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Christine Hacker1†, Ralf D. Kirsch1†, Xin-Sheng Ju1,Thomas Hieronymus1,Tatjana C. Gust1,Christiane Kuhl1*,Thorsten Jorgas1, Steffen M. Kurz1, Stefan Rose-John2,Yoshifumi Yokota3

and Martin Zenke1

Published online 24 February 2003; doi:10.1038/ni903

Dendritic cells (DCs) are potent antigen-presenting cells with a pivotal role in antigen-specificimmune responses. Here, we found that the helix-loop-helix transcription factor Id2 is up-regulatedduring DC development in vitro and crucial for the development of distinct DC subsets in vivo. Id2–/–

mice lack Langerhans cells (LCs), the cutaneous contingent of DCs, and the splenic CD8α+ DC subsetis markedly reduced. Mice deficient for transforming growth factor (TGF)-β also lack LCs, and wedemonstrate here that, in DCs,TGF-β induces Id2 expression.We also show that Id2 represses B cellgenes in DCs.These findings reveal a TGF-β–Id2 signaling pathway in DCs and suggest a mechanismby which Id2 affects the lineage choice of B cell and DC progenitors.

1Max-Delbrück-Center for Molecular Medicine, MDC, Robert-Rössle-Str. 10, 13092 Berlin, Germany. 2Department of Biochemistry, Christian-Albrechts-University,Olshausenstr. 40, 24098 Kiel, Germany. 3Department of Biochemistry, Fukui Medical University, Matsuoka, 910-1193 Fukui, Japan. *Present address:Weatherall Institute of

Molecular Medicine, Headington, Oxford OX3 9DS, UK. †These authors contributed equally to this work. Correspondence should be addressed to M.Z.(zenke@mdc-berlin.de).

Transcriptional profiling identifies Id2function in dendritic cell development

Dendritic cells (DCs) are professional antigen-presenting cells thathave key roles in antigen-specific immune responses and have beenimplicated in determining the balance between immunity and toleranceinduction1–3. DCs originate from hematopoietic precursor cells andoccur throughout the organism in both lymphoid and nonlymphoid tis-sues, and are specialized for uptake, transport, processing and presen-tation of antigens. DC subsets have been identified that differ in phe-notype, function, activation state and location, but their relationship anddevelopmental origins have remained unclear or controversial4–6. Inaddition, the underlying genetic programs that determine lineage com-mitment and differentiation of DCs are largely unknown and likelyinvolve the selective activation and/or repression of specific genes.

DCs are assumed to originate from both myeloid and lymphoid pre-cursors. In mouse, CD11c+CD11b+CD8α– DCs represent the classicalmyeloid tissue DCs. CD11c+CD11b–CD8α+ DCs are frequentlyreferred to as lymphoid DCs, because they resemble the major thymicCD8α+ DC population. However, both myeloid- and lymphoid-com-mitted precursor populations can give rise to CD8α+ and CD8α– DCs,and thus CD8α expression seems not to delineate the origin of DCs7–9.Plasmacytoid DCs (pDCs) represent yet another DC subset and arecharacterized by production of large amounts of type 1 interferon inresponse to virus and bacteria10. In mouse, these cells areCD11c+CD11b–B220+Gr-1+ (refs. 5,6,11,12). Additionally, a DC-com-mitted precursor population has been defined that has the capacity togenerate all DC subtypes present in mouse lymphoid organs, but lacksmyeloid and lymphoid differentiation potential13. Yet, how distinct DCsubsets relate to each other and how DC subset development is regu-lated have remained largely unclear.

Transcription factors such as helix-loop-helix (HLH) proteins have apivotal role in determining lineage choice and differentiation14–16. The

HLH protein E2A, for example, is absolutely required for B cell devel-opment, and E2A-deficient mice lack preB and mature B lymphocytes.Most HLH proteins act as transcriptional activators and are antagonizedby another class of HLH proteins referred to as Id proteins (inhibitorsof DNA binding or differentiation), of which four (Id1–4) have beenidentified14–16. Id proteins contain the highly conserved HLH domain,but lack the adjacent basic region that is required for DNA binding.Thus, heterodimerization of Id proteins with HLH activators results ina complex that is incapable of DNA binding. Gene inactivation studiesin mice have identified functions of Id proteins: Id1–/– mice do notexhibit overt abnormalities, whereas Id2–/– mice are compromised indevelopment of natural killer (NK) cells, mammary gland and nasopha-ryngeal-associated lymphoid tissue (NALT), and lack lymph nodes andPeyer’s patches15–20.

