Identification of TROP2 (TACSTD2), an EpCAM-Like Molecule, as a Specific Marker for TGF-b1-Dependent Human Epidermal Langerhans Cells

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1 ORIGINAL ARTICLE Identifition of (TACSTD2), n -Like Moleule, s Speifi Mrker for TGF-1-Dependent Humn Epiderml Lngerhns Cells Gregor Eisenwort 1, Jennifer Jurkin 1, Night Ysmin 1, Thoms Buer 1, Bernhrd Gessluer 1 nd Herert Strol 1 Lngerin () expression is hllmrk of epiderml Lngerhns ells (s); however, þ ells omprise severl funtionl susets. Murine studies showed tht epiderml, ut not derml, þ ells require trnsforming growth ftor- 1 (TGF-1) for development, wheres humn dt re lking. Using gene profiling, we found tht the surfe moleule (TACSTD2) is strongly nd rpidly indued during TGF-1- dependent ommitment of humn CD34 þ hemtopoieti progenitor ells or monoytes. is onserved etween mouse nd humn, nd shres sustntil mino-id identity with, mrker for murine epiderml s. To our knowledge, neither nor expression hs een nlyzed in humn dendriti ell (DC) susets. We found tht (i) ll humn epiderml s re þ þ ; (ii) humn dermis lks þ or þ DCs, i.e., equivlents of murine derml þ DCs; nd (iii) pulmonry þ ells re. Moreover, lthough ws rodly expressed y pulmonry nd intestinl epithelil ells, s well s y one mrrow erythroid progenitor ells, these ells lked. However, lthough is expressed y humn s s well s y humn nd murine kertinoytes, most murine s, exept of smll suset, lked. Therefore, is mrker for humn TGF-1-dependent epiderml s. Journl of Investigtive Dermtology (211) 131, ; doi:1.138/jid ; pulished online 16 June 211 INTRODUCTION Dendriti ells (DCs) re professionl ntigen-presenting ells (APCs) tht n e found in vriety of lymphoid nd nonlymphoid tissues (Merd nd Mnz, 29). Although they re promising trget for vine development nd tumor immunotherpy, their funtionl spetrum is not fully understood (Pluk et l., 211; Ueno et l., 211). Furthermore, most funtionl studies nlyzing speifi DC susets re onduted in mie. In order to trnslte findings from murine models to the linis, etter interspeies orreltion of DC susets is prerequisite. Aordingly, humn equivlents of murine DC susets suh s CD8 þ DCs resemling humn DNGR1 þ DCs hve reently een disovered (Bhem et l., 21; Crozt 1 Institute of Immunology, Center of Pthophysiology, Infetiology nd Immunology, Medil University of Vienn, Vienn, Austri Correspondene: Herert Strol, Institute of Immunology, Center of Pthophysiology, Infetiology nd Immunology, Medil University of Vienn, Lzrettgsse 19/3, 19 Vienn, Austri. E-mil: herert.strol@meduniwien..t Arevitions: DC, dendriti ell;, Lngerhns ell; modc, monoytederived dendriti ell; mo, monoyte-derived Lngerhns-like ell; TGF-1, trnsforming growth ftor- 1 Reeived 29 Mrh 211; revised 26 April 211; epted 28 April 211; pulished online 16 June 211 et l., 21; Jongloed et l., 21; Poulin et l., 21). Considering inresing evidene for dditionl DC susets identified in mouse (Br-On et l., 21; Henri et l., 21), etter phenotypi nlysis of humn DC susets is neessry. Lngerhns ells (s) re the DCs of the epidermis. In ontrst to other DC su-popultions, they hve trditionlly een defined y their expression of the C-type Letin reeptor Lngerin () (Romni et l., 21). With the reent disovery of þ, derml DCs, nd the ft tht is lso expressed y CD8 þ DCs in lymphoid tissues in mie, the need for previously unreported speifi mrkers eme evident. The epithelil dhesion moleule n e used to study murine skin DC susets (Borkowski et l., 1996). Derml nd epiderml þ DCs n e disriminted y the expression of (Bursh et l., 27; Ginhoux et l., 29; Ngo et l., 29). However, despite these importnt oservtions in mie, dt in the humn system re still lking. In our study we imed to identify previously unreported mrker moleules for humn epiderml s. Trnsforming growth ftor- 1(TGF-1) ddition to ultures of monoyti ells is suffiient to indue n phenotype (Strol et l., 1996). Therefore, we sreened for TGF-1 response genes during linege ommitment using genome-wide rry pproh. & 211 The Soiety for Investigtive Dermtology 249

