© 2012 Nature America, Inc. All rights reserved. authors and affiliations appears at the end of this paper. Correspondence7 should be addressed to S.J.T.Massachusetts, ([email protected]). USA. Rheumatology, Immunology, and Allergy, The Massachusetts,Immune Disease USA.Institute and Program in Cellular and Molecular Medicine,1 Children’s Hospital, Harvard Medical School, Boston, venules endothelial high through nodes lymph enter egress (DCs) and soluble antigens into lymph nodes erance trating immune responses, whereas FRCs and LECs also promote tol (DNCs) cells double-negative (LECs), gp38 gp38 (FRCs), cells reticular tic gp38 subsets: LNSC following the distinguishes tein podoplanin (gp38) and the adhesion molecule CD31 (PECAM-1) and study to difficult are ­constitute ~1% of lymph node cellularity. Expression of they the glycopro yet system, immune the of tioning conditions. matory and inflam steady-state under (LNSCs) cells stromal of node lymph the we transcriptomes pipelines analyzed have this collaboration, meticulously analysis and rigor methodology shared, controlled involves ously generation data The mouse. immune the the of in system networks gene-regulatory and gene-expression of biolo database that aims gists to a accessible build publicly comprehensive, computational and immunologists of multicenter a venture is collaborative Project (ImmGen) Genome Immunological The of characteristics LNSC subsets. as pericytes myofibroblastic that expressed integrin CD31 (gp38)-negative in the acute-phase response and the and machinery.antigen-processing antigen-presentation Poorly studied podoplanin fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic receptors across and hematopoietic stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate elucidated. Here, comparative analyses transcriptomic of mouse LNSC subsets demonstrated the expression of important Lymph node stromal cells (LNSCs) closely regulate immunity and yet self-tolerance, key aspects of their biology remain poorly Genome Project Consortium Michael B Brenner Santiago F Gonzalez Malhotra Deepali resting lymph nodes defines immunological hallmarks Transcriptional profiling of stroma from inflamed and nature immunology nature Received 15 August 2011; accepted 14 February 2012; published online 1 April 2012; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. LNSCs are nonhematopoietic cells crucial for the normal func normal the for crucial cells nonhematopoietic are LNSCs 8 3– , whereas BECs form vessels that allow naive lymphocytes to to lymphocytes naive allow that vessels form BECs whereas , 7 . LECs facilitate the entry of antigen-bearing dendritic cells cells dendritic of antigen-bearing entry the . facilitate LECs − CD31 5 3 Brigham and Women’s Hospital, Boston, Massachusetts, USA. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA. + blood endothelial cells (BECs) and gp38 1 , 5 2

− , , 6 LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified 8 4 aDV , , Michael C Carroll , , Anne L Fletcher , , Kutlu G Elpek 2 A . LNSCs have distinct roles in orches in roles distinct have LNSCs . NCE ONLINE PUBLIC ONLINE NCE + CD31 9 + lymphatic endothelial cells cells endothelial lymphatic 3 and control 1 , Sook , KyungSook Chang 1 , 8 + 4 , , Jillian Astarita CD31 , , David J Mooney A TION 1 3 a . As part of of part As .

. Inside the the Inside . − lymphocyte

7 fibroblas . . Together our data describe comprehensively the transcriptional − CD31 − ------

6 Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. 1 , consist of a generic myofibroblastic phenotype with expression of of expression with phenotype myofibroblastic generic a of consist to understood essentially is it present, at limited; is FRCs of identity outcomes clinical with worsened are or lesions associated cancerous autoinflammatory in identified cells Moreover, FRC-like life. postnatal in regulated are and interact BECs and LECs FRCs, how about known is little tively rela and of interactions, cell immune orchestration responses DC–T tolerance peripheral of maintenance the for or responses immune of phases early during antigens of source important an be may lymph conduit-borne that suggests which antigens, soluble tissue-derived process can network DCs and cells T attract immune response adaptive the promotes which nodes, lymph to leukocytes attracts chemokines with venules endothelial high of decoration the venules endothelial high to sinus subcapsular the from kilodaltons) (<70 factors small other and chemokines duct con rapidly that microchannels of system conduit complex a forms migrate and interact work of used fibers by and lymphocytes DCs as a on scaffold which to net reticular a and specialized regulate construct FRCs lymph node, 2 5 3 , , Veronika Lukacs-Kornek , , Konstantin Knoblich , , Shannon J Turley Despite their maintenance of naive lymphocytes, regulation of of regulation lymphocytes, naive of maintenance their Despite doi:10.1038/ni.226 1 1 . FRCs also secrete survival factors and chemokines that that chemokines and factors survival secrete also FRCs . 8 These authors contributed equally to this work. 2 Division of Medical Sciences, Harvard Medical School, Boston, 13 , 1 3 2 9 4 . . Ensheathed by FRCs, the reticular network also , . Nonetheless, knowledge of the transcriptional of . the knowledge transcriptional Nonetheless, 1 2 2 . , 3 1 . Indeed, DCs in contact with the reticular reticular the with contact in DCs Indeed, . , 4 7 Department of Pediatrics and Division of & the Immunological 1 , , Martin E Hemler 1 , , Prakriti Tayalia 9 , 1 e c r u o s e r 0 . During infection, infection, During . 9 Complete list of 1 ,

3

,  - - - © 2012 Nature America, Inc. All rights reserved. Euclidean Euclidean distance; and Pearson’s correlation by (measured concordance population of plots matrix on evident also were relationships These distinct relationship. transcriptionally a demonstrated which prin component, third cipal the along separated clearly DNCs and FRCs However, DNCs and which FRCs were closer to positioned each other variability, than to components, subset. either endothelial observed the principal of two 83.5% for first accounted the Along tissues. these tightly, genes of conservation which between reflected subset-specific clustered (MLNs) nodes lymph and mesenteric (SLNs) nodes lymph total of ( proportion variability a for accounts princi which of into each data components, pal expression multidimensional simplifies transfor mation mathematical This expression. in difference greatest the with probes of 15% the on based (PCA), analysis component cipal ( markers hematopoietic common of expression lacked samples and markers, expected the of expression demonstrated data the of Inspection Methods). (Online populations within variability low showed and type cell one least at by expressed were they if only comparisons the in probes included normalization to indicate positive expression (at 95% confidence) and 0.205). We assumed the general ImmGen Project threshold of 120 after quality (median inter-replicate coefficient of variation values of 0.086– passed the quality control of the ImmGen Project, with good replicate microarrays ( Project ImmGen the of 1a Fig. ( mice C57BL/6 from nodes of lymph fractions stroma-enriched from purity LECs, high to FRCs, DNCs and sorted BECs we subset, each for transcriptomes obtain To subsets LNSC between distances transcriptional Comparative RESULTS pericytes. FRC-like contractile, of largely previously all unknown were DNCs of markers surface identifying and tion localiza function, lineage, the Finally, whereas specialization. tional func reflected which counterparts, thymic and skin their and FRCs of profiles in transcriptional the differences Wesubstantial observed network. reticular lymph-node the of makeup charac molecular the terized fully more we Next, response. immune an during we that isolated LNSCs profiling by explored further we hypothesis a gers, trig also infectious or inflammatory to LNSCs respond to readiness a suggested steady-state of Analysis partners. stromal and poietic tors and growth to factors that of cognate receptors on likely hemato We media of immune signature. expression the expression matched DNCs studied profiles. transcriptional stromal of database ‘cross-comparable’ comprehensive, a create to We aimed therefore types. cell stromal multiple of specialization the factors. So far,secreted has not transcriptomics used to been examine for further study, including molecular pathways, surface receptors and targets new of numbers large identifying and cell’s a biology mining unstudied. completely almost remains machinery presenting antigen- and receptors pathogen of expression their yet induction, in roles tolerance have may important and that FRCs LECs also gests sug Evidence chemokines. as such elements specialized identified e c r u o s e r  of clustering in probes relative reinforced expression with differences

We examined global relationships among sorted LNSCs by prin by LNSCs sorted among relationships global Weexamined and BECs poorly of LECs, WeFRCs, transcriptomes the examined deter for point starting unbiased powerful, a is Transcriptomics , b ). Through the use of profiling and quality-control pipelines pipelines of and use the ). quality-control profiling Through 2 , 6 Fig. 1 Fig. , 7 Supplementary NotesSupplementary 1 , this in-depth analysis showed that this subset consists consists subset this that showed analysis in-depth , this 2 , 6 , 7 d , identifying and analyzing each subset’s unique unique subset’s each analyzing and identifying , ). For each cell type, samples from skin-draining skin-draining from samples type, cell each For ). Fig.

1 1 Fig. 1 Fig. , we generated gene-expression profiles on profiles gene-expression generated we , e and c Supplementary Supplementary Fig. 1e and Supplementary Fig. 1c Fig. Supplementary i. 1a Fig. and 2 ). ). All data analyzed below , b and and Supplementary Supplementary ). ). Hierarchical , d ). ------­

of fewer probes ( probes fewer of expression in differed LECs and BECs SLN fact, In between BECs. and LECs relationship developmental closer the emphasized which probes; of (>91.3% subset EC other the in expression their to relative upregulated be to tended also subset (EC) cell endothelial expression. in change comparing plots the in diagonal the along probes of distribution the by shown as MLNs, and SLNs in conserved highly were differences ( respectively probes, 1,936 and 2,026 by BECs LECs SLN and from differed FRCs SLN differences; metric simple transcriptional a of as twofold over of expression in difference a with subset- identify to ­specific transcriptional signatures LECs ( and BECs with FRCs compared we Next pathways and genes subset–specific LNSC of Identification BECs. or LECs with than FRCs with and similarity that DNCs differentiation shared more transcriptional site-specific considerable undergo not did locations different from BECs and between LECs and DNCs and FRCs between similarities transcriptional hematopoietic populations ( populations hematopoietic key and stroma by receptors and ligands cognate of expression the with paired of receptors, relevant immunologically and expression factors growth higher for cytokines, of expression FRCs LNSC examining by genes of cytokine-related enrichment the explored We node lymph the in communication Molecular encode. they molecules the of not genes before in providing reported clues while LNSCs to for the likely function lists cell-specific comb simultaneously to us allowed which functions, stromal known confirmed overviews global These was also highly conserved in MLNs ( MLNs in conserved highly also was pattern This FRCs. and ECs between those than smaller magnitude immune mediators such as type I and type II interferons, TGF- II I interferons, and as type such type mediators immune ( cells hematopoietic by recognized factors growth and signals of sources ( permeability and integrity of vessel regulation for the vital adhesion, cell-cell and (VEGFs) factors growth endothelial vascular via signaling in involved molecules encoding genes for enrichment That relevant transmigration. list to molecules also showed leukocyte for encoding genes enrichment showed probes of list the EC-specific ( interactions receptor cytokine–cytokine and between the extracellular matrix (ECM) and interactions receptors, focal adhesion in involved molecules encoding genes for enrichment Visualization Annotation, Discovery) and Integrated for (Database database bioinformatics DAVID the with overrepresentation statistical of basis the on ways (Kyoto Encyclopedia of Genes and Genomes) and ranking those path lists by collating genes into functional pathways in the KEGG database and pathways with associated these cells, we analyzed signature probe variability. intrapopulation for FCs penalizing nentially expo by noise minimizing populations, two between FCs adjusted 1 Tables Supplementary comparisons; other (FC) and change’ comparison, one least ‘fold at for (adjusted more or twofold of expression ( score delta BECs; and LECs in lated (upregu probes EC-specific 513 and probes FRC-specific 594 tified Probes Probes upregulated in SLN FRCs relative to their expression in one To identify previously unknown, immunologically relevant genes genes relevant immunologically unknown, previously Toidentify To better understand how FRCs differed from sorted ECs, we iden Fig. 3a Fig. aDV , b A ). LNSCs shared expression of receptors for common common for receptors of expression shared LNSCs ). 1 NCE ONLINE PUBLIC ONLINE NCE 5 Fig. 2 Fig. ( Fig. P < 0.05 for differences in expression value (EV); (EV); value in expression < for 0.05 differences c

