reserved. or blood basophils (bottom right). Data are pooled from three (skin, tongue and tracheal mast cells; splenic and blood basophils; blood eosinophils), eosinophils), blood basophils; blood and splenic cells; mast tracheal and tongue (skin, three from pooled are Data right). (bottom basophils blood or left) (top eosinophils blood in differentially expressed genes total indicate plots in numbers population; other the in than blue) (dark basophils blood or (red) eosinophils blood in higher or twofold expression and 120 than greater values expression with transcripts indicate colors dot basophils; blood and eosinophils blood ( right). (bottom basophils blood or left) (top cells mast skin in differentially expressed genes total indicate plots in numbers population; other the in than blue) (dark basophils blood or (aqua) cells mast skin in higher or twofold expression and 120 than greater values expression with transcripts indicate colors dot basophils; blood and cells mast skin in expression ( distance. Euclidean indicate in (identified expression variable most the with transcripts 15% top the using calculated populations cell selected between similarity of degree ( basophils. from distinct transcriptionally as 2 Figure gene these, of receptors: pathway encoded members of the Mrgpr family of G--coupled ( experiments independent cells) mast (esophageal ( experiments independent basophil) blood cell; mast esophageal and (tracheal five or cell) mast tongue and (skin four basophil), splenic and cell mast (peritoneal three from pooled are Data component. principal each by described transcripts of frequency indicate parentheses in Numbers expression. variable ( database. ImmGen the of designations replicate biological- indicate (#) numbers populations; linked between homology to correlated inversely is height Bar (Treg.Sp). T cells regulatory and CD8 (NKT.Sp), cells NKT Sp), (NK. cells killer natural (BB.Sp), cells B-2 splenic and (B1ab.PC), cells B-1a peritoneal (DC.Sp), cells dendritic (MF.PC), splenic macrophages peritoneal (GN.Sp), neutrophils splenic (Eo.Bl), eosinophils blood (BA.Sp), spleen and (BA.Bl) blood the from basophils (MC.PC), peritoneum and (MC.Sk) skin Es), (MC. (MC.To), esophagus (MC.Tr), tongue trachea the from cells mast include populations expression; variable most the with transcripts 15% top the using margin) (right populations 10 bars, Scale populations). cell of isolation the for used strategy gating confirm (to spleen from sorted basophils identifying staining blue toluidine and fluid, lavage peritoneal and skin ear tongue, esophagus trachea, from sorted cells mast ( ( mean the indicate lines horizontal small replicate; biological individual an represents symbol Each axis). (horizontal tissues CD45 among (Ba) ( populations. cell assayed other from distinct 1 Figure nature immunology nature experiments. independent populations) other (all two or cells) mast (peritoneal five b a a ) Frequency of mast cells (MC) and basophils basophils and (MC) cells mast of ) Frequency ) Euclidean distance matrix indicating indicating matrix distance ) Euclidean ) Chloracetate-esterase staining identifying identifying staining ) Chloracetate-esterase Five of the genes encoding products in the ‘signal-transduction’ ‘signal-transduction’ the in products encoding genes the of Five + T cells (T8.Sp), CD4 (T8.Sp), T cells MRGPRX2

µ Characterization of mast cells cells mast of Characterization as cells mast of Identification Mrgprb2 m. ( m. Mrgpra4 a Fig. 1 Fig. c ), one experiment ( experiment one ), ) Hierarchical clustering of cell cell of clustering ) Hierarchical c ) in expression ) Gene . The protein encoded mediates mast-cell activation activation mast-cell mediates encoded protein . The + cells in various digested digested various in cells , has been described as the homolog to the human the to homolog the as described been has , c ); numbers in plot plot in numbers ); , , Mrpgrb1 γ

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MC.Sk 0.15 Lymphocyt had high expression specifically in skin mast cells, while while cells, mast skin in specifically expression high had encoding transcripts in the mast cell signature, patients in reactions pseudoallergic with IgE-independent compounds associated pharmaceutical several to venom wasp from ranging stimuli of array broad a to response in

59 45 44 40 0.10 MC.PC Blood Ba PC2 (17%) 0.05 101 95 95 91 96 Tongue MC l BA.Bl Spleen Ba e Neutrophi

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2 0 2 DC.SP –1 113 116 110 118 115 111 114 99 97 96 96 NK.SP c , using the top 15% transcripts with the most most the with transcripts 15% top the , using 106 108 112 120 116 112 114 57 87 93 94 94 NKT.SP z -score 109 111 108 117 113 110 111 1 0 41 55 92 93 93 94 Tgd.SP b c 3

Expression Expression 5 blood eosinophil skin mast cell Mrgpr- five to the . In addition 10 10 10 10 10 10 10 10 1 2 3 4 1 2 3 4 T8.Sp#1 Tgd.Sp# Tgd.Sp# NKT.Sp# NKT.Sp# NK.Sp# NK.Sp# BB.Sp# BB.Sp# B1ab.PC#2 DC.Sp#1 DC.Sp#2 MF.PC#1 MF.PC#2 GN.Sp# GN.Sp# Eo.Bl# Eo.Bl# Eo.Bl# BA.Sp# BA.Sp# BA.Sp# BA.Bl# BA.Bl# BA.Bl# MC.PC#0 MC.PC#4 MC.PC#3 MC.PC#6 MC.PC#5 MC.Sk# MC.Sk# MC.Es# MC.Es# MC.To# MC.To# MC.Tr# MC.Tr# MC.Tr#3 Treg.Sp#2 Treg.Sp#1 T4.Sp#2 T4.Sp#1 T8.Sp#2 B1ab.PC#1 MC.Sk# MC.To# 1,428 503 10 10 Mrgprb8 1 5 2 3 3 1 2 1 2 1 2 1 4 3 1 2 1 3 4 1 2 1 2 2 5 2 1 2 1 2 1 e c r u o s e r blood basophil blood basophil 10 Expression 10 Expression 2 2 and 10 10 3 3 Mrgprb13 Mrgpra6 1,135 10 10 869 4 4



© 2016 Nature America, Inc. All rights reserved. The protease-encoding The transcript protease-encoding factor that directs the differentiation and function of both cell types IgE; for encodes a Ca receptor high-affinity the of respectively, receptor; included These basophils. ( genes shared 24 and cells mast in only characterized been small previously have of which of many consisting a signature revealed transcriptional analysis This analyzed. sub tran population other any mast-cell in than and sets of basophil all expression in scripts twofold-higher basophils, of basis the on and signature shared a cells derived we mast between ship environment. local the influence and with interact might basophil the which Itga1 and ines ( scripts from genes chemok several encoding tran included also signature basophil The transcript, sin a protease-encoding contained gle signature ( basophil cells The populations mast cell including other analyzed, all in the to expression relative populations in basophil expression both twofold-or-greater of basis the on calculated similarly was 66 transcripts of signature transcriptional basophil A expression gene shared and Distinct appreciated. previously than family synthetic enzymes and receptors of the Mrgpr bio proteases, of degree broader program a included transcriptional mast-cell unique ( cells mast in detected being to addition in cells, NKT and cells B and populations, cell reported previously as neutrophils, by predominantly Mrgpra2a ( basophils in expression high had experiments. independent populations) other (all two or cells) mast (peritoneal five eosinophils), blood basophils; blood and splenic cells; mast tracheal and tongue (skin, three from pooled are Data analyzed. populations cell across transcripts encoding ( signature. mast-cell the in enrichment specific showing transcripts ( populations. cell non–mast- all of that than (red) higher tenfold or (blue) higher fivefold expression indicate margin right along symbols gene of colors ( analyzed populations cell other all in expression the to relative populations cell mast all in greater or twofold expression transcript of basis the on derived signature gene ( signature. transcriptional 3 Figure e c r u o s e r 