2 We here identified, n evolutionry highly onserved moleule, whih shows 48% mino-id identity to (El Sewedy et l., 1998), s speifi mrker for humn epiderml s. RESULTS Identifition of s TGF-1 response gene during Lngerhns ell ommitment of CD34 þ ells We reently undertook gene rry profiling of purified humn myeloid progenitor ells undergoing TGF-1-dependent ommitment nd differentition (dt not shown). In this sreen, serum-free ultures of flow-sorted myeloid-ommitted CD34 þ RA þ CD19 ord lood ells were supplemented with omintion of ytokines tht promotes monoyti ell differentition. Addition of TGF-1 redireted monoyte to differentition, s previously desried y our group (Strol et l., 1997; Heinz et l., 26). Among the strongest indued genes t 6 nd 24 hours fter TGF-1 stimultion ws TACSTD2 (). Similr to, is expressed y epithelil tumor ells; in ddition, flow-sorting experiments demonstrted tht expression llows to positively identify prostte stem ells nd hepti ovl ells (Goldstein et l., 28; Oke et l., 29). However, expression y ells of the hemtopoieti system ws to our knowledge not desried so fr. Although ws strongly indued in response to TGF-1 stimultion, showed only wek indution (Figure 1). Congruent with this, progenitor ells show muh higher indution of thn mrna levels in TGF-1- dependent dy 7-generted s (Figure 1). Protein expression nlyses reveled tht in vitro-generted þ s re þ þ (Figure 1). Prllel ultures in whih TGF-1 hd een omitted gve rise to monoyti ells nd CD1 þ ells lking (Figure 1 lower pnel nd dt not shown); these ells lked detetle expression of or (Figure 1, lower gtes). s my grdully lose epithelil-ssoited mrker moleules upon migrtion from epidermis to lymphoid tissues or during in vitro mturtion, s previously demonstrted for E-dherin (Jko nd Udey, 1998; Riedl et l., 2). Therefore, we nlyzed whether tivtion/mturtion is ssoited with hnges in or expression. In vitro-generted CD34 þ progenitor ell-derived s were luster purified s demonstrted previously (Gtti et l., 2), nd susequently indued to undergo mturtion in response to tumor nerosis ftor- plus IL-1 stimultion. mturtion ws mrked y strong upregultion of CD83 nd CD86; verge men fluoresene intensities of nd slightly Reltive upregultion CD1 d Reltive MFI Untreted ells 6 Hours 24 Hours 6 Hours 24 Hours Reltive mrna levels + TGF-β1 TGF-β1 5, 4, 3, 2, 1 *** *** *** *** Reltive upregultion * * * ** + TGF-β1, dy 7 TGF-β1, dy 7 + TGF-β1 TGF-β1 CD1 + + CD1 + CD1 CD1 + CD1 CD1 + / + CD1 + / CD1 / CD1 + CD1 Figure 1. is indued upon TGF-1-dependent ommitment of CD34 þ ells. () Brs represent men upregultion of nd t 6 nd 24 hours reltive to hours fter initition of Lngerhns ells () ultures±trnsforming growth ftor (TGF)-1 (n ¼ 6 donors). () Rel-time PCR nlysis of nd expression fter 7 dys of ultures (n ¼ 4 donors). () Cells were gted fter 7 dys nd were nlyzed for or expression. (d) Dy-7 s were luster purified nd stimulted with GM-CSF, tumor nerosis ftor-, nd IL-1 for 48 hours or GM-CSF lone. Empty histogrms in nd d represent isotype ontrols; rs in nd represent the men nd SEM of three independent experiments. *Po.5; **Po.1; ***Po.1. MFI, men fluoresene intensity; NS, not signifint. Reltive MFI TNFα IL-1β 1, CD83 CD86 *** Untreted ells TNFα + IL-1β NS NS CD86 25 Journl of Investigtive Dermtology (211), Volume 131