Here we used an in vitro differentiation system for DCs and tran-scriptional profiling with microarrays to determine the transcriptionfactor repertoire expressed by DCs. Id2 was strongly up-regulated inDCs during in vitro differentiation. Characterization of Id2–/– micedemonstrated that Id2 was crucial for the development of distinct sub-sets of DCs. Id2–/– mice lacked Langerhans cells (LCs), and splenicCD8α+ DCs were severely reduced. Mice deficient for transforminggrowth factor (TGF)-β also lack LCs, and we show that TGF-β inducedId2 expression.

ResultsTranscriptional profiling of DC differentiationDCs were obtained by in vitro differentiation from CD34+ cells ofhuman cord blood, and the ongoing changes in gene expression duringDC differentiation were determined by DNA microarray analysis. Toobtain sufficient numbers of progenitor cells, a two-step in vitro culture

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system was used in which progenitor cells were first amplified and theninduced to differentiate into DCs, thereby using a strategy that was suc-cessfully applied before21,22. The progenitors obtained were able to dif-ferentiate into different hematopoietic lineages including myeloid anderythroid cells (B. Lemke, X.-S.J., C.H. and M.Z., unpublished data). Toinduce differentiation into DCs, progenitor cells were treated with gran-ulocyte-macrophage colony-stimulating factor (GM-CSF) and inter-leukin (IL)-4 (ref. 22). Cells ceased proliferation and concomitantlyacquired phenotypic markers of DCs such as prominent dendriticprocesses and cytoplasmic projections (data not shown). Cells expressedmajor histocompatibility complex (MHC) class II, CD1a, CD80 andCD86, and their expression was further enhanced by tumor necrosis fac-tor (TNF)-α, which induces DC maturation (Fig. 1a). CD1a+ cells werealso positive for the LC surface markers E-cadherin and langerin(CD207), and the frequency of these cells during in vitro differentiationcould be further increased by TGF-β (Fig. 1b and data not shown).These findings indicate that the DCs obtained resembled LCs. DCs didnot produce interferon (IFN)-α in response to virus (herpes simplexvirus, HSV; Supplementary Fig. 1), which is a hallmark of pDCs. DCswere functional in in vitro migration assays and effective in generatingallogeneic T cell responses in in vitro T cell activation assays, whereasprogenitor cells were, as expected, inactive (X.-S.J. and M.Z., unpub-lished data).

To monitor the changes in gene expression during DC differentiationon a genome-wide scale, we carried out DNA microarray analysisusing Affymetrix GeneChip arrays that contain probe sets for 13,000full-length human genes. Among the genes analyzed, 36–40% wereconsistently found to be expressed in progenitor cells and/or differenti-ated DCs, corresponding to 4,700–5,200 genes, which is consistentwith previous studies on murine DCs23. About 5% of the assessed genesshowed a more than two-fold up- or down-regulation during differenti-ation and about 2% of the genes were regulated by more than five-fold.To allow a systematic analysis of gene expression in DCs, expressedgenes were annotated and grouped by gene families and/or by gene

function in biological processes. This annotation was then used for fur-ther studies.

Specific changes in the expression of cell surface molecules repre-sent one of the hallmarks of DC differentiation, and thus DNA microar-ray data were evaluated for expression of a large panel of CD moleculesincluding molecules involved in antigen uptake and presentation, celladhesion and cell-cell interaction. Hierarchical cluster analysis24 wasused to reveal specific patterns inherent to the DC differentiation pro-gram (Fig. 1c and ArrayExpress accession number E-MEXP-1). Thisstudy provided cell surface ‘signatures’ of the progenitor and DC pop-ulations analyzed. It identified distinct clusters of differentially regulat-ed genes, including a cluster of genes that were highly expressed inprogenitors and down-regulated during differentiation (cluster I), aswell as other clusters of genes highly expressed in DCs or DCs treatedwith TNF-α (clusters II, III and IV). These data are consistent with andfurther extend our knowledge about cell surface antigens on DCs1,2 andrelate well to cell surface marker expression on progenitor cells andDCs analyzed by flow cytometry (Fig. 1a).