1 ), and on average these differences were of a of were differences these average on and ), f and ). These data showed that stromal subsets subsets stromal that showed data These ). 1 6 . The list of FRC-specific probes showed showed probes of . list FRC-specific The Fig. 3a Fig. 2 ). The delta-score module computes computes module delta-score The ). δ Fig. 2 = 0.85 (adjusted FC FC (adjusted 0.85 = , A b Fig. 2 Fig. TION ). We identified LNSCs as rich rich as LNSCs We ). identified δ a ) = 1.0 (adjusted change in in change (adjusted 1.0 = ) ). We used a count of probes

c ). nature immunology nature Fig. 2 Fig. Fig. 2 Fig. d ). In contrast, contrast, In ). ≥ 1.8) for all all for 1.8) a Fig. 2 Fig. Fig. 2 Fig. ). These These ). β ≥ and 2)) 2)) d b ), ), ). ). - - - - © 2012 Nature America, Inc. All rights reserved. yoie iae l3 n t spot h dvlpet mainte development, the support to and Flt3 kinase tyrosine (ref. 1 factor colony-stimulating macrophage for receptor the binds which IL-34, scripts BAFF cell–costimulator T and factor the for IL-7 mRNA expressed DCs and cells T killer natural cells, killer ( not did DNCs and ECs BECs pooled whereas in transcripts, lL-7 detected produced LECs and been FRCs that We also found has transcript much as 10% nodes lymph ( potential costimulatory and recognition, pathogen responsiveness, chemokine and cytokine TNF; however, subsets also showed unique transcriptional profiles for ( scatter. side SSC, right). and (center subset LNSC each and (left) stroma CD45 percent indicate areas outlined to adjacent or in Numbers MLNs. or SLNs pooled of fractions ( 1 Figure nature immunology nature DCs of expansion population and nance one experiment with three to five independent replicates ( replicates independent five to three with experiment one ( replicate per mice 10–30 with experiments five to three of representative are Data data. row-normalized and log for calculated population), each within < 0.5 (CV) variation of coefficient and comparison, population pairwise ( correlation. (log LNSCs among expression in difference greatest the with probes of 15% ( (PC1–PC3). component principal each by for accounted variability total of proportion the indicate log with subset), any for > 120 (EV subsets LNSC among expression in ( data). (row-normalized BECs or LECs FRCs, of characteristic ( throughout. each in cells percent indicate areas outlined to adjacent or in Numbers a b a ) Sorting strategy for the isolation of FRCs, LECs, BECs and DNCs from stromal cell–enriched cell–enriched stromal from DNCs and BECs LECs, FRCs, of isolation the for strategy ) Sorting

Interleukin 7 (IL-7) crucially maintains naive lymphocytes in in lymphocytes naive maintains crucially (IL-7) 7 Interleukin SSC-A SSC-A 250K 100K 150K 200K 100K 150K 200K 250K

50 50 receptor receptor CD45 +Ter119 CD45 +Ter119 K 0 K 0

encoding molecules that support myeloid cells, including including cells, myeloid support that molecules encoding 91.7 89.6 Strategy for sorting LNSCs and confirmation of microarray results. results. microarray of confirmation and LNSCs sorting for Strategy 0 0 99.6 100 100 100 10 10 2 2 f SLN 10 10 SLN ) Unbiased hierarchical clustering of probes with the greatest difference in expression (EV > 120 for any population, FC > 2 for any any > 2 for FC population, any for > 120 (EV expression in difference greatest the with probes of clustering hierarchical ) Unbiased 2 3 3 α 18); the cytokine Flt3L, shown to bind the receptor receptor the bind to shown Flt3L, cytokine the 18); , 3 10 10 , and although IL-7 mRNA has been found in FRCs, FRCs, in found been has mRNA IL-7 although , and -chain ( -chain 4 4 10 10 5 5 Fig. 3 Fig.

10 10 gp38 10 10 gp38 10 10 10 10 Fig. 3a Fig. 0 2 3 4 5 aDV 0 4 5 2 3 CD31 99.0 0 0.94 99.3 0 CD31 Fig. 3 Fig. 10 6.29 0 65.6 a 10 A 2 ). FRCs expressed the B cell–survival B cell–survival the expressed FRCs ). 10 2 NCE ONLINE PUBLIC ONLINE NCE , 10 b 3 10 3 and and a 0.41 0.05 98.0 10 98.0 0.05 0.27 ). Furthermore, T cells, natural natural cells, T Furthermore, ). 4 BEC 1 16.8 12.2 LEC 4 0 1 5 1 0 Supplementary Fig. 2a Fig. Supplementary LEC BEC 5 7 . FRCs also produced tran produced also FRCs . 1

9 SSC-A

CD140a10 10 10 10 100K 150K 200K 250K ; and the monocyte-DC monocyte-DC the and ;

50K SSC-A CD140a 0 100K 150K 200K 250K 2 3 4 5 50K 10 10 10 10 0 gp38 0 0 0 2 3 4 5 10 CD44 99.0 0 CD44 10 gp38 85.8 DNC FRC 0 2 0 10 10 10 2 A 10 2 3 2 TION 10 10 10 c 3 99.2 – 10 4 3 3 e 10 10 10 4 ) or one experiment with three to five independent replicates ( replicates independent five to three with experiment one ) or d 98.5 1 5 4 4

0 ) PCA of LNSC subsets, calculated for the 15% of probes with the greatest difference difference greatest the with probes of 15% the for calculated subsets, LNSC of ) PCA FRC 1 10 5 0 DNC 5 5 (8.77%) d PC3 , b (61.65%) ). 2 PC1 –0.2 –0.2 –0.1 c -transformed and row- and column-standardized data. Numbers in parentheses parentheses in Numbers data. column-standardized and row- and -transformed 0.1 0.2 0.3 0.4 0.5 –0.1 2 f - - Pecam1 0 . b

Ccl21a Pdgfrb Pdgfra Lyve1 Acta2 Prox Ccl19 ) Analysis of the purity of SLN FRCs, LECs, BECs and DNCs after sorting. sorting. after DNCs and BECs LECs, FRCs, SLN of purity the of ) Analysis 2 Pdpn Cdh5

Selp 0.1 Tie1 0 Des -transformed and row-standardized data). Blue indicates the highest highest the indicates Blue data). row-standardized and -transformed Vwf Flt4 Tek SLN BEC MLN BEC Il 0.2 ment ment with published reports ( evant hematopoietic subsets expressed the chemokine receptor CCR7 rel whereas CCL21a, and CCL19 chemokines the for transcripts of resembled FRCs ( ECs. and FRCs between codependence dynamic a suggested which PDGFD, and PDGFC PDGFB, PDGFA, ( tors recep relevant the expressed ECs whereas factors, those of some of ANGPTL2, VEGF-C, HGF, ( and SERPINF1 GREM1 including molecules, regulatory angiogenic as reported VEGF-A, amounts. lower much in albeit factors, these of ( CXCL14 chemoattractant 1 7 Fig. Fig. 3 BEC (MLN) FRC (MLN) DNC (SLN) LEC (SLN) LEC (MLN) BEC (SLN) FRC (SLN) FRCs dominated chemokine production, although here too DNCs although production, chemokine FRCs dominated vasculature node lymph regulate also FRCs –0.3 MLN Fig. 3 Fig. –0.2 c FRC b ) Heat map of hierarchically clustered expression data for genes genes for data expression clustered hierarchically of map ) Heat 0 ). ). Although we little detected CCL19 mRNA in ECs, in agree SLN DNC –0.1 MLN LEC SLN MLN FRC a ). ECs produced fibroblastic growth factors, including including factors, growth fibroblastic produced ECs ). 0 (21.86% SLN FRC Normalized expression SLN LEC 0.1 PC2 DNC SLN

0.2 Fig. 3 ) 0.3 e − MLN 0.13 ) Heat map of coefficients of correlation for the the for correlation of coefficients of map ) Heat

2 0.4 0 LEC b , and we detected transcripts for several other other for several transcripts , and we detected e ). As expected, FRCs produced large amounts SLN DNC –0.92 BEC FRC LEC 2 Fig. 3a Fig. , , BECs showed expression substantial of a Normalized expression MLN MLN MLN ), three to five experiments ( experiments five to three ), SLN SLN SLN SLN Fig. 3 Fig. MLN > 0.53 2 -transformed, row mean-centered mean-centered row -transformed, BEC DNC SLN , b a ). DNCs also expressed many many expressed also DNCs ). SLN ). DNCs shared the expression expression the ). DNCs shared –0.75 0 SLN FRC f ; mean values). ; mean MLN Correlation e c r u o s e r SLN 2 > 0 0.77 0 LEC . They expressed expressed They . MLN b ), ), SLN