3 inresed during mturtion (Figure 1d). We onlude tht is indued y TGF-1 during ommitment/ differentition of myeloid progenitor ells, nd oth nd show stle expression during mturtion of in vitro-generted þ s. Erythropoieti ells express ut not In order to evlute nd s potentil -speifi mrker moleules, we systemtilly nlyzed their expression mong hemtopoieti ells. Norml peripherl lood leukoytes lked detetle expression of oth mrkers (Figure 2). Conversely, minor suset of one mrrow ells expressed ut not (Figure 2). nd E-dherin were previously found to e expressed y humn erythropoieti ells (Lmmers et l., 22). Congruent with these oservtions, most gted þ one mrrow ells represented glyophorin-a þ erythroid progenitor ells (Figure 2). As it is known tht erythropoiesis trnsits through n þ E-dherin þ stge nd tht TGF-1 elertes in vitro erythropoiesis (Zermti et l., 2), we nlyzed whether TGF-1 might indue expression in erythropoieti progenitors. Thus, CD34 þ ord lood ells were indued to differentite to erythropoieti ells in response to erythropoietin, IL-3, nd stem ell ftor in SFM. Prllel ultures were further supplemented with TGF-1. FACS nlyses onfirmed previous oservtions (Zermti et l., 2): In the sene of TGF-1, erythroid progenitors first quired inresing CD71 expression; this ws followed y the indution of glyophorin-a y CD71 hi ells (Figure 2). TGF-1 promoted the trnsition to glyophorin-a þ ells (Figure 2, left pnel), with most ells positive for glyophorin-a nd showing redued CD71 expression (Figure 2, lower pnel). Although erythroid ells o-expressed E-dherin nd s expeted from previous study (Lmmers et l., 22), these ells lked detetle expression (Figure 2). Moreover, TGF-1 filed to indue expression y erythropoieti ells (Figure 2). These dt demonstrte tht is not expressed y one mrrow erythropoieti ells, even when stimulted y TGF-1, wheres erythropoiesis trnsits through n þ E-dherin þ ell stge. is strongly expressed y mos, wheres these ells lk Previous studies reveled tht the ddition of TGF-1 to GM- CSF plus IL-4-supplemented ultures of humn monoytes indues the genertion of DCs showing phenotypi hrteristis of s (mos) (Geissmnn et l., 1998). To test whether mo differentition is ompnied y nd/ or expression, CD14 þ monoytes were indued to differentite to lssil monoyte-derived DCs (GM-CSF/IL- 4) or mos (GM-CSF/IL-4/TGF-1). Alterntively, they were ultured in GM-CSF only to indue mrophge differentition. mos strongly expressed ut lked detetle (Figure 3). Additionlly, minor portion of monoyte-derived DCs in the sene of TGF-1 wekly expressed. Furthermore, mrophges were (Figure 3). Therefore, mos generted in the presene of TGF-1 strongly express, ut lk detetle expression. is expressed y epiderml Lngerhns ells nd kertinoytes in situ Stining of epiderml-ell suspensions reveled tht vile þ HLADR þ þ s (identified in Figure 4) re strongly positive for nd (Figure 4 nd ); HLA-DR kertinoytes (s) showed similr right stining s oserved for s; however, these ells showed weker expression of ompred with s (Figure 4 ). In situ immunofluoresene nlyses of in helthy humn rest skin reveled strong epiderml stining pttern, thus onfirming previous oservtions (Klein et l., 1987). Co-stining with showed tht s express (Figure 4d). In omprison with, showed weker stining of s; however, þ ells o-expressed to similr extent s oserved for (Figure 4e). These dt demonstrte tht s in situ o-express nd. High levels of expression on DCs in derml preprtions re restrited to migrtory s Derml þ ells were reently identified in mie, sed on the expression of in the sene of. Whether humn equivlents of murine derml þ ells exist hs, to our knowledge, not een studied. Humn derml DCs hve previously een extensively studied leding to the delinetion of t lest three DC susets (Klehevsky et l., 28). These inlude þ CD1 hi ells (Angel et l., 26), CD1 int ells, s well s CD1 CD14 þ DCs. Although ll three susets ould lerly e defined y flow ytometry (see gted popultions in Figure 5 nd dt not shown), high levels of nd expression were