Up-regulated genes (clusters II–IV) included those encoding mole-cules involved in antigen uptake and presentation such as various MHCclass I and II alleles, β2-microglobulin, invariant chain Ia, CD1a, CD1b,CD1c, Fc receptor CD16 and mannose receptor CD206 (Fig. 1a,c). Thecostimulatory molecules CD80 and CD86 and the DC activation mark-ers CD83, CD150 (SLAM), CD205 (DEC205) and CD208 (DC-LAMP) were also in these clusters. Another up-regulated gene was thatencoding IL-7 receptor (CD127), in accordance with the potent activi-ty of IL-7 and the IL-7–like cytokine thymic stromal lymphopoietin(TSLP) to induce DC activation25.

GM-CSF and IL-4 receptors were also up-regulated when cells dif-ferentiated into DCs, which is consistent with the potent activity oftheir cognate ligands GM-CSF and IL-4 to induce DC differentiation.Conversely, expression of stem cell factor (SCF) receptor (c-kit,CD117) and Flt3 receptor (CD135) was high in progenitor cells anddown-regulated during DC differentiation (cluster I), in accordance

Figure 1. Differentiation of human DCs from hematopoietic prog-enitor cells in vitro. (a) Progenitor cells (Progs) and DCs at day 6 of in vitrodifferentiation were analyzed for surface antigen expression by flow cyto-metry (filled areas). DCs + TNF-α, DCs treated with TNF-α (10 ng/ml) at day6 for an additional 24 h. Open areas, staining with isotype control antibody.(b) CD1a+ DCs (day 6) were obtained by immunomagnetic bead selectionand analyzed for Langerhans cell markers by flow cytometry (filled areas).Open areas, isotype control antibody. (c) Surface antigen expression in prog-enitor cells, DCs and TNF-α–treated DCs (same cells as in a) was assessedby DNA microarray analysis and subjected to hierarchical cluster analysis24.The analysis of 154 expressed surface antigens is shown for one represen-tative experiment, and each gene is represented by a single row of coloredboxes. For every gene the median expression value for the three samplesshown (Progs, DCs and DCs + TNF-α) was calculated, and its relativeexpression in one sample is given by the color code24. Green, transcriptabundance below median; black, transcript abundance near to median; red,transcript abundance higher than median. Clusters I–IV and selected genesare shown. Cluster I, genes with high expression in progenitor cells only;cluster II, genes that are up-regulated during DC differentiation and furtherup-regulated by TNF-α; cluster III, genes with high expression in DCs andthat are down-regulated by TNF-α; cluster IV, genes that are induced by TNF-α. Microarray data are available in ArrayExpress database (accession numberE-MEXP-1).

HLA-DR

DCs+TN

F-α

CD117, c-kitCD133, AC133CD135, Flt3

CD1bCD11cCD40CD54, ICAM-1CD16, FcRCD206, mannose receptorCD1aCD11bCD80CD83CD127, IL-7 receptorCDw197, CCR7CD205, DEC205CD208, DC-LAMP

Prog

sDCs

IV

III

I

II

CD1a

CD83

CD80

CD86

CD14

100 101 102 103 104 100 101 102 103 104100 101 102 103 104

CD33

Fluorescence intensity

Cel

l num

bers

Progs DCs DCs+TNF-α

CD1a HLA-DR LangerinE-cadherin

Fluorescence intensity100 101 102 103 104

100 101 102 103 104100 101 102 103 104

CD

14

CD1a

100 101 102 103 104

a c

b

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with the activity of SCF and Flt3 ligand (FL) on progenitor cell growth,which is lost during differentiation (X.-S.J. and M.Z., unpublisheddata). Expression of the stem cell antigen CD133 (AC133) was high inprogenitors and down-regulated during DC differentiation. A compre-hensive catalog of cell surface molecules on progenitor cells and DCsis available in the ArrayExpress database (accession number E-MEXP-1). These results validated the microarray approach used and also iden-tified molecules that so far had not been found in DCs and might serveimportant functions in DCs.