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B1ab.PC B1ab.PC BB.Sp BB.Sp mast cells and basophils had high expression of transcripts encoding encoding of transcripts expression had high and basophils cells mast analysis of the monoamine-biosynthetic pathways indicated that both involved in loading histamine into transporter secretory vesicles solute ( a Slc18a2, encoding transcript the included here identified profile shared the mast cell–basophil that, with Consistent by basophils transcript signature, consistent with published reports of high expression of this NK.Sp NK.Sp

Mast cells and basophils are well-known sources of histamine of sources well-known are basophils and cells Mast NKT.Sp NKT.Sp

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T8.Sp T8.Sp T4.Sp T4.Sp Treg.Sp Treg.Sp aDV Osbpl Nckap1 Lxn Kcnq Hs3st3a1 Hpgds Grik2 Fam198b Dnm3 Ctsg Cobl Chst1 Tpsb Tpsab Tph1 Rab27b Plau Olfr920 Mrgprx2 Mrgprb2 Mrgprb1 Mrgpra4 Meis2 Mcpt Mcpt Mao Kcne Hs6st2 Gnai Ednra Creb3l Cma2 Cma1 Ccl2 Ctsg Plau C2 Tpsg Cma2 Mcpt Tpsab Tpsb Cma1 Mcpt Mcpt D330045A20Rik Copz2 Cdh9 Cdc42bpa Ccl7 Ccdc141 Car13 C2 Bmpr2 Bbs10 5730460C07Rik 4930452B06Rik 2310046K01Rik 1700047G07Rik Vwa5a Tpsg Stard13 Slc45a3 Slc29a1 Serpinb1 Rnf128 Rgs13 Pcp4l1 Pcbd1 Papss2 Adamts9 Adamts9 Atl2 Armcx3 Acer3 A4galt 1 b 1 2 1 2 1 9 4 9 4 2 3 A 1 8 1 1 1 NCE ONLINE PUBLIC ONLINE NCE a z -score 0 3 8 , in addition to mast cells. mast to addition in , c –1

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b Treg.Sp Treg.Sp ). Further Mrgpr Mrgpra2b Mrgpra2a Mrgpra6 Mrgprb1 Mrgprb8 Mrgprx1 Mrgprx2 Mrgprb2 Mrgprb1 Mrgpra4 Zfp9 Zfp521 Zdhhc13 Wdr60 Unc13b Trank Tiam2 Tcf7l1 Stk32b St6galnac3 Spred Socs Smpx Smarca1 Slc7a5 Slc6a4 Slc4a4 Slc31a2 Sgce Ror1 Rnf180 Riok Rbfox1 Rapgef2 Pde1c Pcdh Optn Olfr918 Olfr917 Olfr915 Oa Neo1 Ndst Nceh1 Mt Mrgprx1 Mlph Mitf Mfge Mcpt Lsamp Lrrc66 Lima1 Krt Kit Kdelr3 Id Hspa13 Hs2st1 Gpm6a Gp1b Gnaz Gcet Fam84a Fam129b Ext1 Esyt3 Eno2 Enah Dusp1 Dnahc14 Dgki Dd Dclk s 2 4 1 c f 2 3 2 2 2 8 2 7 1 a e 1 8 3 2 . © 2016 Nature America, Inc. All rights reserved. h tasrps osre ars seis nldd hs encod D those prostaglandin included hematopoietic species proteases, ing across conserved transcripts ( The basophils blood human in than cells mast skin human in expression higher twofold having sets data both in genes signature mast-cell- 82 the of 55 with signature, mast-cell mouse the of expres sion for enrichment species. across significant showed cells signatures mast skin Human basophil and mast-cell the evaluate to basophils and blood mast cells of human dermal ing transcriptome rest the defined that set data a FANTOM consortium we used Next Comparison of mast-cell and basophil signatures across species function. in related closely not are types analysis cluster earlier our ( extended basophils and cells mast by proteins expressed and receptors inflammatory-cell tors, cell-surface ( basophils and cells mast by by mitment of progenitor cells to the basophil lineage. GATA-2 (encoded biology basophil in role important an only C/EBP- included nature and c-Kit proteases mast cell encoding genes the of expression regulates encoded factor transcription the as biology, only knowledge, our included ture signa mast-cell The immunocytes. other in than basophils or cells ( receptor a serotonin encoding transcript ( ( transporter receptor re-uptake histamine a encoding transcripts ent with reports published included ( decarboxylase DOPA l ( ( transporter histidine a system. Identifiers inparentheses(leftcolumn)indicatepathway-moduledesignationsthe PANTHER pathway-classification Signal transduction(BP00102) Carbohydrate metabolism(BP00001) Sulfur metabolism(BP00101) (BP00009) Other polysaccharidemetabolism Other (MF00140) (MF00216) Pathway Table 1 nature immunology nature ( c-Kit and family, Mrgpr the of Fig. 1 Fig. i. 4 Fig. -tryptophan transporter ( transporter -tryptophan Several transcription factors had higher expression in either mast mast either in expression higher had factors transcription Several Gata2 P c c

values,modificationofFisher’s exacttest. ) and pairwise analysis ( analysis ) pairwise and ). Mast cells further expressed transcripts encoding an an encoding transcripts expressed further cells Mast ). Functional pathway-enrichment analysis Functional of pathway-enrichment mast-cell signature genes Maob ) was the only transcription factor in the signature shared shared signature the in factor transcription only the was ) α (encoded by Creb3l1 , which encodes a monoamine oxidase ( Sncaip Mitf , , Mitf , , Ddc Slc6a4

Cebpa Slc3a2 has been previously reported in mast-cell mast-cell in reported previously been has aDV 3 Cebpa Slc7a5 9 , , ) ( ) . . Mast both expressed and cells basophils Smarca1 Fig. 4 Fig. A Fig. 4 Fig. ) ( ) , , NCE ONLINE PUBLIC ONLINE NCE ) and histidine decarboxylase ( decarboxylase histidine and ) Supt3h Fig. 2 Fig. ) has been previously reported to have Fig. 4 Fig. ), tryptophan ( hydrolase tryptophan ), Sgce Mrgpra4 Mfge8 Gnaz Ednra Ccl2 Stard13 Mrgprx2 Cdc42bpa Ids Eno2 Ndst2 Ids Papss2 Hs2st1 Ndst2 Hs6st2 Ndst2 Tpsab1 Mcpt2 Ctsg Supplementary Table 2 Table Supplementary d c ). The diverse transcription fac transcription diverse The ). and and ). The mast-cell signature also also signature mast-cell The ). 2 b 8 c and and ) and indicated that these cell cell these that ) indicated and , in which it directs the com the directs it which in , ), while basophils expressed expressed basophils while ), Zfp9 Htr1b Nfil3 Socs2 Pcdh7 Neo1 Hs3st3a1 Grik2 Ccl7 Tiam2 Ndst2 Mrgprb1 Slc45a3 Ext1 St6galnac3 Ndst2 Ids St6galnac3 Hpgds Chst1 Tpsb2 Mcpt4 Cma1 ( Hrh4 2 ) ) ( Fig. 4 Fig. synthase, members members synthase, 4 ( 0 Fig. 4 Fig. . The basophil sig . The basophil Fig. 4 Fig. A ) and serotonin serotonin and ) Fig. 4 TION d Genes ). Of these, to these, Of ). c d ). ). Of these, these, Of ).