restrited to the smll þ CD1 hi suset. The CD1 int suset showed distintively lower, nd oth mrkers were sent from the CD1 CD14 þ suset (Figure 5, histogrms nd dt not shown). Therefore, we demonstrte here tht using similr stining strtegy s desried for mie, humn equivlents of murine derml þ ells nnot e delineted. DCs from lung nd gut re / Similr to the epidermis, þ DCs hve een desried in severl muosl surfes tht re in ontt with the environment, suh s the lung, orl vity, nd vgin (Hldik et l., 27; Allm et l., 21; Vn Pottelerge et l., 21). Conversely, the intestinl muos seems to e devoid of þ ells (Wtne et l., 27). We sreened lung nd olon tissue for the presene of þ þ þ ells. Although þ DCs ould e deteted within the pulmonry interstitil tissue, these ells lked nd expression;, however, ws expressed y the mjority of the ells (Figure 5, histogrms). Intestinl lmin propri did not ontin ny detetle þ DCs (Figure 5). The numerous HLA-DR þ DCs within the lmin propri were similr to their pulmonry ounterprts negtive for 251

4 Grnuloytes MNCs TGF-β1 CD71 SSC Isotype SSC Isotype Gph A + TGF-β1 Gph A E-Cd Figure 2. Lk of ut not expression mong erythropoieti ells. () FACS nlysis of neutrophili grnuloytes nd peripherl lood mononuler ells (PBMCs) for nd expression. () FACS nlysis of one mrrow spirte for nd expression. Empty histogrm represents isotype ontrol. () Hemtopoieti progenitor ells were indued to differentite in vitro from erythroid ells in response to erythropoietin, stem ell ftor, nd IL-3; prllel ultures were further supplemented with trnsforming growth ftor (TGF)-1. FACS nlyses of ells fter 5 dys of ulture. MNCs, mononuler ells; SSC, side stter. 252 Journl of Investigtive Dermtology (211), Volume 131

5 nd, wheres the intestinl ells were þ (Figure 5, histogrms). llows the identifition of minor sufrtion in mie Humn nd murine show 79% identil mino-id omposition (El Sewedy et l., 1998). Although expression within the murine skin hs een extensively studied (Ngo et l., 29), to our knowledge, hs not een nlyzed so fr. By exmining epiderml er sheets GM-CSF, IL-4, TGF-β1 of wild-type C57BL/6 mie, we found to e expressed y the mjority of s, similr to wht hs een desried in the humn system (Klein et l., 1987). However, most s (pproximtely 9%) lked (Figure 6). Anlyses of single-ell suspensions onfirmed the histologil dt: Although s were ll þ, the mjority of s lked SSC 74 7-AAD HLA-DR GM-CSF, IL-4 CD GM-CSF CD1 CD14 * * 12.5 d Reltive MFI Reltive MFI GM, IL-4, TGF GM, IL-4 GM Merge Isotype. Figure 3. is mrker for mos. () Surfe expression of CD1, CD14,, nd on the indited dendriti ell (DC) sutypes fter 5 7 dys of ulture. () Cells were gted s indited nd were nlyzed for nd expression. Empty histogrms represent isotype ontrols. Brs represent the men nd SEM of three independent experiments. *Po.5. GM-CSF, grnuloyte mrophge olony-stimulting ftor; IL-4, interleukin-4; MFI, men fluoresene intensity; TGF-1, trnsforming growth ftor-1. e Figure 4. nd expression y s nd kertinoytes isolted from humn skin nd in situ. () FACS nlysis of humn epiderml singleell suspensions. The gting strtegy to identify s nd kertinoytes () is shown. () nd expression y nd s gted in. Empty histogrms represent isotype ontrols. One representtive experiment of five is shown. () Reltive men fluoresene intensity (MFI) of nd. Cells were nlyzed s shown in nd. Brs represent the men nd SEM of three independent experiments. (d, e) Tissue immunofluoresene stining of humn rest skin ryosetions for (d) or (e) nd. Nulei re visulized with DAPI. Dt re representtive of four independent experiments. Brs ¼ 25 mm. SSC, side stter. Merge Isotype 253