Transcriptional regulators in DC differentiationWe identified 481 transcriptional regulators that were expressed in DCs,including various DNA binding proteins, coactivators and corepressors,chromatin modifying proteins, basic transcription factors and compo-nents of the RNA polymerase complex. Some of these factors under-went specific changes in expression when progenitors differentiated intoDCs, suggesting that they might serve determining functions in self-renewal or cell cycle arrest, and/or in the induction of differentiation andestablishment of the DC phenotype. Therefore, clustering by self-orga-nizing maps (SOMs)26 was used to identify transcriptional regulatorswith a potential function in these processes. SOMs are particularly wellsuited for exploratory analysis of microarray data, as they impose struc-tures on data, with neighboring nodes tending to define related clusters.

Expression data from progenitor cells, differentiated DCs and TNF-α–treated DCs were used, and clustering in nine clusters was found tobe optimal (Supplementary Fig. 2a). Fewer clusters resulted in highvariation within individual clusters, whereas higher cluster numbersyielded clusters with redundant profiles. Clusters 0, 1 and 3 contained40 transcriptional regulators that were up-regulated, and clusters 5, 7and 8 contained 65 down-regulated genes during DC differentiation.Genes related to cell proliferation, stem cell and/or progenitor cell sta-tus were down-regulated, such as those encoding c-myb, B-myb andSCL (Supplementary Fig. 2a,b). EKLF, GATA-1 and GATA-2 werealso down-regulated, relating to the potential of the progenitor popula-tion to differentiate into red cells, which is lost when cells differentiateinto DCs (B. Lemke, X.-S.J., C.H. and M.Z., unpublished data). At thesame time, genes encoding proteins with a known or potential functionin DCs were induced, such as C/EBPα, C/EBPβ, IRF-1, IRF-4 and var-ious members of the NF-κB/Rel transcription factor family like c-Rel,RelB, p105 and IκBα (Supplementary Fig. 2a,b and ArrayExpressaccession number E-MEXP-2).

The analysis revealed specific expression patterns of several HLHtranscription factors. Expression of the HLH protein E2A declined

during DC differentiation, accompanied by an increase in expressionof the HLH proteins Id2, Dec1 and ABF-1. E2A and Id2 have beenimplicated in lineage commitment, and Id proteins antagonize E2Afunction. Furthermore, Dec1 and ABF-1 were also found to act asinhibitors of HLH activators, and thus exhibit an activity similar to thatof Id216,27. Given the prominent increase in Id2 expression during DCdifferentiation, the potential impact of Id2 on DC development wasanalyzed in more detail.

Id2 was up-regulated during DC differentiationWe quantified Id2 expression using the DNA microarray data andshowed that Id2 was effectively up-regulated during DC differentiation(Fig. 2a). Id1, another member of the Id family, was expressed at lowabundance. To further extend the DNA microarray data, human andmouse DCs were analyzed for Id2 mRNA and protein by RNAhybridization and immunoblot analysis, respectively. The amount ofId2 mRNA was low in human progenitor cells and up-regulated whencells differentiated into DCs, and was particularly high in TNF-α–treat-ed DCs and CD1a+ DCs, in accordance with the DNA microarray data(Fig. 2a,b). Id2 protein was essentially absent from progenitor cells andwas abundantly expressed in DCs (Fig. 2c). Next, we generated mouseDCs from mouse bone marrow progenitors using a two-step amplifica-tion and differentiation protocol similar to the one used for human DCs(T.H. and M.Z., unpublished data). Similar to human DCs, amounts ofId2 mRNA and protein were low in mouse DC progenitor cells, where-as they were induced when cells differentiated into DCs (Fig. 2d,e,fand data not shown).

Id2–/– mice were deficient in CD8α+ DCsTo determine the potential impact of Id2 on DC development, Id2–/–

mice were studied17. Bone marrow cells from Id2–/– mice were isolated,and DC progenitors were amplified in vitro and treated with GM-CSFto differentiate them into DCs. DCs were obtained from both Id2–/– andwild-type bone marrow cells (data not shown). RNA hybridization andimmunoblot analysis were used to confirm the absence of Id2 mRNAand protein from DCs of Id2–/– mice (Fig. 2e,f). To investigate whetheran increase in expression of the other Id proteins would substitute forabsence of Id2, mRNA from such in vitro generated Id2–/– DCs was sub-jected to RNA hybridization and microarray analysis. The amount ofId1, Id3 and Id4 mRNA was very low and was the same in Id2–/– andId2+/+ DCs (Fig. 2a and data not shown).