c Tph1 Ror1 Pde1c Kit Gp1ba Dgki Bmpr2 Rab27b Mrgprb2 Hs3st3a1 A4galt Hs3st3a1 Ext1 Hs3st3a1 Tpsg1 Mcpt9 C2 Tph1 ), consist ). Other Other ). Fig. Fig. ) ) and Hdc 5 4 ). ). 1 ------)

Rgs13 Plau Lrrc66 Gnai1 Dusp18 Cdh9 Rapgef2 Mrgprx1 Hs2st1 Chst1 Hs2st1 Hs3st3a1 Hs2st1 Plau Cma2 fold or more in single mast-cell subset relative to their expression in all comparison. this in distinct most the were subsets mast-cell ( genes 957 of expression ential ( of expression 612 transcripts had tongue and differential peritoneum mast in ( expression esophagus the from their cells to relative tongue the from cells mast in ( trachea the from cells mast in expression their to relative tongue the of or from in in twofold expression mast cells greater ing a difference show genes 110 only with esophagus, or the trachea the either from for populations. cell mast other with cells comparison subsequent mast peritoneal treated enzymatically used we treatment, because a subset of genes underwent transcriptional alteration by such ( most subset the distinct were transcriptionally cells mast peritoneal that demonstrated again mast cells peritoneal treated enzymatically with cells mast of clustering hierarchical and genes, signature mast-cell were genes None of these whose expression decreased five- to tenfold ( ( one transcript including cells, untreated in expression two their to relative more or by fold genes 26 of expression the decreased digestion Enzymatic ( tenfold than more increased expression as 17 genes whose expression increased five- to tenfold and 7 genes (such including cells, untreated in expression their to relative more or fold of the expression 137 mast genes by increased cells of two peritoneal of digestion enzymes on mast-cell transcription. Enzymatic treatment population derived from undigested tissue, we first the assessed effect wise comparison. Because peritoneal mast cells were the only mast-cell pair through subsets Next among the diversity we mast-cell assessed populations mast-cell among programs genetic Tissue-specific species. across program transcriptional mast-cell the of nature conserved the demonstrated Fig. 6 Fig. Fig. 6 Fig. We next analyzed transcripts with a difference in expression of four cells mast with homology high showed tongue the from Mast cells Ccl3 , , c a Il13 ), and mast cells from the peritoneum and skin had differ had skin and peritoneum the from cells mast and ), ), and only 122 genes showing a difference in expression expression in difference a showing genes 122 only and ), 2.6 ×10 1.1 ×10 3.8 ×10 1.9 ×10 2.3 ×10 1.3 ×10 P and the transcription-factor-encoding gene and the transcription-factor-encoding value Myl1 −2 −2 −3 −3 −6 −6 , which encodes the myosin -chain protein) protein) light-chain myosin the encodes , which included included these roles; less-well-defined with products across encoded species conserved transcripts human mast cells than in human basophils basophils human in than cells mast human Gata2 Cpa3 mast as mouse such transcripts with by basophils, and cells shared signature the for (relative to its expression in human basophils) mation. Human mast cells showed enrichment to sites other leukocytes recruiting of inflam in species across basophils for role shared a ( CCL4 and CCL3 ines chemok the encoding those were basophils mouse and human in conserved transcripts mast ( cells skin human in than basophils blood sets data in human- higher twofold expression both having in present genes 44 the signature of 10 only with signature, basophil mouse the for enrichment significant show ( protein) G ( upeetr Tbe 4 Table Supplementary In contrast, human basophils did not not did basophils human contrast, In Fig. 6 Fig. having expression 7.6-fold higher and and higher 7.6-fold expression having Supplementary Table 3 Table Supplementary having expression 5.2-fold higher in in higher 5.2-fold expression having Fig. 6 Fig. b Maob Supplementary Fig. 5 Fig. Supplementary ). In contrast, mast cells from the the from cells mast contrast, In ). Fig. Fig. c and and ), which indicated these two two these indicated which ), 5 ). Supplementary Fig. 4a Fig. Supplementary Supplementary Supplementary Fig. 4b Gnai1 Fig. Fig. e c r u o s e r (which encodes a encodes (which ), which again again which ), 5 ), suggestive of of suggestive ), ). Among the the Among ). Egr2 ). However, ). ) whose ) whose ). ). ). ).  ------

© 2016 Nature America, Inc. All rights reserved. factor IL-3 and the transcription factor SOX7. Skin mast cells also also cells growth mast Skin SOX7. mast-cell factor transcription and the and IL-3 cytokine factor the ADAMTS5, and ADAMTS1 metalloproteases the encoding transcripts of expression increased of expression higher ( subsets cell mast other the expres in their sion to relative genes 18 of expression lower fourfold and ( cells mast other in than cells mast toneal was more receptor), lipoprotein than lower oxidized fourfold in peri and inhibitor) membrane-attack-complex including transcripts, ten of expression ( cells mast other in than cells mast peritoneal in higher Bmp2 three of expression The including transcripts, shown). not other (data in expression populations its cell to mast relative esophagus or trachea the from ( transcript, single a of expression higher ( and subset) this to limited was sion transcripts encoding in than esophagus the protease- included these other mast cells; the from cells mast in of was atfive transcripts least fourfold higher cell (data subsets not shown). The expression relative to in expression their the other mast- a difference in expression of fourfold or more from cells the tongue with had no transcripts cells from the trachea, mast esophagus and of tongue, mast similarity transcriptional the with Consistent populations. mast-cell other experiments. independent populations) other (all two or cells) mast (peritoneal five eosinophils), blood basophils; blood and splenic cells; mast tracheal and tongue (skin, three from pooled are Data signatures. gene basophil and mast-cell shared and distinct the in factors transcription encoding transcripts ( basophils. and cells mast by shared signature the or signature cell-specific than other transcripts all basophils; or cells mast in receptors, neurotransmitter and biosynthesis monoamine in involved products encoding ( populations. non-basophil or non–mast-cell all of that than higher tenfold expression indicates margin) ( ( analyzed populations cell other all in expression the to relative populations basophil and mast-cell all in greater or twofold expression transcript of basis the on derived basophils, ( populations. cell non-basophil all of that than (red) higher tenfold or (blue) higher fivefold expression indicate margin right along symbols ( analyzed populations cell other in expression the to relative populations basophil both in greater or twofold expression transcript of basis the on ( cells. mast and basophils of patterns expression 4 Figure e c r u o s e r  showed enhanced expression of t b a Fig. 6 Fig. Fig. 6 Fig.