6 Dermis Lung Colon 5.8 HLA-DR CD1.5 HLA-DR CD1 HLA-DR CD CD1 + CD1 + CD1 CD HLA-DR + Figure 5. nd re not expressed y humn derml-resident nd interstitil DCs. FACS nlysis of humn derml (), lung (), or olon () singleell suspensions. 7-Aminotinomyin D-negtive ells were gted on seprte FACS digrm (not shown) nd were nlyzed for versus HLA-DR expression. þ HLA-DR þ ells were gted nd were nlyzed for versus CD1 expression; histogrms represent phenotypilly defined ell susets nlyzed for or ; empty histogrms represent isotype ontrols. Dt re representtive of n ¼ 5 (dermis), n ¼ 4 (lung), nd n ¼ 2 (olon) experiments, eh performed with seprte donor smple. MHC-II expression. A minor frtion of þ ells were þ (i.e., 9.8±.9%). Furthermore, epiderml gd-tells, known to reside within þ MHC-II epiderml ell frtions, lk (Figure 6 nd ). MHC-II Merge TC Isotype TC Figure 6. expression in the murine epidermis. () Tissue immunofluoresene stining of murine epiderml er sheet for nd mjor histoomptiility omplex-ii (MHC-II). Nulei re visulized with DAPI. The rrowhed indites þ MHC-II þ ell. Brs ¼ 25 mm. () FACS nlysis of murine epiderml single-ell suspension. Gtes were set to otin ell frtions enrihed for kertinoytes (, ), gd-t-ells (TC, þ MHC-II ), nd ( þ MHC-II þ ). () expression y su-popultions gted in ; empty histogrms represent isotype ontrols; dt re representtive of 1 experiments., kertinoytes;, Lngerhns ells. DISCUSSION Anti- stinings reently enled the identifition of previously unreported murine DC suset, the so-lled derml þ DCs. expression is restrited to epiderml s; therefore llowing to sudivide derml þ ells into migrtory epiderml s ( þ ) nd derml-resident ells ( ). To our knowledge, hs not een evluted s ndidte mrker moleule for the lssifition of humn DC susets. Here we show tht nd re o-expressed y epiderml s. Moreover, we provide evidene tht humn dermis lks n nlogous popultion to murine derml þ ells, nd tht þ DCs from lung differ from s in tht they re mostly ; similrly intestinl DCs were. We furthermore showed tht, ut not, is strongly indued during TGF-1-dependent linege ommitment of monoytopoieti ells. Finlly, on the sis of nd expression, TGF-1-dependent CD34 þ ell-derived CD1 þ þ ells resemle s in vivo, therefore supporting other dt tht these ells re model ells of s. Among the DC susets nlyzed in this study, only skin DCs expressed nd. These þ þ DCs represent epiderml-resident s s well s derml-migrtory s, the ltter onstituting minor derml 254 Journl of Investigtive Dermtology (211), Volume 131

7 DC suset hrterized y high expression levels of (Angel et l., 26). Although derml-resident CD1 þ DCs were lso þ, these ells lerly showed lower expression levels ompred with s. Furthermore, these ells lked. Similrly, mos generted in the presene of GM-CSF plus IL-4 nd TGF-1 were þ. Therefore, mong DC susets, might e more speifi mrker for s thn. A key finding of our study is tht ll derml þ DCs o-expressed nd. The lk of þ ells in humn dermis, therefore, implies tht humn equivlents to murine derml þ DCs (previously identified s þ derml ells) do not exist. In this ontext, it is interesting tht ells with this phenotype ( þ ) ould redily e deteted in lung. Our dt therefore indite tht þ stinings in skin identify epiderml s, wheres þ lung DCs phenotypilly differed from s in tht these ells lked nd. This study is to our knowledge the first to demonstrte tht humn pulmonry nd epiderml þ ells show distint phenotypi hrteristis. These ells might represent humn equivlents of murine CD13 þ CD11 þ pulmonry DCs (Sung et l., 26), possiility tht might e tested using gene rry profiling. mrna is strongly indued within 24 hours during TGF-1-dependent linege ommitment of myeloid progenitor ells. Aordingly, the ddition of TGF-1 to GM-CSF/IL-4-supplemented monoyte-derived DC ultures resulted in the indution of long with the quisition of other mrkers. indution ws ssoited with TGF-1-dependent progenitor ell ommitment to s nd ws not generlly indued