> BEC 0.83 ML N  - - - © 2012 Nature America, Inc. All rights reserved. cells as well as each other via complex regulatory networks. regulatory complex via other hematopoietic each as well as support cells subsets stromal that suggested data These chains integrin the expressed uniquely DNCs LNSCs, chains integrin of the expression shared ( level protein at the evident also was this and BECs, and LECs FRCs, ( and as integrins such chains, integrin of certain expression shared subsets Although profiles. integrin-expression LNSC fore examined regulating in organs. lymphoid important were LNSCs hemat that indicated data Together these populations. these to relevant be may CCL20 that gested sug which transcripts, CCR6 of expression highest the had cells B nodes lymph from cells T uniquely activated of egress LECs DNCs. ( and CCL20 BECs in expressed that as twice expression than much more as with CXCL12, chemoattractant lymphocyte the ( DCs and cells T memory of or receptor-expressing organization the recruitment may facilitate me as of transcripts, CXCL13 abundance a had similar SLN FRCs and bulk ( PCR by assessed as CXCL13, of expression similar had subsets Both MAdCAM-1 and h osre CC1 epeso b srig MAdCAM-1 sorting by expression CXCL13 for responsible observed were the cells reticular marginal whether determined Fc and CD35 CD21, markers canonical of their of expression basis on the preparations, cell our in cells dendritic follicular detect not Wedid cells. reticular marginal MAdCAM-1 minor a and cells dendritic follicular to restricted reportedly chemoattractant cell B a CXCL13, of scription CCL21a ( replicates. independent to five four with experiment of one representative are Data mmu04670). (TEM; migration transendothelial leukocyte and (mmu04360) guidance axon (mmu04370), pathway signaling VEGF (mmu04520), junction adherens (mmu04514), molecules cell-adhesion (mmu00480), metabolism glutathione (mmu05200), pathways cancer (mmu04060), interactions receptor cytokine–cytokine (mmu04512), interactions ECM-receptor (mmu04510), adhesion focal in parentheses): code accession KEGG procedure; (Benjamini enrichment significant most the with pathways and comparison, one at least for ( versa or in vice ECs expression to their relative in FRCs upregulated ( as in values (gray; or LECs (blue) in BECs upregulated probes indicate colors profiles; ( that side of the vertical axis. indicate total number of probes of that color on in quadrants Numbers (bottom). BECs or SLN (top) LECs SLN and FRCs of SLN plots volcano ( versus. vs, in each. of probes number indicate in quadrants Numbers LNSCs). CV < all for 0.5 and subset, LNSC one EV > at least for 120 (gray, (gray, FC > LECs or SLN 2, top) bottom; or BECs in SLN (blue) FRCs in SLN upregulated probes indicate Colors sites. in both expression similar have diagonals along probes MLNs; and SLNs for (bottom), or LECs (top) BECs of those with of FRCs profiles expression the ( function. FRC into insight 2 Figure e c r u o s e r  d c b Supplementary Supplementary Fig. 3c Fig. Fig. 3 Fig. 3 Fig.

) Comparison of BEC and LEC expression expression LEC and of BEC ) Comparison ) Enrichment in KEGG pathways for probes probes for pathways in KEGG ) Enrichment in identified of probes ) Projection Integrins help shape cell-cell and cell-ECM interactions; we there interactions; and cell-ECM help cell-cell shape Integrins such as other chemokines, CCL2 and that FRCs CCL7, transcribed β a 1 o c , the expression of other chains had a more restricted pattern pattern restricted more a had chains other of expression the , sured by microarray ( by microarray sured c poietic-cell recruitment to and localization within secondary secondary within to and localization recruitment poietic-cell

and ). In fact, DNCs expressed little integrin integrin little expressed DNCs fact, In ). Unbiased analysis of LNSCs provides provides of LNSCs analysis Unbiased Fig. 3 Supplementary Fig. 2c Supplementary b ). ). Unexpectedly, in FRCs we found considerable tran + δ gp38 = 1.0 (adjusted FC = (adjusted 1.0 i. 3 Fig. ). ). Consistent with the PCR data, MAdCAM1 + a CD140a P ) Comparison of ) Comparison < 0.0001 ( b Fig. 3 Fig. ), which is suggested to promote the the promote to suggested is which ), Supplementary Fig. 3d Fig. Supplementary δ ε = 0.85 (adjusted FC (adjusted = 0.85 RII ( RII b a + onto onto CD31 ). FRCs were the main source of of source main the were FRCs ). , d Supplementary Fig. 3a Fig. Supplementary χ ). Meanwhile, FRCs ). and Meanwhile, DNCs a ≥ 2 ). ). test). 2)

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2 1 . Memory T cells and and cells T Memory . β a ≥ 5 α Expression change Expression change , whereas among among , whereas 1.8) for all other comparisons; comparisons; other all for 1.8) 2 ). in MLN in MLN + , in contrast to to contrast in , population of population α (log10 fold) (log10 fold) –2 –1 –2 –1 7 0 1 0 1 2 , , α –2 Expression changeinSLN Expression changeinSLN –2 8 and and − , b FRCs FRCs –1 α ). We ). FRC vsBEC FRC vsLEC –1 1025 1 (log (log 1054 , , α α 2 9 4 10 10 1 0 − 5 - - - . 995 2 1 0

4 fold) fold) 870 3 membrane-attack complex of the complement system and prevents prevents and system complement the of complex membrane-attack which promotesvitronectin, cell adhesion and spreading, inhibits the glycoprotein the for transcripts expressed FRCs function. and ture struc conduit to contribute also may laminins other that suggested chains laminin the expressed FRCs I to membrane and with growth the fibrils basement interact collagen bridge fibrillogenesis, collagen regulate SLRPs (SLRPs). proteoglycans leucine-rich small as such molecules of secretion the the to it localized ( zone microfibrillar which ER-TR7, with together localized mostly ( ment of XIV collagen by ER-TR7, it placed which in the core collagen ensheath showed microscopy Immunofluorescence XII. collagen of expressionhighest of the collagen XV, had BECs whereas LECs and molecules. DNCs shared these expression of expression their in FRCs scripts for collagens III, V, VI, XIV and XVI ( ( FRCs by components conduit of secretion and ( interactions ECM-receptor in involved molecules encoding of genes expression for higher ment by FRCs ensheathed membrane basement 4a Fig. Supplementary (ref. ER-TR7 antibody monoclonal by the recognized antigen the and fibrillins contains that zone microfibrillar a by enveloped core, collagen-rich a lies center conduit the At allow. would tissue dense cell- through filtration than faster cortex to the factors lymph-borne small, deliver that conduits studied poorly yet unique create FRCs Identification of previously unknown conduit-network components laminin-10 ( laminin-10 and were surrounded by ER-TR7 and localized to the conduit core ( ( immunofluorescence microscopy, prolargin decorin, biglycan and and fibromodulin biglycan decorin, glycin, FRCs had high expression of the SLRPs lumican, fibromodulin, osteo Fig. 4 Fig. FRCs can regulate conduit structure and organization through through organization and structure conduit regulate can FRCs In addition to expressing collagens I and IV The The conduit basement membrane contains laminin-8 ( Supplementary Figs. 4c Figs. Supplementary 2 c and and b P Significance < 0.05 for differences in expression). Blue indicates KEGG KEGG indicates Blue in expression). differences for < 0.05 Significance aDV (log P) (log P) –11 10 –11 α 10 Supplementary Figs. 4b 4b Figs. Supplementary –9 –7 –5 –3 –1 –9 –7 –5 –3 –1 5 A –2 β 905 896 73 NCE ONLINE PUBLIC ONLINE NCE 86 –2 1 γ FRC vsBEC(SLN Expression change Expression change FRC vsLEC(SLN) 1) –1 Fig. 4 Fig. –1 9 (log (log , which were expressed by all LNSCs ( by LNSCs all were expressed , which 10 10 1 0 ). Surrounding the microfibrillar zone is a is zone microfibrillar the Surrounding ).

1 0 d fold) fold) and Fig. 2 Fig. – e , 7 7 ) 798 913 156 Supplementary Figs. 4f 4f Figs. Supplementary 131 2 and 2 d ), we examined the transcription transcription the ), we examined A α TION 8 and 2, 2, d Expression change c ).

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© 2012 Nature America, Inc. All rights reserved. picture of conduit structure and ECM maintenance by FRCs than than FRCs by maintenance ECM and structure conduit of picture MFAP-5 ( and FBLN1 in matrix and elastic fiber assembly, including LOX, LOXL1, MFAP-2, involved molecules for transcripts of amounts large produced FRCs ( DNCs or FRCs did than ADAM10 metallopeptidase the of protein and mRNA of expression higher had also BECs and LECs ( LECs had substantial expression and of DNCs transcripts forFRCs, MMP9 and whereas MMP14 DNCs, and FRCs in greatest was MMP3 ( LNSCs by well as tissues extranodal in ECM the of turnover these changes. accommodate Matrix metalloproteinases (MMPs), which areto important for ECM the of which remodeling hours, extensive of suggests matter a within often responses, immune during ( membrane basement the to it Figs. at ( points branch conduit structures like ER-TR7 of surface the decorated vitronectin zone cell T the in pattern reticular a shows also it the and inhibitor-1 activator ECM plasminogen between interactions in as integrins of expression T,memory. ( Mem, naive; T cell; Nve, T cell; killer log for population), stromal any in ligand or receptor the for > 120 (EV subsets hematopoietic node lymph 11 and LNSCs by receptors and factors growth cytokines, of expression the of analysis 3 Figure nature immunology nature realized. previously Fig. 4 Fig. interferon cytokines Common a PDGF IL-10 Lymph nodes undergo considerable expansion and contraction contraction and expansion considerable undergo nodes Lymph γ factors growth VEGF TGF- -chain family family family family family family Other orgrowthfactor TNF IL-6 IL-1

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1 2 1 T 6 7 4 5  - - -

© 2012 Nature America, Inc. All rights reserved. Although FRCs had much higher expression of of expression higher much had FRCs assay Although wrinkle a by demonstrated been has contractility their and toward contractile ability than were SFs ( residents. lymph-node of these specialization functional the ( FRCs in greater much was scripts for similar chemokines and cytokines, thetran transcript produced abundanceThFs and SFs FRCs, Although nodes. lymph within of cells of the immune response to lymph nodes and their homeostasis ( cells presenting SFs, antigen- did ‘professional’ in that than than lower pathway much was II expression this class MHC the of components some of expression higher had FRCs Although genes. these of transcription enhance might environments lymphoid-tissue that suggested which of transcripts, such had abundance a greater also cells hematopoietic MHC class I molecules ( noncanonical and canonical and machinery antigen-processing ing the major histocompatibility complex (MHC) class I pathway, includ ( interactions ECM-receptor involving functions fibroblast classical serve that molecules encoding genes of e c r u o s e r  ( ThFs or SFs did than chain light myosin muscle Collagens a

Laminins KEGG analysis suggested that FRCs and ThFs were more biased biased more were ThFs and FRCs that suggested analysis KEGG In addition to presenting self antigens, FRCs regulate the recruitment of of many components had SFs than expression higher had FRCs MMP Adamts15 Adamts17 Adamts12 Adamts10 Lamb1−1 Adamtsl Adamtsl Adamts8 Adamts1 Adamts9 Adamts4 Adamts7 Adamts2 Adamts3 Adamts5 Adam17 Adam19 Adam33 Adam10 Adam15 Adam12 Col27a1 Col23a1 Col16a1 Col14a1 Col18a1 Col15a1 Col12a1 Mmp24 Mmp13 Mmp14 Mmp15 Mmp23 Mmp11 Mmp19 Adam9 Col9a3 Col9a2 Col7a1 Col4a4 Col2a1 Col5a3 Col6a3 Col6a2 Col6a1 Col5a1 Col5a2 Col3a1 Col1a2 Col1a1 Col4a2 Col4a1 Col4a5 Lamb2 Lamc2 Lamc1 Lama4 Lama5 Lama3 Lamc3 Lama2 Mmp3 Mmp2 Mmp9 Emid2 Dvwa 4 1