-test)); red (in gene symbols along right right along symbols gene (in red -test)); ) Basophil-specific gene signature derived derived signature gene ) Basophil-specific ) Gene signature shared by mast cells and and cells mast by shared signature ) Gene Skin mast cells showed fourfold higher expression of 28 genes genes 28 of expression higher fourfold showed cells mast Skin Hdc (which encodes a bone-morphogenic protein), was fourfold fourfold was protein), bone-morphogenic a encodes (which e d

). No transcript had expression fourfold lower in mast cells cells mast in lower fourfold expression had transcript No ). were included in either the mast- the either in included were ). Tracheal mast cells had fourfold fourfold had cells mast Tracheal ). Distinct and shared transcriptional transcriptional shared and Distinct P <0.05 ( <0.05 c ) Expression of transcripts transcripts of ) Expression t -test)); colors of gene gene of colors -test)); Mrgprb8 Mcpt1 Itgb2 d (which encodes integrin integrin encodes (which (whose expres (whose ) Expression of ) Expression and and CD59a Mrgprb13 P < 0.05 < 0.05 Fig. 6 Fig. Mcpt2 , , which suggested substantially Lipf Fig. 6 Fig. Cd59a g Olr1 -

). In addition to having having to addition In ). , skin mast cells showed showed cells mast , skin

f (which encodes an an encodes (which ). wih noe a encodes (which –1 a

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terms of the (GO) Consortium most ‘enriched’ in in ‘enriched’ most Consortium (GO) Ontology Gene the of the terms Among (GSEA). analysis gene-set-enrichment by populations these compared we expression, gene differential of degree greatest proteins on their untreated counterparts ( face staining of either CD34 or ItgB2 relative to surface staining of these Enzymatically treated peritoneal mast cells showed no decrease in sur ItgB2 expression only on mastwas detected ( cells peritoneal and cells, mast onperitoneal undetectable was and cells mast onskin highest was expression CD59a cells, mast skin for except subsets cell indicated that the adhesion molecule CD34 was expressed on all mast- respectively). protein, 5-lipoxygenase-activating and marker) included cells mast of subsets other in expression their to relative cells mast skin in more or lower fourfold expression with Transcripts peritoneum. the from of expression different gene this in mast from cells the skin and those BB.Sp NK.Sp Because the skin and peritoneal mast-cell populations showed the the showed populations mast-cell and peritoneal skin the Because cytometry flow by analysis data, transcriptional the of support In NKT.Sp Tgd.Sp T8.Sp T4.Sp Treg.Sp 1 aDV Gpr183 Gm9895 Gabbr1 Fbxo9 Far2 Fads1 E2f8 Defb40 Cebpa Ccl Ccl Ccl Cables Bmp4 Bace1 6030469F06Rik 4930503B20Rik 2010002N04Rik 1110067D22Rik 1110032A13Rik Mcpt Itga Grm Gpr56 Gpc Fads2 Dnase2b Mrgpra Igfbp7 Htr1b Cdh Wrn Unc93a Tulp3 Tec Supt3 St6galnac5 Sncai Slc26a8 Slc26a3 Rnf39 Plg Plekhg5 Pig Per Pdzd Pdk3 P2ry1 Otub2 Osbpl5 Nt5c3 Npl Nfil3 Mboat Lama5 L1cam Inf2 Ifitm Id Hist1h2ac Hist1h1d Hgf Gpr9 Jazf Aqp9 Alox8 Adora2 Acss2 1 Alox5 y 9 4 3 p 1 1 1 1 6 4 A 7 8 4 p h 1 NCE ONLINE PUBLIC ONLINE NCE 1 b 6 and and

Cd34 b –1 Alox5ap –1 –1 d c MC.Tr MC.Tr MC.Tr MC.Es MC.Es MC.Es (which encodes a canonical mast-cell mast-cell canonical a encodes (which MC.To MC.To MC.To MC.Sk MC.Sk MC.Sk (which encode 5-lipoxygenase and and 5-lipoxygenase encode (which MC.PC MC.PC MC.PC BA.Bl BA.Bl BA.Bl A

TION BA.Sp BA.Sp BA.Sp Eo.Bl Eo.Bl Eo.Bl z z z -score -score

GN.Sp GN.Sp -score Supplementary Supplementary Fig. 6 GN.Sp 0 0 0 MF.PC MF.PC MF.PC

nature immunology nature DC.Sp DC.Sp DC.Sp B1ab.PC B1ab.PC B1ab.PC BB.Sp BB.Sp BB.Sp NK.Sp NK.Sp NK.Sp NKT.Sp NKT.Sp NKT.Sp Tgd.Sp Tgd.Sp Tgd.Sp T8.Sp T8.Sp T8.Sp T4.Sp T4.Sp T4.Sp Treg.Sp Treg.Sp Treg.Sp 1 1 Fig. 6 1 Nfil3 Supt3h Cebpa Sncaip Gata2 Zfp9 Smarca1 Mitf Creb3l1 Htr1b Hrh4 Slc6a4 Slc18a2 Maob Hdc Ddc Tph1 Slc7a5 Slc3a2 Syde Slc41a3 Pafah2 Mt Mcpt Lat Itga2b Hrh4 Gstm Gstm Gnb4 Gna14 Far2 Fam110c Slc6a4 Slc24a3 Slc18a2 Ms4a2 Gata2 Fcer1a Cyp11a1 Cpa Cd200r3 Ad 2 k 2 ). 3 8 2 3 1 h ). ). - © 2016 Nature America, Inc. All rights reserved. suggested by transcript data). Gray shaded curves, isotype-matched control antibody. Data are pooled from three (skin, tongue and tracheal mast cells), cells), mast tracheal and tongue (skin, three from pooled are Data antibody. control isotype-matched curves, shaded Gray data). transcript by suggested expression gene differential confirm (to ItgB2 and CD59a CD34, of lines) (black expression the assessing margin) (left tissues various from cells mast ( esophagus the from cells mast in lower or higher ( right). (bottom axis horizontal or left) (top axis vertical the along genes expressed differentially total indicate plots in numbers plotted; population other the in than (aqua) cells mast skin or blue) (dark cells mast peritoneal (pink), cells mast esophageal (red), cells mast tracheal green), (light cells mast tongue in expression higher or twofold and 120 than greater values expression with transcripts 6 Figure ago, their contribution to immunological defense and disease has been chordates uro as back far as found are cells mast-cell-like Heparin-containing DISCUSSION inflammation. of absence the in populations both in expression Ki67 notable and population peritoneal-mast-cell the in skin mast cells ( peritoneal mast cells were positive for Ki67, compared with only 4% of in skin mast cells, which also expressed Ki67 ( mast than cells was the G-0 greater in Ki67 phase. staining peritoneal during a but protein mitosis nuclear present during rapidly degraded cellular undergoing be turnover. Thus, we expression ofevaluated peritoneal-mast-cell might Ki67, cells mast peritoneal that suggested ( phase’ ‘M and ‘Mitosis’ were cells mast peritoneal basophils) and human cells mast (human donors independent three or basophils) and mouse cells mast (mouse experiments independent three ( blue) (44 transcripts; signature basophil mouse of the for expression do not enrichment show basophils blood human ( green) (17 transcripts; and by basophils cells mast shared and the signature ( red) (82 transcripts, signature of mast-cell the mouse for expression enrichment significant show cells mast Human expression. in difference for a cutoff twofold indicate lines blue axis); (vertical basophils blood mouse versus or cells mast axis) in skin mouse (horizontal basophils blood human versus cells mast in skin human (human), sets data and the FANTOM set data (mouse) consortium consortium the ImmGen in both represented transcripts of all 10,773 Expression cells. mast human 5 Figure nature immunology nature ( experiments independent cells) mast (esophageal two or cells) mast (peritoneal five c a Expression Expression tongue mast cell tracheal mast cell 10 10 10 10 10 10 10 10 P 1 2 3 4 1 2 3 4