upon TGF-1 stimultion of progenitors; e.g., TGF-1 promoted erythroid progenitor ell differentition, however, this effet ws not ompnied y expression. Therefore, is mrker moleule for TGF-1-dependent s. Future detiled studies should ddress the moleulr mehnisms underlying TGF-1-dependent indution onomitnt with differentition. We demonstrted tht CD34 þ ell-derived TGF-1- dependent þ ells re þ þ ; from these hrteristis they resemled epiderml s. We previously demonstrted numerous ytoplsmi Birek grnules in these ells, indistinguishle from ex vivo isolted s (Strol et l., 1996). Together, these findings support tht these in vitro-generted DCs n e used s model ells for s. As mentioned ove, mos expressed ut lked. Therefore, these mos lk ertin hllmrk fetures of s. Our dt on -ssoited expression re omptile with previous oservtions tht TGF-1 is essentil for the development of s (Strol et l., 1996; Borkowski et l., 1996; Kel et l., 21), wheres it is dispensle for the development of derml DCs (Ngo et l., 29; Kel et l., 21). Therefore, right expression y s might e inditive for the requirement of s on onstitutive-tive TGF-1 signling. A positive reltionship of expression nd TGF-1 signling my lso exist for epithelil ells, s llowed the seletion of epithelil stem ells (Goldstein et l., 28) nd TGF-1 stimulted sphere formtion of epithelil stem ell (Wng et l., 211). It will e interesting to further study the smll suset of murine þ s. These ells might e enrihed in proliferting s, in nlogy to proliferting hepti progenitors (Oke et l., 29). Alterntively, þ s might ntomilly differ from s. Interestingly, humn epidermis is multilyered tissue, with s residing in sl/ suprsl lyers, wheres murine epidermis ontins only few lyers. Furthermore, expression is diminished in sl lyers (Klein et l., 1987), therefore showing similr expression pttern s reported for TGF-1 (Shuster et l., 29). Finlly, the question of the funtionl role of nd in iology remins to e nswered. nd show non-redundnt funtion in humn s reveled y the geneti disorders gelntinous drop like onrel dystrophy, used y muttions (Tsujikw et l., 1999), nd tufting enteropthy nd Lynh syndrome, used y muttions (Sivgnnm et l., 28; Kempers et l., 211). Funtionl dt in ner ell lines indite pro-prolifertive role for oth moleules (Wng et l., 28; Cus et l., 21). my shre downstrem effetor pthwys with ; however, unlike, the intrellulr domin of ontins onserved PIP 2 inding domin (El Sewedy et l., 1998). The intrellulr til of trnslotes to the nuleus to tivte -tenin signling (Metzel et l., 29), nd overexpression ounter regultes E-dherin dhesion (Winter et l., 23). As E-dherin nd -tenin hve een implited in tolerogeni DC funtions (Jing et l., 27), it will e interesting to further study nd/or funtion in ex vivo-generted s. Beuse full knokout of in mie is emryonilly lethl (Ngo et l., 29), DC-speifi knokout models will e essentil for in vivo studies. With potentil linil pplitions of our study in mind, it is noteworthy tht expression ws not detetle in one mrrow or peripherl lood. Together with the reported strong overexpression of on metstti tumor ells, studies should inlude nti- stinings to detet nd quntify irulting tumor ells; this strtegy urrently mostly relies on nti- stinings (Crisitiello et l., 21). MATERIALS AND METHODS Cells nd tissues CD34 þ hemtopoieti progenitor ells nd CD14 þ monoytes were isolted from ord lood s desried (Tshner et l., 27). Humn nd mouse epidermis nd dermis were seprted following overnight inution on Dispse II (1.2 U ml 1 ; Rohe, Vienn, Austri) nd inuted for 1 hour t 37 1C in phosphte-uffered sline ontining DNAse I (2 mgml 1, Sigm-Aldrih, Vienn, Austri) nd trypsin (2 mg ml 1, Sigm-Aldrih) for epidermis or ollgense IV (4, U ml 1, Sigm-Aldrih) for dermis. Single-ell suspensions were prepred y filtering the digested tissues through 7-mm ell striner (BD Biosienes, Shweht, Austri). Single-ell suspensions of lung nd intestinl tissue were prepred s desried (Suer et l., 26; Ng et l., 21). Lung nd olon speimens were 255