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FRCs interacted closely with each other as they ensheathed the the ensheathed they as other ( that secreted fibers they ­reticular each with closely interacted FRCs FRCs between junctions cell-cell identifies Cadherin-11 microenvironment. they unique but their to specialized counterparts, nonetheless were thymic and skin their with of number characteristics large unexpectedly an shared FRCs that demonstrated fibr and ECM the of also and MMP23 FRCs expression MMP9. of highest the transcription had considerable had molecules ThFs those and of ( expression ThFs shared with matrix- or or ECM molecules certain ­regulatory expressed uniquely FRCs ­components, ( ECM of most expression shared functions populations fibroblastic all whereas these in that specialized suggested most analysis were SFs pathway KEGG fibroblasts. of acteristics ( myoblasts mouse C2C12 of that on than did mouse fibroblasts, NIH3T3 par function with contractile Fig. 10d ( genes muscle-associated smooth other for scripts Fig. 10d NK

CD4 B 8 + The production and maintenance of ECM are fundamental char fundamental are ECM of maintenance and production The Nve CD4 NKT Nve CD8 Mem CD4+ T Mem CD8+ T + ), ), both FRCs and ThFs produced a greater abundance of tran ). ). Consistent with their expression profile, FRCs had stronger o T + modulin ( modulin ( log of expressed probes (EV > 120 for any LNSC population) for by gene ontology (AmiGO database), presented as heat maps network. ( Figure 4 experiments ( experiments with seven mice ( with three to five independent replicates ( Scale bars ( identified as being localized to the basement membrane. for the ER-TR7 antigen (red) and vitronectin (green), newly collagen VI (green). ( ER-TR7 antigen (red) and the microfibrillar zone constituent right). ( (top right), biglycan (bottom left) and fibromodulin (bottom collagen core components (green): collagen XIVzone stained for(top the ER-TR7 antigenleft), (red) and the followingdecorin immunofluorescence microscopy of reticular fibers in the T cell (gray shading), isotype-matched control antibody. ( FRCs, LECs, BECs and DNCs, assessed by flow cytometry. Isotype T aDV b ) Expression of ADAM10 (black lines) by freshly isolated SLN 2 upeetr Fig. Supplementary

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) Confocal microscopy as in Transcriptional insights into the lymph-node conduit a ) Identification of proteinaceous ECM components Supplementary Fig. Supplementary c – c e – ), 2 ER-TR7 c e b ER-TR ). Events (% of max) µ 10 ADAM10 m. Data are representative of one experiment e 0 Collagen XI 7 10 ) Confocal microscopy as in

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remodeling molecules molecules remodeling Decori ). Together these data data these Together ). a ), three independent n Fig. 5 Fig. n BEC Supplementary Supplementary Isotype Basement membrane ER-TR d e ER-TR Microfibrillar zone c c , with staining d ) Confocal 7 ). Notably, Notably, ). 7 ); ); however,

Collagen VI Vitronectin

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© 2012 Nature America, Inc. All rights reserved. subset known to transcribe even small amounts of Aire of amounts small even transcribe to known subset do not express the adhesion molecule EpCAM regulator Aire transcriptional encompass the express that DNCs cells extrathymic whether tolerogenic highly unclear is It localization, lineage, function. and unknown ­phenotype have yet LNSCs of 5–10% stitute DNCs of existence the reported have groups Several pericytes contractile are DNCs FRCs suggested that it may contribute to FRC-FRC interactions. fibroblast-like synoviocytes microscopy, in junctions between adjacent FRCs ( this protein ( FRCs expanded cell of the same type in adjacent cells and interacting laterally within a single ( molecule for this negative distinctly were cells hematopoietic and expression some had DNCs cadherin-11, of In accordance with the microarray data, ECs had little to no expression ( FRCs isolated on freshly of cadherin-11 expression surface mesenchymal origin of these cells DNCs; extent, lesser a to (and, Notably, we also transcripts observed for in cadherin-11 SFs and ThFs observed been has node before lymph the in cadherin-11 of Expression molecules adhesion of family cadherin calcium-dependent the of expression of high cadherin-11 by FRCs observed ( and LNSCs by molecules cell-adhesion candidate of expression the examined We junctions. these about known is little ( replicates independent five to three with experiment one of representative are Data procedure). ( (mmu04510). adhesion focal E, (mmu04512); interactions receptor ECM- D, (mmu04270); contraction muscle smooth vascular C, (mmu04060); interactions receptor cytokine–cytokine B, (mmu04612); presentation and processing antigen A, parentheses): in code accession (KEGG results notable biologically indicate letters Colored (right). ThFs or (middle) SFs (left), FRCs in enrichment pathway of terms in presented are P ( analyses multiplot and delta-score pairwise from generated lists type–specific cell of analyses enrichment pathway- KEGG pooled from results significant ( distance. smallest in as in identified expression in difference greatest the with probes of 15% the for distances Euclidean of map ( correlation. highest indicates Blue data. row-standardized and transformed in identified expression in difference greatest the with probes of 15% the for coefficient) (Pearson values correlation ( EVs. column-standardized log for ThFs, and SFs FRCs, MLN FRCs, SLN among expression in difference greatest the with probes of ( SFs. and ThFs FRCs, 5 Figure nature immunology nature noted been not has protein Aire < 0.05). 0.05). < Cadherin molecules form homotypic interactions, binding cadherins 2 6 b . We. populationsonlocalization cadherin-11 ofevaluatedthe of ). Blue indicates samples with the the with samples indicates Blue ). 2 7

; however, its specific localization has remained unknown. unknown. remained has localization specific its however, ; Transcriptional specialization of of specialization Transcriptional P values (Benjamini procedure) procedure) (Benjamini values Fig. 6 5 . . Unlike those extrathymic Aire-expressing cells, DNCs in vitro 2 -transformed and row- and and row- and -transformed c d δ ). We observed cadherin-11, by immunofluorescence = 1 (adjusted FC FC (adjusted 1 = ) Scatter plots of of plots Scatter ) a ) PCA of the 15% 15% the of PCA ) and found they maintained surface expression of a a (data transformed transformed (data log with calculated , e

) Comparison of the contractile activity of FRCs, NIH3T3 fibroblasts and C2C12 myoblasts myoblasts C2C12 and fibroblasts NIH3T3 FRCs, of activity contractile the of Comparison ) b aDV 2 ) Heat map of of map Heat ) 6 . The specific expression of cadherin-11 in A NCE ONLINE PUBLIC ONLINE NCE Fig. 6 Fig. 2 6 6 c . Among LNSCs, DNCs uniquely uniquely DNCs LNSCs, Among . . We confirmed, by flow cytometry, ) Heat Heat ) Fig. 6 ≥ 2), 2), a

), which reflected a common common a reflected which ), a ). Cadherin-11 is a member Fig. 6 Fig. 2 - 6 but are the only LNSC Fig. 6 b a e d (5.19%) ).

Enrichment, SLN FRC PC3 A vs ThF (log P) 2

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Contraction (matrix/tube) –6 7 ), as shown in 0.1 0.2 0.3 0.4 0.5 0.4 6 , which con which , 0 0. 0. 0. 0. , (36.23%) 7 B 0.3

0 2 4 6 8 , although although , –5 PC2 0.2 MLN FRC 0 fibroblas Fig. 6 Fig. 0.1 Skin –4 0 –0.1 C 1 –0.2 t –3 SLN FR b 2 –0.3 6 ). ). - . –2 A 2 Time (d fibroblast –0.3 Thymic C –0.2 * –1 population, hoping to assign it a lymph-node microniche and and microniche calponin-1 lymph-node arose: candidates a two following The it function. probable assign to hoping population, heter ( protein for this of transcripts expression highest the had whereas Notably, types. cell multiple to common is others of expression the Myh11 and ( chains myosin subtypes actin including contraction, smooth-muscle for tant ( muscle cells smooth- and cardiomyocytes of those of reminiscent profiles KEGG encoding molecules with structural and contractile functions and had receptor interactions. In contrast, DNCs cytokine-cytokine had high expression of in genes involved molecules encoding genes for ment enrich showed probes FRC-specific of list the DAVIDanalysis, By in the expression of genes measured by these probes (data not shown). Table 4 Supplementary (adjusted FC type pheno in surface differences FRCs despite were considerable similar ( probes 1,880 than more of expression in varied ECs and DNCs contrast, In (FC probes 834 of DNCs expression the in differed plots. FRCs and EV-versus-FC two-class by LNSCs other with DNCs CD45 contaminating excluded combination this strategy, we DNCs chose to define as further CD44 As of of LNSCs. other part our sorting proteins characteristic surface antigens expression of tissue–restricted peripheral (TLR3) 3 receptor Toll-like of expression lack –0.1 E 0 3 ) We next sought potential markers to delineate the conceivably conceivably the delineate to markers potential sought next We compared We FRCs. resembled DNCs analysis, our Throughout 10 0.1 (51.86%) D 0

0.2 P 6 * PC1 ) . We identified probes specific to SLN DNCs and FRCs ( FRCs and DNCs SLN to specific probes We. identified o 0.3 0.4 eos bl-eaie (gp38 ‘bulk-negative’ genous 4 is described as being Fig. 7b Fig. α Enrichment, SF vs ThF -SMA is often used as a surrogate marker for FRCs, DNCs DNCs for FRCs, marker a surrogate as used often is -SMA FRC b C2C12 SLN FR NIH3T3 5 (log10 P) –9 –8 –7 –6 –5 –4 –3 –2 –1 ≥ Fig. Fig. 7 Enrichment, SFvsSLNFRC(log 0 MLN 2); –6 SL ThF SF Fig. 7 Fig. , C N –0.68 c ). Thus, the transcriptional profiles of DNCs and and DNCs of profiles transcriptional the Thus, ). –5 P a d < 0.05 for differences in EV in DNCs versus FRCs; MLN – D Day ). We assessed genes encoding molecules ). impor We molecules genes encoding assessed –4 d FRC e ) or two experiments ( experiments two or ) ). Of those genes, the expression of of expression the genes, those Of ). 4 Correlation ). MLN DNCs closely resembled SLN DNCs DNCs SLN resembled closely DNCs MLN ). SLN –3 0.04 E

specific specific to smooth muscle cells –2 NIH3T3 SF * –1 ThF B >0.84 10 C 0

P A * ) − in vitro in CD31 Enrichment, ThF vs SF FRC c (log10 P) –9 –8 –7 –6 –5 –4 –3 –2 –1 e Enrichment, ThFvsSLNFRC(log 0 ML SLN ThF ; mean mean ; FR –6 SF N − . * . C CD45 D C A E B 0 –5 6 P e c r u o s e r MLN lo Focal adhesion ECM-receptor VSM contraction Cytokine-receptor Antigen processing and have different different have and < 0.05 (Benjamini (Benjamini 0.05 < 6 CD44 − Euclidean distanc FR . . DNCs lack many –4 ± , , as we found that s.d.). C − SLN CD44 –3 51.97 ≥ + 2; 2; cells. –2 2 E Fig. 7 Fig. C SF 8 Actg2 2C12 , whereas − Fig. 7 Fig. * –1 ) DNC DNC ) D e C ThF δ >82.16 = 1.0 1.0 = B 06.21 e 10 and and 0 ).