= 0.0028, hypergeometric cumulative distribution upper tail); tail); upper distribution cumulative hypergeometric = 0.0028, Enrichment for expression of the mouse mast-cell signature in signature of mast-cell the mouse for expression Enrichment Tissue-specific mast-cell gene expression. ( expression. gene mast-cell Tissue-specific 29 65 3 10 10 , , and although mast cells were first identified over 100 years 3 peritoneal mastcell 1 1 tongue mastcel Expression Expression 10 10 Fig. 7 2 2 10 10 3 3 c ), ), which indicated a much higher rate of mitosis l 10 10

45 31 4 4 9 aDV

Expression Expression b A skin mast cell esophageal mast cell NCE ONLINE PUBLIC ONLINE NCE 10 10 10 10 10 10 10 10 1 2 3 4 1 2 3 4 73 551 10 10 peritoneal mastcell 1 1 tongue mastcel Expression Expression 10 10 2 2 P Fig. Fig. 7 10 10 = 0.33). Data are = Data from 0.33). d ), trachea ( trachea ), 3 3 a l – P 10 10 b 406 c = 5.5 × 10 49 A 4 ). ). In total, 16% of ) Comparison of gene expression in various mast-cell subsets (axes); dot colors indicate indicate colors dot (axes); subsets mast-cell various in expression gene of ) Comparison 4 TION Fig. 7 Fig. d –1 –1 –1 MC.Tr f MC.Tr e MC.Tr e

), peritoneum ( peritoneum ), MC.Es MC.Es MC.Es a z z MC.To z

MC.To -score MC.To -score -score ), which which ), 0 0 MC.Sk 0 MC.Sk MC.Sk 4 −16 MC.PC MC.PC MC.PC 1 . Sprr2a1 Mcpt Mcpt Cyp2c55 Car Olr1 Nr4a2 Il1 Gch1 Emp1 Ctla2a Cd59a Car C3ar Itgb2 Cyp51 Olr1 Atp6v0d Lip ) a 1 8 f 1 1 1 1 2 1 - 2 g –1 MC.Tr transcriptionally distinct cell type, as they clustered independently independently clustered they as type, cell distinct transcriptionally most the as cells mast Weidentified populations. cell myeloid and lymphoid other 14 with comparison in cells mast mouse of analysis poorly defined. Here we have provided a comprehensive transcriptional f

) or skin ( skin ) or MC.Es

MC.To z Expression (log fold)

-score 2

MC.Sk 0 mouse mast cells vs mouse basophils +10 +11 +12 –12 –11 –10

MC.PC +1 +2 +3 +4 +5 +6 +7 +8 +9 –9 –8 –7 –6 –5 –4 –3 –2 –1 Tbc1d Stat4 Slc6a12 Prss34 Pla2g Pecam P2ry14 Nphs2 LOC641050 Itk Il18r Il12rb2 Gm8979 Erv Cd34 BC005685 Sox7 Sema6a Rpp2 Mrgprb8 Mrgprb1 Il3 Icos Gpm6 Gfra Fermt Fam115c Etv1 Emp2 Efcab Cyp2j Ctnnal Cnr Clec12b Clec10a Cdh9 Cd59a Car5b 9330182L06Ri 1700025G04Ri Alox5ap Alox5 App Aff2 Adamts5 Adamts1 a 0 – 3 1 –12 1 2 g 1 5 2 6 3 7 b 8 1 ) or are representative of three independent experiments ( experiments independent three of representative are ) or g 1 –11 ) than in any other mast cell population. ( population. cell mast other any in ) than 3

–10 Ccl3 k k –9 h Skin Peritoneum Tongue Esophagus Trachea –8

–7 Ccl4 d

– –6 g human mastcellsvsbasophils ) Transcripts with expression at least fourfold fourfold least at expression with ) Transcripts Events (% of max) –5 100 100 100 100 100

20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 –4 0 0 0 0 0 CD34 0 0 0 0 0 –3 Expression (log 10 10 10 10 10 2 2 2 2 2 –2 10 10 10 10 10 3 3 3 3 3 –1 10 10 10 10 10 4 4 4 4 4 10 10 10 10 10 0 5 5 5 5 5 +1 100 100 100 100 100 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 0 0 0 0 0 2 +2 fold) CD59a 0 0 0 0 0 1 1 10 1 1 +3 0 0 0 0 2 2 2 2 2 10 10 10 10

10 +4 3 3 3 3 e c r u o s e r 3 Gnai1 h 10 10 10 10 10 +5 ) Flow cytometry of cytometry ) Flow 4 4 4 4 4 10 10 10 10 10 +6 5 5 5 5 5

100 100 100 100 100 +7 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 0 0 0 0 0

ItgB2 +8 0 Maob 0 0 0 0 1 1 1 10 1 0 0 0 0 +9 2 2 2 2 2 10 10 10 10 10 +10 3 3 3 3 3 h 10 10 10 10 10 +11 ). 4 4 4 4 4 10 10 10 10 10 +12 5 5 5 5 5 

© 2016 Nature America, Inc. All rights reserved. tors. Thus, our transcriptional analysis of mast cells and basophils basophils and cells mast of analysis transcriptional our Thus, tors. and expressed different combinations of soluble mediators and recep histamine-biosynthetic lacked basophils the enzymes, diversity of and proteases seen in mast cells receptors activating several encoding While mouse mast cells and basophils shared expression of transcripts cells, as is the closer relationship between basophils and eosinophils observed in this study is similar to that previously observed for human ligands. native as-yet-unidentified and agents pharmacological both to response in cells mast of the activation mediating innate in role critical a have might family this of members diverse in reactions humans that pseudoallergic induce IgE-independent a agents and therapeutic including 48/80 compounds, basic venom, other of wasp array to response in degranulation line cell cells mast blood-derived human-cord- in 48/80 compound mast-cell-activating to classical the response in degranulation cell mast mediate to shown MRGPRX2 been system. has immune innate the in role important an have and analysis showing expression of cells and six were expressed in the other mast-cell populations. Further neurons broader than previously appreciated. Originally discovered in sensory family. Mrgpr the of including cells, proteases, mast of encoding those as and such genes, mast-cell well-known included genes conserved highly These functionality. mast-cell core a retain to pressures evolutionary of suggestive signature, mast-cell mast Human for of expression the mouse enrichment showed substantial also cells mediators. of repertoire unique a generate an to indicate ability findings these Together chemokines. and cytokines of D prostaglandin as such eicosanoids of cells mast expressed transcripts Furthermore, encoding productssulfate. that heparin allow the and acute generationserotonin histamine, including of mediators, a of range preformed broad generation other sion of genes encoding products in metabolic pathways required for the stantial contributor. Mast cells also showed enrichment for the expres were a proteases sub serine encoding those among which 128 genes, a provide function. sentinel their should understanding for framework this cues; environmental to responding and ing sens in involved proteins encoding genes expressed cells mast that Wefound transcriptome. mast-cell the of regulation recognized tissue-specific further and a signature described We transcriptional mast-cell basophils. shared including populations, other all from of a total with experiments independent of a total with experiments ( experiments QQ of representative are Data * ( mean the indicate lines horizontal small mouse; individual Ki67 ( antibody. control isotype-matched curve, shaded Gray ( (nominal similarly treated cells mast skin in abundance their to relative enzymes digestion with treated cells mast peritoneal-cavity in enrichment significant ( 7 Figure e c r u o s e r  regulating in roles independent have cells these that suggested b a P