8 otined from individuls undergoing surgery for tumor removl. Smples were tken from unffeted tissue t the resetion mrgin. All proedures were rried out in ordne to the guidelines from the Medil University of Vienn Institutionl Review Bord for these studies. Informed onsent ws provided in ordne with the Delrtion of Helsinki Priniples. Cytokines nd regents Humn stem ell ftor, thromopoietin, tumor nerosis ftor-), GM-CSF, fms-relted tyrosine kinse 3 lignd (FL), IL-1, IL-3, nd IL-4 were purhsed from PeproTeh (London, UK); TGF-1 ws purhsed from R&D Systems (Wiesden, Germny); erythropoietin ws purhsed from Jnnsen-Cilg (Vienn, Austri). In vitro ell ulture CD34 þ ord lood ells were ultured serum-free for 2 3 dys under progenitor expnsion onditions (fms-relted tyrosine kinse 3 lignd, stem ell ftor, nd thromopoietin, eh t 5 ng ml 1 ) efore suulturing with linege-speifi ytokines. ultures were desried efore (Strol et l., 1997). For some experiments, lusters were purified s desried efore (Gtti et l., 2) nd then further ultured in the presene of GM-CSF (1 ng ml 1 ), tumor nerosis ftor- (25 ng ml 1 ), nd IL-1 (2 ng ml 1 ) to indue mturtion. Erythrolst ultures nd CD14 þ monoytederived DC, nd mo ultures were desried efore (Geissmnn et l., 1998; Zermti et l., 2). Rel-time PCR Rel-time PCR nlysis ws performed s previously desried (Pltzer et l., 29). The following primers were used: HPRT, forwrd 5 -GACCAGTCAACAGGGGACAT-3, reverse 5 -AACACT TCGTGGGGTCCTTTTC-3 ;, forwrd 5 -TGCTCTGAGCGA GTGAGAA-3, reverse 5 -TGCAGTCCGCAAACTTTTA-3, nd TR OP2, forwrd 5 -ACAACGATGGCCTCTACGAC-3, reverse 5 -AGTT CACGCACCAGCACAC-3. Flow ytometry Flow ytometry stining nd nlysis ws performed s desried (Pltzer et l., 24). Mouse nti-humn mas, speifi for (Alex-Fluor 488-onjugted), CD324 (APC-onjugted), CD1, nd glyophorin A (Pifi Blue-onjugted), s well s rt nti-mouse mas speifi for mjor histoomptiility omplex-ii (phyoerythrin (PE)-onjugted) nd (APC/Cy7-onjugted) were purhsed from Biolegend (Uithoorn, The Netherlnds); mas speifi for CD86 (PE-onjugted), HLA-DR (iotinylted), nd CD83 (APConjugted), s well s streptvidin-pe/cy7 were purhsed from BD Biosienes; ma speifi for (PE-onjugted) ws purhsed from Immunoteh (Mrseille, Frne); ma speifi for (APConjugted) ws purhsed from E-Biosiene (Vienn, Austri); unoupled ma speifi for CD71 (lone VIP1) ws kindly provided y O. Mjdi, Vienn, Austri; seond step regent ws polylonl PE-onjugted got nti-mouse ntiody (Dko, Glostrup, Denmrk); ws deteted using got nti-humn or nti-mouse unoupled polylonl ntiody (R&D Systems); seond step regent ws polylonl donkey nti-got Alex-Fluor 488-onjugted ntiody (Invitrogen, Lofer, Austri). Cells were routinely stined with 7-minotinomyin D (Sigm-Aldrih) efore nlysis to exlude ded ells. Flow ytometri nlysis ws performed using n LSRII instrument (BD Biosienes) nd the FlowJo softwre (Tree Str, Ashlnd, OR). Immunohistology Tissue speimens were stined s desried (Göel et l., 29). For detetion of the ntiody, slides were proed with polylonl donkey-nti-got Alex-Fluor-488-onjugted ntiody (Invitrogen). Nulei were stined with DAPI nd slides were mounted using fluoresent mounting medium (Dko). Pitures were tken using n Elipse 8i mirosope (Nikon, Amstelveen, The Netherlnds) nd Lui G softwre (Lortory Imging, Prgue, Czeh Repuli). Murine epiderml er sheets were prepred s desried (Ngo et l., 29). Sttistil nlysis Sttistil nlysis ws performed using the two-tiled Student s t-test; P-vlues of less thn.5 were onsidered signifint. CONFLICT OF INTEREST The uthors stte no onflit of interest. 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