A P a * ) ). ).  - - -

© 2012 Nature America, Inc. All rights reserved. al 5 Table on; so and mice, untreated from FRCs SLN versus FC ( study further for probes such 113 identified We ( counterparts untreated corresponding their in and subsets 2, > (FC expression different with probes of BECs considerably, clustering 373 by as demonstrated hierarchical analysis. further for 13b Fig. here; mice’ h ‘12 or cells’ h ‘12 (called purity high to cells the sorted and ovalbumin and lipopolysaccharide with mice ( SLNs from BECs ( ovalbumin 30 of from injection saccharide intravenous mice gave cell we T after h transfer, 18 At mice. C57BL/6 5-week-old into intravenously receptor) antigen cell T ovalbumin-specific an of expression genic 10 × immune 1.5 ongoing transferred we and responses, inflammation to cells the stromal investigate of To responses insults. infectious to or poised are inflammatory to they respond suggested profiles their and transcriptional molecules, steady-state lymph-borne encounter to node lymph the throughout sites key at positioned are IAPs and BECs LECs, FRCs, LNSCs in changes transcriptional triggers Inflammation (IAPs). pericytes’ most identified data These ‘ITGA7 we called which pericytes, contractile DNCs. DNCs as fibroblastic, of population main the were ( DNCs of >50% stained anti-ITGA7 whereas DNCs, of (<5%) minority small a identified anti-CNN1 cytometry, with flow staining that By apparent was it however, shown). not (data was anti-ITGA7 subset by This stained node. not lymph the of face medullary the subset to DNC limited subcapsular elongated, an identified also anti-CNN1 with Staining cells. or these with without vessels between differences and cortical vessels ( medullary some surrounding cells, pericyte-like as DNCs identified ( matrix basement- of membrane consisting proteins) and gel formed strong contractile (a attachments to the Matrigel with supplemented when to ECM the fibers muscle linking for important is and membrane basement chain cells integrin contractile in production force of lator to and muscle cells is smooth protein a specific regu chief associated integrin and (CNN1) ( replicates independent 10 vitro in expanded FRCs of a population on microscopy of (green) cadherin-11 vitro in ( cytometry. flow by assessed cells, hematopoietic bulk and DNCs BECs, ( log for cells, hematopoietic and stromal in molecules ( cells. reticular fibroblastic 6 Figure e c r u o s e r  and antigen-processing KEGG the for enrichment substantial b c

) Surface expression of cadherin-11 on a population of FRCs expanded expanded FRCs of a population on cadherin-11 of expression Surface ) ) Expression of cadherin-11 (black lines) in freshly isolated FRCs, LECs, LECs, FRCs, isolated freshly in lines) (black cadherin-11 of ) Expression Treatment changed the transcriptional profiles of FRCs, LECs and of LECs FRCs, profiles Treatment transcriptional the changed anti-ITGA7 and (anti-CNN1) CNN1 to antibody with Analysis µ ≥ m. Data are representative of one experiment with three to five five to three with experiment one of representative are Data m. 2); 2); (red, F-actin; blue, DAPI (DNA-intercalating dye)). Scale bar, Scale dye)). (DNA-intercalating DAPI blue, F-actin; (red, ( cytometry. flow by , assessed

Fig. 7 Fig. ). Probes upregulated in 12 h FRCs and BECs showed showed BECs and FRCs h 12 in upregulated Probes ). ). Because of the rarity of IAPs, we did not sort this subset subset this sort not did we IAPs, of rarity the of Because ). P Cadherin-11 identifies junctions between lymph-node lymph-node between junctions identifies Cadherin-11 2 9 < 0.05 for differences in EV for SLN FRCs from 12 h mice h mice 12 from FRCs EV SLN for in differences for < 0.05 Supplementary Fig. 12 Fig. Supplementary . Indeed, we found that sorted DNCs grew in culture only only culture in grew DNCs sorted that we found . Indeed, Supplementary Fig. Supplementary g β ). 1 to bind laminin-1, laminin-2 and laminin-4 in the the in laminin-4 and laminin-2 laminin-1, bind to shrci coli Escherichia n = 5–6 mice per replicate) 12 h after injection of injection after h 12 replicate) per mice 5–6 = Fig. 7h a ) or four independent experiments ( experiments independent four ) or α a 7 ) Expression of candidate cell-adhesion cell-adhesion candidate of ) Expression (ITGA7; (ITGA7; , i ). ). There were no apparent morphological

13a (serotype O127:B8) and 500 500 and O127:B8) (serotype d ), which suggested that pericytes pericytes that suggested which ), 6 ) Confocal immunofluorescence immunofluorescence ) Confocal OT-I T cells (which have trans have (which cells T OT-I ). We isolated FRCs, LECs and and LECs FRCs, We ). isolated i. 7 Fig. P < 0.05) in 12 h stromal stromal h 12 in 0.05) < f . N1 s n actin- an is CNN1 ). 2 8 . ITGA7 pairs with with pairs ITGA7 . 2 -transformed data. data. -transformed δ Supplementary Supplementary Supplementary Supplementary = 1.0 (adjusted (adjusted 1.0 = µ b lipopoly g – d ). Fig. 8 Fig.

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n H2-E and ficantly higher expression of LGMN ( LGMN of expression higher ficantly H2-DM (H2-A molecules II class MHC of pathway, chains presentation II including class MHC the of components many of expression we 12 that upregulated h had found and BECs FRCs lysis, and LECs in degree lesser a to ( trend BECs this ovalbumin, observed and also we and lipopolysaccharide with mice of FRCs in treatment after POSTN, downregulated MMP9, significantly as was LAMA2) (such and COL6A6, molecules ECM-associated of variety 1.9-fold, (2.6-fold, FRCs h 12 in upregulated significantly for not of part IL-7 list, transcripts and the 113-probe IL-33 were also negative a NR1D1, for signaling mRNA of TLR4 regulator less had subsets stromal h 12 The (ref. LCN2 and encoding genes that molecules or regulate these pathways, such as IRF7 (ref. signaling TLR4 or interferons by inducible are ( CXCL9 and sub CCL5 chemokines three inflammatory the more has mRNA encoding all sets whereas SERPINA1B), and A2M, SAA3, (including of mol expression encoding enhanced genes demonstrated FRCs h 12 milieu, matory for largest ( subset generally this were FCs as point, time this at respond to strongly seemed most FRCs although distributions, probe similar in resulted mice untreated and 12 h from mice isolated subsets stromal pathways. KEGG particular for enrichment show not did ( LECs h 12 in upregulated probes to contrast in pathway, antigen-presentation d Glycam1 a Pecam1 Pcdhb4 Pcdh17 Pcdh12 Pcdh19 Pcdh18 Pcdh15 Cldn11 Cldn10 Cldn12 Vcam1 Cdh13 Cdh11 Pcdh1 Pcdh7 Mcam In accordance with the results obtained by KEGG pathway ana pathway KEGG by obtained results the with accordance In FC–versus– onto probes 113 the of Projection Icam1 Icam2 Bcam Cldn1 Cldn5 Jam3 Cdh4 Cdh6 Jam2 Cdh5 Cdh2 Dsc3 Dsc2 F11r F-actin Fig. 8 Fig.

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© 2012 Nature America, Inc. All rights reserved. tially higher expression of LCN2 by FRCs, LECs and BECs after after BECs and LECs FRCs, by LCN2 of expression higher tially pneumoniae against defense host to linked been has ( LECs did than expression II class MHC in higher increases larger the demonstrated with BECs and significantly FRCs Consistent data, DNCs. microarray was on not II but BECs and MHC LECs of FRCs, on expression surface ment, ( ovalbumin and injec lipopolysaccharide of SLN after h tion on 18 mice II from class isolated DNCs MHC and BECs of LECs, FRCs, expression surface the investigated we ( experiments independent three in as DAPI 15 bars, Scale DNCs. are cells indicates CD31 and gp38 for staining of ( ×100. exclusion magnification, Original contraction. matrix visible indicate Arrows Matrigel. ( CNN1 markers ( DNC FRCs. to candidate relative DNCs in upregulated genes ( muscle–associated smooth (mmu04060). vascular 12 of interactions ThFs and receptor cytokine–cytokine and cardiomyopathy (mmu05414) hypertrophic cardiomyopathy (mmu04260), dilated contraction (mmu05410), muscle cardiac vascular (mmu04510), (mmu04810), adhesion focal cytoskeleton (mmu04270), actin of contraction regulation muscle smooth parentheses): in code accession KEGG procedure; (Benjamini enrichment significant most ( versa vice or FRCs SLN to relative DNCs SLN in upregulated probes total indicate EV. ( mean corners in MEV, two-class Numbers probes. 0.5). < CV 2, > FC upregulated subset, LNSC any for 120 > EV (gray; FRCs or (blue) DNCs in upregulated probes indicate 7 Figure nature immunology nature respectively), higher, 4.8-fold and 3.5-fold (17.5-fold, treatment d c b a

1 0 DNC Expression change Expression change Expression change LCN2 is a multifaceted, lipopolysaccharide-inducible molecule that Significance (–log FRC in SLN (log10 fold) in SLN (log10 fold) in SLN (log10 fold) Cytokine–receptor Dilated cardiomyopathy Hypertrophic cardiomyopathy Cardiac muscle Focal adhesion VSM contraction Actin cytoskeleton –1 –1 –1 0 1 2 0 1 0 1

DNCs are contractile pericytes. ( pericytes. contractile are DNCs Expression inSL Expression inSLN Expression inSLN h 3 2 DNC vsBEC 3 2 3 2 DNC vsFRC DNC vsLEC .1 . Scale bars, 15 15 bars, Scale . (log (lo (lo and and g g 10 10 10 MEV) MEV) MEV) 10 Mycobacterium tuberculosis Mycobacterium >3.62

P ) 1,085 4 4 4 986 901 841 N 407 427 aDV f µ Expression (log EV)

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Cnn1 Kcnmb1 Merge CNN1 h gp38 DAPI a ) Comparison of gene expression by SLN DNCs and FRCs (two-class EV-versus-FC plots). Colors Colors plots). EV-versus-FC (two-class FRCs and DNCs SLN by expression gene of Comparison ) Fig. 8d Fig. Escherichia coli Escherichia Mylk b , Fig. 8d Fig. pericytic Medulla c 3 t 2 ) Analysis of SLN DNCs and LECs ( LECs and DNCs SLN of Analysis ) Actg2 Itga7 . Given the substan the Given . t

, Ppp1r12a e ). A ). ( ). TION , Itga7 SLN LE SLN FR e ). After treat After ). g Cald1 ) Brightfield microscopy of sorted FRCs and DNCs grown for 5 d in a three-dimensional three-dimensional a in d 5 for grown DNCs and FRCs sorted of microscopy Brightfield ) reticular Medulla , , Ppp1r14a C C Klebsiella Klebsiella