) GSEA identification of GO Terms ‘Mitosis’ and ‘M phase’ as showing showing as phase’ ‘M and Terms GO of ‘Mitosis’ identification ) GSEA ) Intracellular Ki67 expression in peritoneal and skin mast cells (key). (key). cells mast skin and peritoneal in expression Ki67 ) Intracellular = 0.0000062 (two-tailed unpaired unpaired (two-tailed = 0.0000062 The low homology between The mouse low mast between cells homology and mouse basophils was cells mast in expressed members Mrgpr-family of array The Mast cells from various tissues shared a signature transcriptional of Hpgds Mrgpra2b + mast cells in peritoneum and skin. Each symbol represents an represents symbol Each skin. and peritoneum in cells mast

4 4 Transcriptional analysis indicates peritoneal-mast-cell turnover. turnover. peritoneal-mast-cell indicates analysis Transcriptional 4 2 . The mouse homolog of MRGPRX2, Mrgprb2, mediates mediates Mrgprb2, MRGPRX2, of homolog mouse The . , eight members of this family were expressed in skin mast mast skin in expressed were family of this members , eight , , as well as several that are poorly understood in the context in neutrophils suggested that the Mrgpr family might might family Mrgpr the that suggested neutrophils in P value, <0.001; false-discovery rate Q value, <0.005). <0.005). Q value, rate false-discovery <0.001; value, Maob 4 n 3 = 9 mice ( = 9 mice and the transformed human LAD2 mast- LAD2 human transformed the and , , Mrgpra6 Gnai1 t -test with Welch’s correction). Welch’s correction). with -test b n and genes encoding members members encoding genes and ) or are pooled from three three from pooled are ) or = 9 mice ( = 9 mice in basophils and of a 2 ) or three independent independent three ) or and the rapid production production rapid the and c ). c ) Frequency of ) Frequency ± s.d.). s.d.). Mrgpra2a 3 5 . . Thus,

4 1 - - - - .

different tissue compartments. The basophil signature included included signature basophil The compartments. tissue in roles different similar serving than rather defense, host and homeostasis Methods and any associated references are available in the the in v available are references associated any and Methods M disease. and defense host homeostasis, in cells ancient of the role of evolutionarily for definition these better a provide framework findings These inflammation. of tissue absence for capacity varying and subsets mast-cell connective-tissue distinct three revealed heterogeneity mast-cell of is product not function Analysis whose gene yet families understood. novel several and included of of a range mediators, broad generation and factors involved in pathways, biosynthetic which would allow the ment for the expression of genes encoding a diverse array of proteases core showed Their enrich signature level. at tinct the transcriptional compartment. this of nance mainte and renewal the in role a have might proliferation local that suggested which cells, mast skin in levels low at detectable also was and maturation cell staining Notably, Ki67 cells. neighboring from both signaling differential reflect might compartments mast-cell other and cells mast peritoneal between differences transcriptional profound the Thus, Ki67. for positively stained cells of mast peritoneal fraction substantial a cellu that finding with the to led which associated turnover, lar pathways transcriptional for ‘enrichment’ showed they that We observed wall. tissue gut the serosal the in line instead but embedded not are cells mast peritoneal populations, fibroblasts neighboring by cells mast of regulation demonstrating studies with and tissue peripheral in tion able tissue-specific gene expression, consistent with mast-cell matura in type species. these cell this of divergence drove pressures might evolutionary signature reflect basophil the in genes other of expression their in the poor conservation between human basophils and mouse basophils However, recruitment. cellular directing in chemokines encoded the role in expression fora human suggested mast conserved which cells, transcripts, these of two of expression Ccl3 ersion of the pape the of ersion b a et

In conclusion, we found that mast cells were extraordinarily dis extraordinarily were cells mast that found we conclusion, In consider revealed populations mast-cell of analysis Comparative Enrichment score Events (% of max) 0. 0. 0. 0. 0. 0. , , 1 2 3 4 5 6 0 10 h Ccl4 20 40 60 80 peritoneal MC 0 0 ods Enriched in Ki67 and and aDV 0 10 2 A Ccl9 NCE ONLINE PUBLIC ONLINE NCE Mitosi 10 r . . Human basophils showed enrichment for the the for enrichment showed basophils Human . 3 s Skin Peritoneal 10 4 Enriched in skin MC 10 5

+ c A Ki67 cells (%) Enrichment score TION 0. 0. 0. 0. 0. 0. 10 15 20 25 Ccl4 0 5 6 1 2 3 4 5 0 21 peritoneal MC Enriched in ,

4 in situ in and and 5 . Unlike other mast-cell mast-cell other Unlike . nature immunology nature Ccl4 proliferation in the the in proliferation M , relative to their their to relative , phase * Skin Peritoneal Enriched in skin MC online online ------

© 2016 Nature America, Inc. All rights reserved. Bingfei Yu Daniel F Dwyer The complete list of authors is as follows: 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm R The authors declare no competing interests.financial andcollection assisted in design. experimental design; and experimental The ImmGen Project Consortium contributed to data analyzed the data; N.A.B. and K.F.A. wrote the manuscript the and supervised D.F.D. wrote the manuscript, conceived of and conducted experiments, and YoungJudy Kaye and Steven Award the Innovators (N.A.B.). D.F.D.)to AI007306 and T32 and N.A.B.; to AI095219 N.A.B.; to U19AI095219 and HL120952 R01 Consortium; ImmGen the to (R24AI072073 Health of Institutes National US by the Supported cells. of isolation the with assistance for Chicoine A. and assistance; technical for Rothamel, K. and Rhodes A. especially team, ImmGen core T. the and Shay,Benoist C. discussions; for especially Consortium, ImmGen the of members Weother the thank online version of the pape Note: Any Supplementary Information and Source Data files are available in the codes. Accession nature immunology nature COMPETING FINANCIAL INTERESTS COMPETING AUTH A eprints and permissions information is available online at at online available is information permissions and eprints ckn

Gersch, C. Xing, W., Austen, K.F., Gurish, M.F. & Jones, T.G. Protease phenotype of constitutive T.G. Jones, D.S. Friend, Crapper,Schrader,tissues & normal R.M. in J.W. precursors cell mast of Frequency of specialization functional and origin K.F.Developmental Austen, M.F.& Gurish, G. Younan, J.P. Abonia, pathogens. to immunity cell-orchestrated Mast A.L. John, St. & S.N. Abraham, Rodewald, & M. Wunderlin, T.B., Feyerabend, S.M., Schlenner, L.A., Schneider, M. Metz, E. Razin, Y.A.Mekori, & D. cells. Baram, Mast D.D., Metcalfe, G.W.Wong, basophils. and M.C. cells Cavalcante, mast of roles pathological and Protective D. Voehringer, The Consortium. Project Genome Immunological M.W. Painter, & T.S. Heng, Cell Biol. Cell the by regulated tissue. is mice in cells mast mucosal induced of and tissue connective cells. NKT CD1d-restricted and (2009). 5251–5260 IL-9 on depend numbers phenotype. and ultrastructure with infected mice cells. mast and precursors cell P of frequency the in increases parallel induces antigen assay: vitro in an by determined subsets. cell mast (2010). 5. and 4 proteases cell mast mouse of activities the muscle. skeletal mouse Immunol. Rev. Nat. sarafotoxin. venom snake and H.R. Molecular mechanism of mast cell mediated innate defense against endothelin Science cells. mast mouse proteoglycan, (1997). 451 defense? in role ( Immunol. Rev. Nat. cells. immune in expression gene Immunol. Nat. of networks Project: Genome Immunological Tunicata owle O , 314–318 (2014). 314–318 , R R C Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc.