Next, we analyzed whether the absence of Id2 would affect DC devel-opment in vivo. Id2–/– mice lack lymph nodes and Peyer’s patches17.

Figure 2. RNA hybridization and immunoblot analysis of Id2 expression in human and mouse DCs. (a) DNA microarray data were evaluated for Id1 and Id2expression in human progenitor cells (Progs), DCs,TNF-α–treated DCs and CD1a+ DCs (day 6). Cells were prepared as in Fig. 1.Average values of three experiments areshown. (b and c) RNA hybridization and immunoblot analysis of Id2 expression in human progenitor cells (Progs) and DCs (same cells as in a). Asterisk, unspecific band.rRNA is shown as loading control. (d) Immunoblot analysis of Id2 in differentiating mouse DCs (day 0–9). (e and f) RNA hybridization and immunoblot analysis of Id1 andId2 expression in bone-marrow–derived DCs of Id2+/+, Id2+/– and Id2–/– mice.Asterisk, unspecific band.

DCs+TNF-α

rRNA

Id2

CD1a D

Cs+Pr

ogs

DCs

Id2*

Prog

sDCs

*Id2

+/– –/–

Id1

Id2

rRNA

+/– –/–+/+

Id2

d0 d2 d9d6 d8

CD1a D

Cs

Id2Id1

2,000

6,000

0

Rel

ativ

e ex

pres

sion

10,000

+Prog

sDCs

DCs+TNF-α

a b c d e f

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Therefore, DCs were prepared from spleen, in which different DC sub-types can be distinguished by differential expression of surface markerssuch as CD11c, CD11b and CD8α1,2,4–6,28. Single-cell suspensions ofsplenic DCs from Id2–/– and Id2+/+ mice were then analyzed by flowcytometry, and the percentage of CD11chiCD11b–CD8α+ DCs was foundto be reduced from 15% in Id2+/+ mice to 4% in Id2–/– mice.

To further extend these observations, CD11c+ cells from spleen ofId2–/–, Id2+/– and Id2+/+ mice were isolated by immunomagnetic beadselection and analyzed by flow cytometry. Id2–/– mice are on the129/Sv/NMRI background17, and 129/Sv mice are known to generatetwo DC subtypes, CD11chi and CD11cint DCs, with high and intermedi-ate surface expression of CD11c, respectively12 (Fig. 3a and Table 1).CD11chi DC subtypes are further subdivided by the expression of CD4and CD8α, thus generating three major splenic DC subtypes (namely,CD4–CD8α+, CD4+CD8α– and CD4–CD8α–) that also differ in theirability to produce certain cytokines28,29. The CD4–CD8α+ DC subsetwas nearly absent from Id2–/– mice (Fig. 3a and Table 1). The fre-quency of CD4– CD8α– DCs was also reduced, and CD4+CD8α– DCsrepresented the predominant splenic DC subtype in Id2–/– mice. Theoverall number of CD11chi DCs was similar for Id2+/+, Id2+/– and Id2–/–

mice (4.4 ± 0.8 × 106 CD11chi cells per spleen in six independent exper-iments). In addition, both CD8α+ and CD8α– DCs in Id2+/+ miceexpressed Id2 as determined by RT-PCR analysis (data not shown).Thus, deletion of Id2 severely affected the proportion of splenic DC

subsets: CD4–CD8α+ DCs were markedly reduced, and CD4+ CD8α–

represented the major splenic DC subtype.We proceeded to investigate whether the CD11cint subpopulation of