Ednra δ SLN DE SLN BEC pDC CD8 CD4 SLN DEC SLN BEC SLN LEC SLN FRC Skin fibroblast Thymic fibroblast = 1 (adjusted FC FC (adjusted 1 = subcapsular Gucy1a3 Medulla - - - + +

DC DC C during ongoing immune responses. immune ongoing during matory or infectious triggers and may contribute as active participants to inflam to respond are poised LNSCs that idea the reinforced data not (data These shown). origin were probably of these hematopoietic LYVE-1 desmin-negative, in LCN2 detected we LCN2 of 18 h ( treatment after expression detectable had subsets stromal three all mice, untreated from sections SLN in BECs and LECs to FRCs, In contrast LYVE-1 (to LECs) and identify anti-pNAd (to BECs; identify anti- FRCs), identify (to anti-desmin anti-LCN2, with tissues these from sections stained and ovalbumin and of lipopolysaccharide tion we obtained SLNs from untreated mice and from mice 18 h after injec Myh11 subcapsular µ Myl9 Medulla m. ( m. h ) Immunofluorescence microscopy of ITGA7, F-actin and DAPI; DAPI; and F-actin ITGA7, of microscopy Immunofluorescence ) b Mem CD8 Mem CD Nve CD Nve CD CD NK Follicular Bcell MF ) or BECs ( BECs or ) Acta2 i ≥ ) Immunofluorescence microscopy of CNN1, F-actin and and F-actin CNN1, of microscopy Immunofluorescence ) 4 2), 2), + NK 8 4 4 + + T P Tcell Tcell + + Tcell Tcell < 0.05). Blue indicates KEGG pathways with the the with pathways KEGG indicates Blue 0.05). < c f ) as in in as ) ) Stromal and hematopoietic expression of the the of expression hematopoietic and Stromal ) Fig. Fig. 8 DN FRC g C & CD31 a F-actin f Merge ITGA7 . ( . ). In agreement with published reports ). In published with agreement gp38 DAPI i d ) Enrichment in KEGG pathways for for pathways KEGG in Enrichment ) e ) Expression by LNSCs, SFs SFs LNSCs, by Expression ) pericytic Medulla a – f ; mean and s.d. in in s.d. and mean ; pericytic Corte e c r u o s e r − and pNAd and x subcapsular Corte e Fig. Fig. 8 , x f − ) or or ) cells; cells; 3 f 2 ). ).  - - ,

© 2012 Nature America, Inc. All rights reserved. factors in regulating fibril density and diameter in lymph nodes. collagen XIV and SLRPs. Future studies should define the roles by regulated of tightly core these collagen I type the of property a is conduits in size by molecules of exclusion We the network. that the suggest of ecules to the lymph-node parenchyma and alter the structural integrity control pore size; dysregulation could impair the delivery of small mol structure fibrils have pore-like permeability dependent on tertiary and quaternaryment arrange low-diameter the locking bonds, XIV I–collagen ­collagen diameter fibril of regulator key decorin, which also has high expression in the conduit core, is another diameter by preventing lateral binding of adjacent fibrils fibril limiting I, collagen crosslinks which XIV, collagen contained into chains, long providing fibrillar core collagen the strength, tensile potential mechanism for regulating conduit structure and function. a identified has FRCscomponents by ECM expressionof the of here function, yet its molecular basis is poorly understood. Our delineation cortex idly delivers low-molecular-weight lymph-borne molecules Thedeep lymph-nodeinto conduitthe network is a complex molecular sieve thatDISCUSSION rap ( replicates independent to four three with experiment of one representative LCN2 indicate Arrows BECs). LYVE-1 (red; or pNAd FRCs), LECs) (red; (red; desmin and (green) LCN2 for stained ( mean indicate lines horizontal cytometry. by flow ( 18 h, for assessed ( II. class MHC MHCII, of probes. categorization biological ( (mmu05310). asthma and (mmu04940) mellitus I diabetes type (mmu05332), disease graft-versus-host (mmu04623), pathway DNA-sensing cytosolic (mmu04612), presentation and processing antigen in parentheses): code accession KEGG procedure; (Benjamini enrichment significant most the with pathways KEGG ( data. row-normalized and log for population), FC each > 2 CV < and within 0.5 population, > or either 12 (EV for h 120 (UT) mice untreated from Figure 8 e c r u o s e r 1 be poised to respond to lymph-borne infectious or inflammatory cues. b a –0.65 Normalized expression 0 Significance (–log 0 FRC The transcriptional profiles of LNSCs suggested that these cells may We found that in addition to containing collagen I, which assembles

LEC UT FRC

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. Tight regulation of fibril diameter and packing conceivably h UT BEC 10 >0.61 >

2.74 P 12 c ) h ) Projection of the 113 probes identified in identified probes 113 of the ) Projection c Significance (log10 P) Significance (log10 P) Significance (log10 P) –5 –4 –3 –2 –1 –5 –4 –3 –2 –1 –6 –5 –4 –3 –2 –1 0 0 0 0.2 0.1 b ) Enrichment in KEGG pathways for probes upregulated in 12 h LNSCs ( in 12 h LNSCs upregulated probes for pathways in KEGG ) Enrichment

0.3 0.3 0.2 ±

s.d.). ( s.d.). 0.3 0.4 0.4 3

5 0.4 . Finally, LOX covalently crosslinks crosslinks covalently LOXFinally, . 0.5 0.5 0.5 e

) Median fluorescence intensity (MFI) of the expression in expression of the (MFI) intensity fluorescence ) Median 0.6 0.6 Expression change(fold) Expression change(fold) Expression change(fold) f ) Confocal immunofluorescence microscopy of sections of SLNs from untreated mice and mice treated for 18 h, for treated mice and mice untreated from of SLNs of sections microscopy immunofluorescence ) Confocal 0.7 0.7 0.8 12 hvsUT(FRC) 12 hvsUT(LEC) 12 hvsUT(BEC) a 0.8

) ) Unbiased hierarchical clustering of 373 probes with different expression in the stromal corresponding subsets 1 0.9

1 1

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) Surface expression of MHC class II on SLN LNSCs from untreated mice and mice treated treated mice and mice untreated from II LNSCs on SLN class of MHC expression ) Surface 7

b 8 3 3 onto volcano plots of FRCs, LECs and BECs from untreated or 12 h mice. Key indicates indicates Key or 12 h mice. untreated from BECs and LECs of FRCs, plots volcano onto 4 3 10 . . The SLRP

4 4 5 20 5 6 30 7 6 BEC FR LE Other associate Extracellular matrix Acute phasereponse Cytokines andchemokines Interferon orTLR4 MHCI Biologically interestin 113-probe delta-scorelis Part ofthepooled - - - f

C C a – I c unchanged on DNCs. The presentation of antigens by LNSCs via via LNSCs by antigens of presentation The DNCs. on unchanged remained expression its whereas ovalbumin, and lipopolysaccharide of injection after h 18 BECs and LECs FRCs, on higher indeed was II MHC of expression Surface LGMN. upregulated also FRCs tion. DM and H2-E and (H2-A molecules II class MHC of chains various of sion antigen-processing and antigen-presentation pathway. Notably, expres chemokines inflammatory CCL5 the and CXCL9,response, and acute-phase key the components in pathways, involved of these the molecules MHCregulate class that II molecules encoding genes as well as genes, TLR4-inducible of interferon-and/or expression upregulating the steady state, responded vigorously to the onset of inflammation by stromal resistance to mediated destruction by the immune system promoting by architecture lymph-node preserve to hypothesized is activation of T cells by FRCs less in results receptor, virus-recognition another TLR3, of Ligation virus Dengue and Westvirus Nile virus, influenza against defense poietic cells. IFITM-1, IFITM-2 and IFITM-3 have been linked to host LNSCs had higher expression Furthermore, profiled. of populations IFITM-2other did andthan IFITM-1 IFITM-3protein than did hemato produced more transcripts for the interferon-inducible transmembrane masome that recognizes influenza and adenovirus inflam the of part NALP3, for transcripts lacked LNSCs Although ) or two independent experiments with five to six mice per condition ( condition per mice to six five with experiments ) independent or two Lipocalin Lipocalin Lipocalin d We found that LECs and FRCs, of all BECs, express TLR4 in which β 2 was higher in FRCs and BECs after the onset of inflamma of onset the after BECs and FRCs in higher was 2 2 2 2 g β aDV t 1), the invariant chain (CD74), CTSS, H2-DM CTSS, (CD74), chain invariant the 1), d LYVE-

Desmin Events pNAd A UT (% of max) 100 NCE ONLINE PUBLIC ONLINE NCE 20 40 60 80 0 10 1 d MHCII Isotype UT 18 0 . Each symbol represents an individual mouse; mouse; an individual represents symbol . Each 10 δ h 1 = 1.0 (adjusted FC = (adjusted 1.0 BEC FR 10 C 2 10 3 10 Merge Merge Merge 4 6 . . Exposure of LNSCs to infectious agents DNC LE + C stroma. Scale bars, 25 bars, Scale stroma. Lipocalin Lipocalin Lipocalin A 2 TION e -transformed, row-mean-centered row-mean-centered -transformed,

≥ MHC II expression

2); 2); (MFI) 20 40 60 2 2 2 0

P UT nature immunology nature FR < 0.05). Blue indicates indicates Blue < 0.05). * * 18 L C LYVE- Desmin pNAd h 18 3 8 UT h , , FRCs and DNCs EC 1 18 µ U h m. Data are m. Data BEC T α α * 18 , H2-A , and H2- and Merge Merge Merge U h T DNC NS d 18 6 – , β f 3 4 h ). 9 0 1 - - - ­ . .