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Mast cells can enhance resistance to snake and honeybee venoms. honeybee and snake to resistance enhance can cells Mast me l -, and histamine from cultured bone marrow-derived bone cultured from histamine and -hexosaminidase, 9 . Mast cells and macrophages in normal C57/BL/6 mice. g-eitd ees o luoree 4 codotn uft E sulfate chondroitin C4, leukotriene of release IgE-mediated , 1091–1094 (2008). 1091–1094 , J. Biol. Chem. Biol. J. The inflammatory response after an epidermal burn depends on depends burn epidermal an after response inflammatory The Ancient origin of mast cells. mast of origin Ancient nie-nue icess n umnr ms cl progenitor cell mast pulmonary in increases Antigen-induced GEO: microarray data, data, microarray GEO: t al. et at el ta rsd a dfeet oain i te euu of jejunum the in locations different at reside that cells Mast at el rtae mdae icei-eefso ijr of injury ischemia-reperfusion mediates 5 protease cell Mast nt Immunity J. Exp. Med. Exp. J.

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© 2016 Nature America, Inc. All rights reserved. Missouri, Missouri, USA. Medical Massachusetts School, Worcester, USA. Massachusetts, USA. Pennsylvania, Philadelphia, USA. Massachusetts, USA Massachusetts, and Department of Cancer Immunology, Genentech, San Francisco, California, USA. California, USA. Harvard Medical School, Boston, USA. Massachusetts, California, USA. 5 Keke Fairfax Yuesheng Brian Brown YoshidaHideyuki e c r u o s e r 1 Division Division of Biological Sciences, University of California San Diego, La Jolla, California, USA. 0

L 18 10 7 i Department Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA. Broad Broad Institute of MIT and Harvard, Cambridge, USA. Massachusetts, 14 17 Icahn Icahn Medical Institute, Mount Sinai Hospital, New York, New York, USA. 10 , , , , Gwendalyn J Randolph , , 13 S M usan usan A Skirball Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA. iriam iriam 8 , , Diane 15 S M Department Department of Life Sciences, Ben-Gurion University of the Negev, Be’er Sheva, Israel. hinton erad M athis 10 14 , , Viviana Cremasco , , Richard R Hardy 8 , , Christophe Benoist 9 Department Department of Microbiology & Immunology, University of California San Francisco, San Francisco, 17 , , M ichelle ichelle 17 Department Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, L 14 11 Robinette , , Tal , , S 8 , , Tsukasa Nabekura hannon Turley S hay 8 Division Division of Immunology, Department of Microbiology & Immunobiology, 17 15 11 6 Computer Computer Science Department, Stanford University, Stanford, , , Anja Fuchs Dana-Farber Dana-Farber Cancer Institute and Harvard Medical School, Boston, , , Yilin Qi aDV A 11 NCE ONLINE PUBLIC ONLINE NCE 12 , , Paul Department Department of Medicine, Boston University, Boston, 16 9 , , Katelyn , , Viola 18 & M onach M 16 L arco Colonna Department Department of Pathology, University of am S ylvia 12 A 9 TION , , , , M L 16 ewis ewis ichael ichael

14 , , Joonsoo Kang nature immunology nature Fox Fox Chase Cancer Center, L 17

L L anier Dustin 9 , 13 16 , , © 2016 Nature America, Inc. All rights reserved. protocols targeting a minimum of 10,000 cells per microarray sample. microarray per cells of 10,000 a minimum targeting protocols standard ImmGen on based determined were sizes Sample used. animals of number total minimize to mouse each from harvested were tissues multiple ( ( blood from the were from collected 8 Basophils pooled cells mice). licate was 15,000 ( trachea and mice), 24 from pooled cells 10,000 was replicate ( esophagus mice), 10 from pooled cells 10,000 was replicate each cavity ( the skin ( replicate. microarray per animals and Cells basophils. of identification the for blue toluidine with stained were cytospins and cells, mast of fication identi the for reactivity section esterase Cut chloracetate for stained were overnight. cytospins and dried and slides glass charged onto spun were cells sorted evaluation, For cytospin in glycolmethacrylate. dehyde and embedded paraformal 4% in overnight fixed were sections tissue tissues, peripheral in Cytospins and microscopy. eBioscience. from obtained were staining Ki67 for used set buffer staining FoxP3 and mAbs, control isotype-matched were 1:100), (SolA15, anti-Ki67 (mAbs) 1:250), (23G3 Anti-IgE BioLegend. from obtained antibodies monoclonal control isotype-matched and 1:250), (M18/2, anti-ItgB2 1:250), (mCD59.3, anti-CD59b 1:250), (MEC14.7, CD34 anti- 1:250), (PK136, anti-NK1.1 1:250), (DX5, anti-CD49b 1:250), (53-6.7, anti-CD8 1:250), (GK1.5, anti-CD4 1:250), (6D5, anti-CD19 1:250), (N418, anti-CD11c 1:250), (M1/70, anti-CD11b 1:250), (30-F11, anti-CD45 1:250), anti-Fc used: were concentration) with FlowJo (Treestar).software (clone, The antibodies monoclonal following on a FACSCantoBD analyzed and II acquired was data analysis, intracellular and marker surface For sorter. cell Fusion FACSAria BD a using Core Flow D49b D117 CD45 as identified were cells Mast (Sigma). gradient Percol 44%/67% a using depleted were erythrocytes and puncture cardiac through obtained B220 was Blood against (Invitrogen). Thy1.2 directed and beads Dynal using depleted were lymphocytes lysis, Following ACK (Sigma). using lysis buffer by followed erythrocyte the spleen 500 RPM. Spleen suspensions were obtained through mechanical disruption of collagenase IV and 20 U/ml 600 with min 30 for incubated and blades scalpel two between minced finely was tissue remaining epidermis, the away pulling After epidermis. the 2.5 with HBSS in min 20 for bated tocol pro described previously a of modifications using obtained were digests Ear and (Gibco) dispase 0.1% 20 (Worthington), IV collagenase U/ml 600 with finely for by 30 at min 37 and °C incubating blades two scalpel between trachea tissue mincing and esophagus, tongue, from obtained Single-cell were TA. EDcell suspensions mM 1 containing HBSS ml 7 with the of cavity lavage peritoneal by obtained were suspensions cell Peritoneal below. noted as time digestion for increased modifications with below) (identified antibodies m ( protocol operations standard ImmGen and sorting. isolation Cell DFCI. of Committee and with approval review by guidelines Care the and Animal institutional Use light/ h with 12 accordance in a was studies these under for mice all of (DFCI) use The cycle. dark Institute h 12 Cancer Farber Dana the at facilities pathogen-free specific in cage) per mice (four housed were Mice Laboratory. Jackson the from mice C57BL/6J 10-week-old to 6- male from obtained was histology for used tissue and mice C57BL/6J 6-week-old male from obtained Mice. MET ONLINE doi: n m = 3, each replicates was 25,000 cells pooled from 4 mice). Whenever possible, µ 10.1038/ni.3445 G g/ml g/ml DNAse 1 (Roche) in RPMI supplemented with 10% FBS at 500 RPM. 4 + + e All cells used for transcriptional and flow cytometric analyses were were analyses cytometric flow and transcriptional for used cells All 6 . Basophils were identified as CD3 as identified were Basophils . . Cells were sorted at the Brigham and Women’s and Brigham the at Immunology Human sorted were Cells . n . Briefly, dorsal and ventral halves of the ear were separated and incu and separated were ear the of halves ventral and dorsal Briefly, . n n % = 5, each replicate was 30,000 cells pooled from 4 mice), tongue ( = 3, each replicate was 10,000 cells pooled from 5 mice) and spleen spleen and mice) 5 from pooled cells 10,000 was replicate each 3, = n 2 = 3, from each replicate was 8 cells pooled 25,000 peritoneal mice), 0 C e l l % H 2 ODS 0 p µ r e g/ml g/ml DNAse 1 in RPMI supplemented with 10% FBS at p % 2 Cells were purified according to the standardized to standardized the according were purified Cells For evaluation of histochemical mature mast cells 0 a + n CD11b d % ε 2 R1 0 µ s − o g/ml dispase at 300 RPM to separate separate to RPM 300 at dispase g/ml α CD11c r (MAR-1, 1:250), anti-CD117 (2B8, (2B8, anti-CD117 1:250), (MAR-1, h t i t n − t p CD19 g : % / Mast cells were collected from from collected were cells Mast / − 2 w CD19 0 w S − O NK1.1 w P . i . m p − CD4 d m f − ) ) using the indicated g CD117 e − n CD8 . o n r = 3, each rep = each 3, g / − − P n Fc Fc r = 2, each each 2, = o ε ε t R1 R1 o c n o α α = 3, l + + s / C C - - - - - I