splenic DCs also displayed a phenotype in Id2–/– mice. This DC subtypewas described as comprising the mouse equivalent to human pDCs,which are characterized as CD11cintCD11b–B220+Gr-1+ (refs. 11,12).DCs from spleens of Id2–/– and Id2+/+ mice were prepared, depleted of Bcells, T cells and CD11b+ myeloid-related DCs, and stained for expres-sion of CD11c, CD11b, B220 and Gr-1. The proportion ofCD11cintCD11b–B220+Gr-1+ pDCs in Id2–/– mice was found to be higherthan that in wild-type controls (11.2% versus 7.2%; Fig. 3b). This find-ing is in accordance with an increased number of pDCs in Id2–/– micemeasured in cell sorting experiments. pDCs of both Id2+/+ and Id2–/– micewere competent in producing IFN-α in response to HSV and influenzavirus, with pDCs of Id2–/– mice being two to four times more potent inproducing INF-α than pDCs of Id2+/+ mice (Supplementary Fig. 3).

Absence of epidermal LCs from Id2–/– miceId2–/– mice were analyzed for the presence of LCs in ear epidermis usingimmunofluorescence and MHC class II, langerin (CD207) and CD86specific antibodies. Id2–/– mice essentially lacked LCs, whereas Id2+/+

control mice contained high numbers of LCs per high-power field (3.0 ±3.6 and 111.3 ± 3.5 MHC class II+ cells per field, respectively; Fig. 3c).This result was obtained for all antibodies used. Thus, similar to splenicCD8α+ DCs, the development of LCs was also dependent on Id2.

TGF-β induced Id2 expression in DCsThe absence of LCs from Id2–/– mice is reminiscent of the absence ofLCs from TGF-β–/– mice30,31. This observation prompted us to investigatewhether, in DCs, TGF-β and Id2 are components of the same biochem-ical pathway. Analysis of the microarray data showed that DCs and theirprecursor cells express the components of the TGF-β signaling pathway,such as TGF-β receptor and SMAD2, SMAD4 and SMAD7 transcrip-tion factors (data not shown). Thus, human DC precursors and DCs,generated from CD34+ cells with the cell culture system describedabove, were treated with TGF-β, and Id2 expression was analyzed byRNA hybridization. TGF-β effectively induced Id2 transcription in DC

Figure 3. Id2–/– mice lack CD8α+ DCs and LCs. (a) CD11c+ splenic DCs from Id2+/+, Id2+/– and Id2–/– mice were isolated by immunomagnetic bead selection and CD11chi

DCs (gate in left panel) were analyzed by flow cytometry for CD4 and CD8α expression (right panel). (b) For analysis of pDCs, single-cell suspensions of spleen from Id2+/+

and Id2–/– mice were depleted of CD3+, CD11b+ and CD19+ cells, and cells were analyzed for CD11c, B220 and Gr-1 expression by flow cytometry. (Left) R2, pDCs,CD11cintGr-1+; R3, myeloid DCs, CD11chiGr-1–. (Right) pDCs, CD11cintB220+Gr-1+ cells of R2 in left panel. (c) LCs in ear sheets of Id2+/+ and Id2–/– mice stained with MHCclass II antibody (top), and phase contrast images of the respective fields (bottom).

Cel

l num

bers

FSC

CD

11c

R3

88.4%

7.2%

9.0%

11.2%

2.4%

CD11c B220

89.8%

Gr-

1G

r-1

CD11c

Id2+/+ Id2–/–

Id2 –/–

R2

R2

R3

Gr-

1

Id2 –/–

Gr-

1

Id2 +/+Id2 +/+Id2+/+

CD

8-α

26.5%

13.7%

CD4

0.5%

Id2 +/+

Id2 +/–

Id2 –/–

CD

8-α

CD

8-α

a b c

Table 1. CD4– CD8α+, CD4+CD8α– and CD4–CD8α–CD11chi

DC subtypes in Id2+/+, Id2+/– and Id2–/– mice

Id2 CD4– CD4+ CD4–

CD8α+ CD8α– CD8α–

+/+ 26.2 ± 4.1 49.7 ± 3.3 17.8 ± 3.3+/– 14.1 ± 1.0 65.9 ± 4.5 15.9 ± 5.3–/– 1.1 ± 0.6 86.7 ± 5.0 7.4 ± 2.7CD11c+ splenic DCs of Id2+/+, Id2+/– and Id2–/– mice were isolated by immunomag-netic bead selection. CD11chi cells were analyzed for CD4 and CD8α expression,and the percentages of the respective DC subtypes are shown (average values ofthree independent experiments).