© 2012 Nature America, Inc. All rights reserved. Alternatively, FRCs in the T cell zone may also produce CXCL13 CXCL13 produce also may zone cell T the in FRCs subset. Alternatively, CXCL13-expressing discrete a form may zone cell B the in ER-TR7 ing bright CXCL13 were associated with desmin-positive FRCs surround ensheath sparse conduits of CXCL13 localization to B where confirmed rare cell follicles, FRCs Unexpectedly, CXCL13 we expression observed in FRCs. Microscopy CXCL12. chemokine homeostatic the of expression high had DNCs encoding gene the IL-7, in contrast transcribed to published reports LECs and FRCs both addition, In before reported as produced substantial quantities of transcripts for CCL19 and CCL21a, FRCs Although cells. B or cells T memory of egress steady-state the stroma thymic to similar DCs, node–resident lymph maintaining in FRCs for role unknown a previously suggested which profiled, subsets etic ­expression of transcripts for Flt3L among highest the stromal the and hematopoi had FRCs survival. and organization cells, recruitment, B their DCs, cells, T memory on macr act may that chemokines and restricted. FRCs and, subset to a lesser extent, be DNCs produced transcripts to for cytokines thought genes many of expression stromal shared was finding A notable of crosstalk. aspects undescribed ously previ for niches hematopoietic and stromal lymph-node examined processes. similar regulates FRCs in signaling cadherin-11 whether arthritis rheumatoid in cytes synovio of ECM by production fibroblast-like and altered cytokines and bone adhesion, division, infiltration of production inflammatory more with associated is cadherin-11 of Upregulation origin. chymal mesen common a reflected which SFs, and ThFs in cadherin-11 for transcripts observed Wealso FRCs. between junctions to localized cadherin-11 that Wefound contacts. cell-cell these about known is ensheath they that conduits the along other each specialization. immune-related demonstrated further molecules involved in receptor cytokine–cytokine inte uniquely showed enrichment for higher expression of genes encoding tors and lymph flow for antigen presentation and contractility. Exposure to immune media distinctive profile. Both FRCs and ThFs were better equipped than SFs a had population each characteristics, many sharing Despite organs. lymphoid in fibroblasts of specialization the study to SFs and ThFs FRCs, of transcriptomes the We compared. examined systematically been have not tissues various from populations however, fibroblastic aim; therapeutic important an therefore is organs lymphoid tertiary ated with poor outcomesclinical environments node–like lymphoid organs) and lymph in support tumors, they where (tertiary inflammation chronic of sites at appear fibroblasts specialized larly of iron availability the by decreasing of infection stages early during expansion bacterial limit may LNSCs by LCN2 of secretion molecule iron-sequestering antimicrobial potent a LCN2, produced BECs and LECs FRCs, that found also We conditions. inflammatory under LNSCs in pathway role of the MHC class II and antigen-processing antigen-presentation during chronic immune responses. Future studies should elucidate the ance or the generation of regulatory T cells in inflammatory settingsCD4 of or induction the to contribute may II class MHC nature immunology nature regulated. tightly be may synthesis protein but transcripts, LNSCs interact closely with hematopoietic cells. Accordingly, we we Accordingly, cells. hematopoietic with closely interact LNSCs with closely interact FRCs fibroblasts, interstitial to contrast In simi organs, lymphoid of secondary are residents FRCs Although o phages and natural killer cells, which probably contributes to to contributes probably which cells, killer natural and phages 4 4 . Meanwhile, CCL20 expression by LECs may contribute to to contribute may LECs by expression CCL20 Meanwhile, . + conduits (data not shown), which suggested that FRCs FRCs that suggested which shown), not (data conduits 2 4 , we observed lower expression in other LNSCs. LNSCs. other in expression lower observed we , 1 probably regulates the FRC transcriptome. FRCs

13 4 5 aDV . . We found that 11.6% , 1 3 4 2 . These FRC-like cells are often associ often are cells FRC-like These . 26 , after the onset of inflammation. The The inflammation. of onset the after , A NCE ONLINE PUBLIC ONLINE NCE , 4 13 3 . Future studies should determine determine should studies Future . , 1 4 . . Controlling the development of 2 . . Furthermore, FRCs, BECs and ± 8.2% of areas with 4 A 2 TION ; however, little little however, ; r actions, actions, which + T cell toler cell T

3 2 . ------

analysis of lymphoid organ stroma. Our data simultaneously sup and providing while ported published ready findings extended access simultaneously data Our stroma. organ lymphoid of analysis specialization. functional demonstrate vessels IAP-ensheathed whether and FRCs and IAPs between ship relation developmental any elucidate to needed are IAPs. studies Further called we that cells contractile highly as identified sequently sub DNCs, of >50% for marker surface effective an was ITGA7 that matrices. collagen three-dimensional contracted of FRCs, and cultured DNCs formed elongated networks that strongly those to relative function contractile in involved molecules for ment chemokines and growth factors. DNC-specific probes showed enrich FRCs in terms of gene global expression and production of cytokines, ECs. of proliferation and survival the promote FRCs origin, hematopoietic of cells of homeostasis the regulating to addition in that likely it is Thus, receptors. relevant the sion of Notably, ANGPTL4. for BECs and transcripts LECs produced GREM1, including and HGF VEGF-C molecules, lymph-angiogenic and angiogenic our analysis showed FRC-restricted expression of transcripts for other nance of ECs. In addition to confirming FRC expression of VEGF-A reprints/index.htm R Published online at The authors declare no competing interests.financial and assisted with the analysis of individual experiments. experiments; and S.K.C., M.B.B., D.J.M., M.C.C. and M.E.H. contributed reagents data; V.L.-K., P.T., S.F.G., J.A., K.G.E. and K.K. did and analyzed individual results, and wrote the manuscript; D.M. did analysis primary of the microarray the manuscript; S.J.T. anddesigned directed the study, analyzed and interpreted D.M. and A.L.F. the designed study, did and analyzed most experiments, and wrote European Union (Marie Curie International Outgoing Fellowship 220044 to S.F.G.). Cancer Institute (K.K. and V.L.-K.) and the Seventh Framework Programme of the R01 DE019917 to D.J.M.; and GM38903 to M.E.H. and K.K.), the Dana-Farber S.J.T.; R24 AI072073 to the ImmGen Consortium; R01 AI063428-06 to M.B.B.; by the US National Institutes of Health (R01 DK074500 and P01 AI045757 to Project; and K.W. Wucherpfennig for reading critical of the manuscript. Supported and data; ExpressioneBioscience, Affymetrix Analysis for support of the ImmGen stromal-cell populations; J.B. forLewis discussions during analysis of microarray polyclonal anti–collagen XIV; M. forCurry technical assistance in sorting anti-fibromodulin and anti-biglycan; M. Koch (University of forCologne) We thank L. Fisher (US National Institutes of Health) for polyclonal anti-decorin, Note: Supplementary information is available on the GSE15907. etic), codes. Accession online the of at­version paper in the http://www.nature.com/natureimmunology/. available are references associated any and Methods M study.of avenues new many for data supportive tion, support structural and regula stromal-cell homeostasis, which provides immune of facets many in involved closely are LNSCs that an resource, immunological this analysis suggests expression-patterning As apparent. became interaction of webs plausible cells, poietic LNSCs. By comparison of stromal data with data gathered for hemato to by a expressed comprehensive determinants of database molecular C AUTH A eprints and permissions information is available online at at online available is information permissions and eprints ck O ethods Here we aimed to provide the first comparative transcriptome transcriptome comparative first the provide to aimed we Here Little is known about the DNC niche. DNCs most closely resembled mainte the to contribute FRCs that suggested data our Notably, MPETI now O R R C N ledgme ON G G FI TRIBUTI N l . http://www.nature.com/natureimmunology A GEO: ImmGen raw data (stromal and hematopoi and (stromal data raw ImmGen GEO: N n 4 CIAL CIAL I ts 6 , whereas FRCs, LECs and DNCs shared expres and shared DNCs LECs FRCs, , whereas ON N S TERESTS N a t u r e

e c r u o s e r I m http://www.nature.com/ / In vivo In m .

u n o l o g , we found found we , y website.

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© 2012 Nature America, Inc. All rights reserved. Boston, Massachusetts, Boston, USA. Massachusetts, 22 University, Boston, USA. Massachusetts, Science Department, Brown University, Providence, Rhode Island, USA. Disease Institute, Children’s Hospital, Boston, USA. Massachusetts, Immunobiology, Harvard Medical School, Boston, USA. Massachusetts, Institute, Boston, USA. Massachusetts, California, USA. 10 Ananda Goldrath JakubzickClaudia Tal Shay David Laidlaw Joonsoo Kang Angelique Bellemare-Pelletier Lewis L Lanier Yan Zhou The complete list of authors is as follows: 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. e c r u o s e r 12 University, Boston, USA. Massachusetts,

Department Department of Medicine, Harvard Medical School, Boston, USA. Massachusetts, Fox Chase Cancer Center, USA. Pennsylvania, Philadelphia,

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© 2012 Nature America, Inc. All rights reserved. Expression value probabilities. value Expression platform. analysis genomic GenePattern the of module ExpressionFileCreator the in algorithm average multichip robust the with Raw data were normalized manufacturer’swith accordance instructions. in Assay Labeling Target Sense (WT) Transcript Whole GeneChip the with array ST 1.0 MoGene Affymetrix the to hybridization for prepared and fied hybridization and Microarray data normalization. described as isolated was RNA FACSAria. a with samples of analysis immediate by followed PBS) and EDTA mM 2 FBS, (2% buffer cytometry flow into cells of by sorting assessed 20 psi). cells Dead were excluded with propidium (5 iodide Purity was ng/ml). ( (140 mM NH sliced with a razor blade and digested as described per group) were placed in ice cold RPMI medium, 2% FBS, then were split and stroma and sorted as described ( mice C57BL/6 from isolated were nodes nodes lymph lymph mesenteric and brachial) and Skin-draining axillary Project. (inguinal, ImmGen the of protocols operating sorting. and enrichment Cell Invitrogen). (S-32354; A488 Fluor streptavidin–Alexa and Invitrogen) (R415, TRITC-phalloidin Invitrogen), Fluorstreptavidin–Alexa 555 (S-32355; Invitrogen), DAPI (D3571; R&D), (AB946; ADAM10 anti–mouse polyclonal Fluor BD), IgG–Alexa anti–mouse 488 biotin (550331; (A-11029; Invitrogen), IgG1– anti–mouse Invitrogen), (A-10518; IgG–biotin anti–goat Invitrogen), (A-11055; 488 Fluor IgG–Alexa anti–goat Invitrogen), (A-31572; 555 Fluor anti-rabbit IgG–Alexa Fluor 647 (A-31573; Invitrogen), anti (A-21247; Invitrogen), immunogl anti–rabbit IgG–Alexa Fluor 488 (A-11034; Invitrogen), 647 anti–hamster Fluor IgG–Alexa anti–rat Invitrogen), (A-21110; 488 Fluor Systems), (IgG)–Alexa R&D (AF1857; anti-LCN2 pol eBioscience), (ALY7; 488 Fluor anti-LYVE-1–Alexa (MECA-79; Biolegend), anti-pNAd–biotin Abcam), (ab15200; polyclonal-anti-desmin LF-113; (batch Fisher L. polyclonal–anti-mouse-decorin Fisher), L. anti-fibromodulin LF-150; polyclonal (batch Epitomics), (EP7984; anti-CNN1 Systems), anti– Abcam), (ab51824; smooth muscle ER-TR7 actin (1A4; Sigma-Aldrich), anti-ITGA7-FITC (3349908; R&D Abcam), (ab28023; anti-vitronectin Koch), (M. XIV anti–collagen polyclonal Abcam), (ab6588; VI anti–collagen I-E (M5/114.15.2; Biolegend), anti–mouse CD44 (IM7; eBioscience), polyclonal and I-A anti–mouse M.B.), MOPC21; antibody control isotype-matched and science), anti-MAdCAM-1 (MECA-367; eBioscience), anti-cadherin-11 (13C2 from the Developmental Studies Hybridoma Bank), anti-CD140a (APA5; eBio from a hybridoma in-house purified (8.1.1; anti-gp38 Biolegend), (MEC13.3; anti-CD31 (Ter119; Biolegend), anti-Ter119 Biolegend), (30-F11; anti-CD45 conjugates. and Antibodies the of analysis for protein. LCN2 and II injection class MHC of expression after h 18 collected were SLNs analysis. array micro for BECs and LECs FRCs, of sorting for injection after h 12 collected 500 and intravenous Sigma-Aldrich) received mice later, h 18 30 of injection Then, mice. C57BL/6 to venously inflammation. of Induction Institute. Cancer Farber Experiments approval received atfrom Care Animal the subcommittee Research the Dana- guidelines. Health of Institutes National US under and for cared institutional were Mice Institute. Cancer Dana-Farber the at (Taconic) bred 2 were gene recombination-activating in deficient mice OT-I ditions. con pathogen–free 1 specific under shipped week before use and maintained from Laboratory, the Jackson mice C57BL/6 male were 6-week-old used mice Mice. METHODS ONLINE doi:10.1038/ni.2262 the across expression of distribution the from determined were expression single round of sorting) directly into TRIzol with a FACSAria (100- According to standard operating protocols of the ImmGen Project, all all Project, ImmGen the of protocols operating standard to According 4 7 , oylnlat-os-ilcn bth F19 L Fisher L. LF-159; (batch polyclonal-anti-mouse-biglycan ), 4 Cl and 17 mM Tris-base, pH 7.4) and cells were stained and sorted µ g lipopolysaccharide from from lipopolysaccharide g n = 10–30), then were digested, enriched for CD45 for enriched digested, were then 10–30), = 4 µ 9 g ovalbumin (A5503; Sigma-Aldrich). SLNs were were SLNs Sigma-Aldrich). (A5503; ovalbumin g . The following antibodies and stains were used: used: were stains and antibodies following The OT-I T cells (1.5 × 10 × (1.5 cells OT-I T 6 , Cells were prepared according to standard standard to according prepared were Cells 4 8 Thresholds for likelihood of positive gene gene positive of likelihood for Thresholds . . For skin fibroblasts, mouse ears ( E. coli E. 6 (serotype 0127:B8; L3129; L3129; 0127:B8; (serotype . . Red blood cells were lysed 6 Isolated RNA was ampli ) were transferred intra transferred were ) – rabbit IgG–Alexa µ n o m nozzle; = 5 mice ui G bulin y clonal clonal 4 α 7 ), ), − - - - - - ­