to tongue mast cells also showed increased expression in tracheal mast cells cells mast tracheal in expression increased showed also cells compared mast tongue to cells B splenic in expression increased 16-fold than greater with transcripts All cells. B splenic and cells mast esophagus between expression in changes fold to cells mast esophagus and cells mast tracheal between expres sion in changes fold comparing by identified were cells mast tracheal in noglobulin genes, suggesting B cell contamination. Contaminating B cell genes mast were cells found B for to immu including cell genes, be several enriched Tracheal Institute). (Broad software of GenePattern on module Studio Multiplot the using based characterized was collapsed expression gene were differential and values probes median multiple through identified Transcripts ImmGen%20 ( protocols consortium’s the standard to and quality-control documentation was also conducted by ImmGen according Data generation levels. transcript on impact detected minimal demonstrating ( significant statistically twofold a by reduced were assayed ( heparinase with treated RNA cell mast peritoneal of Comparison extraction. TRIzol of round second a to subjected then and temperature, room at h 2 for (Sigma) 5 Tris in buffer containing incubated 50U of was RNAsin RNA plus (Promega) and extraction, 0.02 U of chloroform heparinase of round initial an ing described as previously RNA heparinase with was treated 20with%20Trizo protocols ( to 1.0 ST Mouse consortium’sthe Gene array by according ImmGen standard lected directly into TRIzol. RNA was amplified and hybridized to the Affymetrix and analysis Microarray data evaluation. in the individual and shared mast-cell and basophil signatures that also also that signatures basophil and mast-cell shared transcripts and identifying individual by the determined in were factors transcription specific with system classification PANTHER the software DAVID using determined were pathways enriched the signatures, calculating After in than and any cell non-basophil. non–mast basophil and cell mast both in higher fold two least at expressed transcripts all by determining was calculated signature and basophil the mast-cell shared and similarly, calculated was signature basophil The treatment. dispase and non-digested peritoneal mast cells to exclude any genes induced by collagenase student’s by expression there which in all was no statistically significant difference were between mast-cell and non–mast-cell as in excluded, units were subsets relative mast-cell 120 two below than more expressed transcripts All population, basophils. non–mast-cell including any in than populations mast-cell all in higher twofold least at expressed were signature mast-cell the in Transcripts values. median on based collapsed were population cell each for replicates Multiple analyzed. populations cell all to comparison in generated was signature cell signatures. transcriptional and basophil of mast-cell Derivation expression. marker surface cell for stained mast treatment, cells peritoneal were either isolated for oranalysis microarray DNAse 1 in (Roche) RPMI supplemented with 10% FBS. Following enzymatic 20 and (Gibco) dispase 0.1% (Worthington), IV collagenase U/ml 600 with °C 37 at min 30 for incubated were EDTA mM 1 containing HBSS ml 7 with cavity peritoneal the lavaging by obtained suspensions cell Peritoneal Controlling for the effects of collagenase treatment on peritoneal mast cells. sets. gene (www.geneontology.org) Consortium Ontology Institute) (Broad program software Analysis Enrichment Set Gene the using compared further were transcriptomes peritoneal-mast-cell and enzyme-treated skin The populations. cell analyzed all across transcripts on variable top the most 15% based pro School) Medical Harvard Davis, PCA (S. gram PopulationDistances the using calculated components principal component Principal analysis was using visualized MatLab software Gene-E. (MathWorks) using using constructed also were maps heat transcript all and using Spearman’s was tering calculated matrix distance Euclidean correlation. Pearson’susing transcripts most variable clus and population cell correlation ( com pairwise Gene-E using was conducted all for transcripts clustering Hierarchical from parisons. excluded were and cells mast tongue to compared http://broadi https://www. n nstitute.org/cancer/ = 3) or control showed that 4 transcripts among the 21,775 21,775 the among transcripts 4 that showed control or 3) = QC%20Documentation_A l.pd f ) ) with Tomodification. improve microarray success rate, immgen.org/Protocols t -test. The mast-cell signature was calculated using using calculated was signature mast-cell The -test. software/GENE- ht Samples were twice sorted and col tps://www.immgen.org P LL-DataGeneration_06 < 0.05. Mast-cell- and basophil- and Mast-cell- 0.05. < /Total%20RNA%20Extra nature immunology nature E ) based on the top 15% 15% top the on based ) 47 , 4 P 8 49 . . Briefly, follow < 0.05) degree, degree, 0.05) < 51 , 5 0 , 5 using Gene Gene using /Protocols/ 2 The mast- based on on based 12.pd ction% µ g/ml g/ml µ f m ). ). ------

© 2016 Nature America, Inc. All rights reserved. t unpaired a two-tailed with (GraphPad) 6.0 Prism using evaluated were levels Ki67 intracellular in Differences (Mathworks). Matlab in tail upper tribution dis cumulative hypergeometric the using evaluated was signatures basophil and mast-cell mouse the for basophils and cells mast human of Enrichment DAVID. in test exact Fisher’s modified a using determined was enrichment pathway PANTHER of Significance statistically. predetermined not was size Statistics. highlighted. then were signatures combined and basophil cell, mast mouse the in found Genes datapoint. each in which numerous transcript levels had a set, value of zero, data a value of 1 was added to sequencing CAGE the in comparisons change fold for Toallow plot. change fold vs change fold a on visualized were sequencing CAGE via cells human in and array 1.0 Mouse Affymetrix the both in identified scripts of Comparison human and mouse mast cells and basophils. gsc.riken.jp/TFdb ( database factor transcription institute Riken the in appeared nature immunology nature -test -test with Welch’s that after correction determining the samples represented a There was no randomization, blinding, or exclusion of data. Sample / ). All 10,773 tran htt p://genome. - - 52. 51. 50. 49. 48. 47. 46. P D’Agostinothe using test. distribution gausian normality & omnibus Pearson values of < 0.05 were considered statistically significant. statistically considered were 0.05 < of values

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