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precursor cells within 4 h and Id2 mRNA declined after 36 h (Fig. 4a).TGF-β also induced Id2 in DCs, although the induction was less pro-nounced than in DC precursors due to the high basal Id2 expression inDCs (data not shown). This induction of Id2 also occurred in the pres-ence of the protein biosynthesis inhibitor cycloheximide, indicating thatTGF-β signaling had a direct effect on Id2 transcription (Fig. 4b). Theamount of Id2 mRNA with TGF-β plus cycloheximide was even higherthan with TGF-β alone, probably due to stabilization of Id2 mRNA.Thus, TGF-β effectively induced Id2, identifying a TGF-β dependentpathway in DCs that acts through Id2. In B lymphocytes, Id2 and alsoId3, another member of the Id family, are targets of TGF-β signaling32,33.

Id2 suppressed B cell genes in DCsId2 might act in DCs by influencing the balance of inhibitory and acti-vating HLH proteins, thereby directly affecting the expression of HLHprotein target genes. LCs and CD8α+ DCs are absent from Id2–/– mice.Accordingly, to identify genes downstream of Id2 in DCs,CD11c+CD11b+ DCs were isolated from spleens of Id2+/+ and Id2–/–

mice by cell sorting and subjected to DNA microarray analysis; ∼ 3,000genes were found to be expressed. We found that 257 genes were dif-ferentially expressed in Id2+/+ and Id2–/– DCs by more than two-fold:183 genes were expressed at lower abundance in Id2+/+ DCs comparedwith Id2–/– DCs, which is consistent with Id2 functioning as a repressorof gene transcription. Also, 74 genes were expressed at higher abun-dance in Id2+/+ DCs compared with Id2–/– DCs. The analysis of differ-entially expressed genes revealed several immunoglobulin genes thatwere expressed in Id2–/– cells, but not (or at lower abundance) in Id2+/+

cells. These data provide initial evidence that Id2 suppresses theexpression of B cell genes.

DiscussionTranscription factors have a central role in lineage choice and cell dif-ferentiation. Here we have studied more than 400 transcriptional regu-lators that were expressed in DCs, by transcriptional profiling withDNA microarrays. Cluster analysis revealed that up-regulation of theHLH transcription factor Id2 was one of the most prominent changesthat occurred when hematopoietic progenitors differentiated into DCs.This finding was unexpected given the fact that Id2 is down-regulatedupon cell cycle arrest and differentiation in several cellular systems,and that ectopic expression of Id proteins inhibits differentiation14–16.

Id2 was not the only inhibitory HLH protein that was up-regulated dur-ing DC differentiation. Our cluster analysis demonstrated an effective up-regulation of Dec1 and ABF-1, which also encode inhibitory HLH pro-teins15,27. Coregulation of Id2 and ABF-1 is noteworthy because therespective proteins have been demonstrated to directly interact in a yeasttwo-hybrid assay34. Furthermore, our analysis also demonstrated that indifferentiating DCs, simultaneously with up-regulation of Id2, Dec1 and

ABF-1, the expression of the activating HLH proteins E2A, SCL andLyl1 declines. These findings suggest that up-regulation of inhibitoryHLH proteins and down-regulation of HLH activator proteins might beinherent components of the DC differentiation program that are criticalfor DC development and/or maintenance of the DC phenotype.

Targeted gene inactivation represents a powerful approach to deter-mining the impact of a given gene in development in its physiologicalenvironment in vivo. Accordingly, Id2–/– mice were used to elucidateId2 function in DC development. We demonstrated that Id2–/– micewere deficient in LCs, and that the splenic CD11c+CD11b–CD8α+ DCswere markedly reduced. CD11c+ CD11b+ DCs (also referred to asmyeloid DCs) were apparently not affected, indicating that Id2 had aselective impact on the development of LCs and CD8α+ DCs. This isconsistent with our observation that DC development in vitro frombone marrow progenitors of Id2–/– mice with GM-CSF (which yieldsCD11c+CD11b+ myeloid DCs) was apparently normal and was notdependent on Id2. Thus, Id2 affects development of distinct DC subsetsrather than development of all DC types. This is in accordance with theobservation of only a partial overlap between the genes affected by Id2and the gene regulation pattern associated with DC differentiation(T.H., R.-D.K., C.H. and M.Z., unpublished data).