Probes were ranked according to the following ratio: s.d. across/s.d. within. within. across/s.d. s.d. ratio: following the to according ranked were Probes across). (s.d. populations cell all across deviation standard single a calculate to values expression mean uses and within), (s.d. type cell each vari within ability reflects that value deviation standard single a generate to values these values to compute the standard deviation within each population and averages replicate uses module the subset), any for 120 > (EV probes expressed manner: all for unsupervised an in identified were probes variable most 15% The for log PCA PCA. used populations across expression in difference greatest the with probes of 15% the and used identified module This among populations. variability maximal for account to components principal earlier with uncorrelated is component principal Each components. principal into data multidimensional complex, simplifies PCA GenePattern. for module Plot PCA Population ImmGen the Population distance measurements. Project. ImmGen the from sion, was as used the standard cutoff for gene for expression most populations to a of corresponds 95% probability expres which of normalization, 120 after could be applied to all samples from the ImmGen Project. An expression value value a single whether determine to examined was of cutoffs and distribution the sample, each for calculated were Thresholds expression. of probability at a single to arrive combined were model the in of components Probabilities T lymphocytes and B lymphocytes RNA from same the from obtained sets data of parallel by comparison confirmed an initially approach of expression, trols, a Gaussian mixture model was con used to negative determine probabilistic reliable thresholds contain not do arrays ST1.0 the Because microarrays. Pathway chart was selected. Count requirements for genes were set as 0 and and 0 as set were genes for requirements Count selected. was chart Pathway KEGG_ the option, Pathways In the chip. MoGene-1_0-st-v-1 the to set was for symbols gene official as uploaded were DAVID of Tool Annotation Functional the with analyzed were lists signatures. gene in enrichment of functional Analysis ( by Excel. analysis subsequent for combined were expression in differences with probes of Lists of FC adjusted an A populations. two the between differences ( adjusted score delta resulting The populations. both of deviation standard geometric the of sum the of square the of subtraction by replicates within for noise are penalized values FC to FC). of a 2 power corresponds (so of means class the between FC the populations. two the of respectively, where follows: as defined were expression in differences effective stromal populations, two For identify manner. pair-wise a to in signatures used cell–specific was GenePattern for module Score Delta Project analysis). (delta-score determination Stromal gene-signature GenePattern. of module HierarchicalClusteringViewer the with visualized then normalized, log were Data GenePattern. of module HierarchicalClustering the with linkage) average and (Pearson’scorrelation clustered then one population), EV for > at 120 least comparison; population probes with different expression were identified (FC > 2 in at least one pair-wise analysis. clustering Hierarchical GenePattern. of module HeatMapImage the with generated were computed with R software (the R project for computing). statistical Heat maps log cients and Euclidean distances were with calculated the same probes. Data was coordinate-axes onto which samples were projected. Pearson correlation coeffi top 15% of were these The used. components first three principal were used as Probes with the highest ratios were determined to be the most variable, and the P < 0.05; 0.05; < Probes subsequently identified as having significantly different expression expression different significantly as having identified subsequently Probes 2 transformed and row standardized. Correlation and distance values were values and distance Correlation and row transformed standardized. µ and and t -test) were used (Multiplot module of GenePattern). of module (Multiplot used were -test) σ are the geometric mean and geometric standard deviation, deviation, standard geometric and mean geometric the are ≥ 2, a a 2, d 2 , ( δ -transformed and row- and column-normalized data. and row- -transformed and column-normalized b a of 2 corresponds to an adjusted FC of of FC adjusted an to corresponds 2 of l ) = og With the Multiplot of module GenePattern, 2    a and and m PCA was done on expressed probes with m

b a The ratio in the first term represents represents term first the in ratio The 2    transformed, row centered and row and centered row transformed, + − b . This value was log was value This . ( Mus musculus Mus s s b a nature immunology nature ) 2 δ of 1 corresponds to to corresponds 1 of Population . The background background The . δ ) reflects noise- reflects ) 2 The ImmGen ≥ transformed transformed 4 and so on. on. so and 4 - a 1 specific specific 6 and and . Lists Lists . 4 b 9 ­ - - - , .

© 2012 Nature America, Inc. All rights reserved. FACSAria (BD Biosciences) and analyzed with FlowJo v.8.7.3 software. saline with 2% FBS and 1 mM CaCl ing of cadherin-11, cells were prepared and stained Flowin cytometry.EDTA-free HEPES-buffered described as microscopy. Electron CS. Photoshop Adobe and ImageJ (NIH) with and SP5X) analyzed (Leica microscope confocal scanning laser- a with visualized were Sections conjugates. secondary with incubated with 4% PFA, with 0.2% permeablized Triton-X-100 fixed (T-9284; were Sigma) and then Cells antibodies. primary with incubated and above described as coverslips coated with fibronectin Invitrogen).(33016-015; Cells were blocked × 2.5 10 FRCs, on cultured ing of cadherin-11 For stain (Sigma). ABC chondroitinase with 1 for h °C at 37 pretreated were with primary and antibodies. secondary For staining of fibromodulin, sections were cut and blocked with FcR block (2.4G2) and 2% BSA before 7.5 being stained Sections then frozen. being and before sucrose paraformaldehyde 30% in 4% overnight in h 4 for fixed and were CNN1 nodes lymph ITGA7, LCN2, of staining For compound. temperature cutting mum microscopy. Immunofluorescence visualization. for used was distance) Euclidean in mean the to probe probe closest (the a representative probes, multiple with genes single details. analysis microarray general Other procedure). (Benjamini ‘hits’ with significant biologically presented or are Top-ranked 1. as set was score EASE the nature immunology nature 5 0 . Lymph nodes were digested and stained as described P Popliteal lymph nodes were prepared and analyzed analyzed and prepared were nodes lymph Popliteal values corrected by the multiple-hypothesis test test multiple-hypothesis the by corrected values 2 . Samples were acquired on a FACSCalibur or Lymph nodes were snap-frozen in opti in snap-frozen were nodes Lymph For bar graphs and heat maps of maps heat and graphs For bar 4 cells were plated overnight on overnight were plated cells µ m in thickness thickness in m 6 . For stain - - -

49. 48. 47. procedure. Hochberg and Benjamini with the ComparativeMarkerSelection module of GenePattern, which uses the with GraphPad Functional Prism 5. DAVIDFalse-discovery-rate-adjusted the of option Annotation Tool. Benjamini the with testing hypothesis lists, gene in enrichment functional For Statistics. CCAGAACACCTACA. TTGAAATCACT CXCL13R, and TGGCCAGCTGCCTCTCTC; CXCL13F, described been all RT-PCR. width. tube by total divided matrix collagen of width the as measured was were obtained with a Nikon camera and analyzed with ImageJ, and contraction in Biosciences) (BD described 10 as × cultured were (5 SLNs from FRCs confluent. 70% until assay. Contraction 50.

Painter, M.W., Davis, S., Hardy, R.R., Mathis, D. & Benoist, C. Transcriptomes of Transcriptomes C. Benoist, & D. Mathis, R.R., Hardy, S., Davis, M.W.,Painter, A. Fletcher, human to probes cDNA and Antisera M.F. Young, & III J.T. Stubbs, L.W.,Fisher, Gonzalez, S.F.Gonzalez, dependent differences in fibroblastic reticular cells. reticular fibroblastic in differences dependent (2011). Suppl. and certain animal model bone matrix noncollagenous proteins. s seta fr uoa imnt i dann lmh nodes. lymph (2010). draining 427–434 in immunity humoral for essential is 186 profiling. microarray multiplatform by compared lineages T and B the , 3047–3057 (2011). 3047–3057 , 5

RNA extraction, cDNA preparation and PCR cycling conditions have ) were suspended in 500 500 in suspended were ) Multiplot (GenePattern) was used for computation of 266 , 61–65 (1995). 61–65 , et al. et et al. et P 6 values for the . Primers (Integrated DNA Technologies) were as follows: follows: as were DNA Technologies) (Integrated . Primers Reproducible isolation of lymph node stromal cells reveals site- reveals cells stromal node lymph of isolation Reproducible C2C12 myoblasts and NIH3T3 fibroblasts were cultured cultured were fibroblasts NIH3T3 and myoblasts C2C12 Capture of influenza by medullary dendritic cells via SIGN-R1 via cells dendritic medullary by influenza of Capture α MEM) and seeded into tubular constructs. Images Images constructs. tubular into seeded and MEM) χ 2 test with Yates’ were correction computed µ gl 1 gm rt al olgn I collagen tail rat mg/ml (1 gel l P values were corrected for multiple multiple for corrected were values P rn. Immunol. Front. values were calculated doi:10.1038/ni.2262 Acta Orthop. Scand. a. Immunol. Nat. t -test J. Immunol. J. P 6

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