During hematopoiesis, the developmental potential of multipotentprogenitor cells, including a common lymphoid progenitor (CLP) forT and B cells, NK cells and DCs, becomes increasingly restricted,leading to the establishment of a specific cell lineage from the choiceof several35–37. Both activating HLH proteins and Id proteins have beendemonstrated to control cell fate and lineage choice15,16. E2A, forexample, is a determining factor for B cell development, and Id2 con-trols NK cell development15–17. Thus, a model presents itself in whichthe relative abundance, for example, of E2A and Id2 determines lin-eage choice by affecting the propensity of such a common progenitorto develop into B cells, or NK cells and DCs: low or no expression ofId2 and high expression of E2A supports B cell development, where-as high expression of Id2 blunts E2A activity and B cell development,and thus allows differentiation into NK cells and DCs. Accordingly, adevelopmentally controlled up-regulation of Id2 (and/or the inductionof Id2 by TGF-β) is suggested to represent a determining step for dif-ferentiation of LCs and the splenic CD8α+ DC subset. In Id2–/– micethis differentiation potential is compromised or lost. The result thatspleen of Id2–/– mice contains more mature B cells, as compared withId2+/+ mice38, is consistent with this idea. Our finding that Id2–/– DCsexpressed several B cell genes, such as various immunoglobulin genes,that were absent or expressed at lower abundance in Id2+/+ DCs pro-vides further support for this model.

Forced expression of Id2 (and also Id3) in CD34+ human fetal livercells blocks development of B cells, T cells and plasmacytoid DCs, butnot myeloid DCs, and supports NK cell development39. These resultsrelate well to the findings described here, as ablation of Id2 in mice leftdevelopment of myeloid DCs unaffected, but led to an increased fre-quency of plasmacytoid DCs. The results are also in accordance withthe observation that mature B cells are increased in Id2–/– mice38.

The Id2 dependence of CD8α+ DCs and LCs now links these cellsnot only with NK and B cell development19,38, but also with develop-ment of lymph nodes and Peyer’s patches that also depend on Id217. Theprogenitors used in the in vitro DC differentiation system expressed IL-7 receptor (CD127), which represents a marker for lymph node andPeyer’s patches initiating cells40, and IL-7 receptor expression was fur-ther up-regulated during DC development.

One of the most surprising findings in this study was the absence ofLCs from Id2–/– mice. TGF-β–/– mice also lack LCs30,31, and we have

Figure 4.TGF-β induces Id2 transcription. (a) RNA hybridization analysis of Id2expression in human DC progenitor cells that were treated with TGF-β (10 ng/ml)for 4, 16 and 36 h, or left untreated. DC progenitors derived from CD34+ cells at day10 of culture were used. 18S rRNA is shown as a loading control. (b) RNA hybridiza-tion analysis of human DC progenitor cells treated with TGF-β (10 ng/ml) and cyclo-heximide (CX, 20 µg/ml) for 2 h as indicated.

TGF-β (h)4 16 360

Id2

18S RNA

Id2

18S RNA

TGF-βCX

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www.nature.com/natureimmunology • april 2003 • volume 4 no 4 • nature immunology 385

demonstrated here that TGF-β effectively induced Id2, thus placingTGF-β signaling upstream of Id2. Therefore, it is tempting to speculatethat, in DC development, TGF-β acts through Id2 in vivo, as ablation ofeither component in this signaling pathway leads to loss of LCs.Furthermore, many reports using several in vitro systems demonstratedthat TGF-β skews DC differentiation toward the LC phenotype4,31. Inaccordance with these reports, the DCs used in the transcriptional pro-filing study described here yielded DCs with a LC phenotype that wasenhanced by TGF-β treatment (X.-S.J. and M.Z., unpublished data).Thus, induction of TGF-β in inflammatory processes and up-regulationof Id2 might be a determining step that shifts differentiation towardLCs by affecting the balance of HLH transcriptional regulators in DCprecursors and/or DCs.

Id2 influences distinct subsets of DCs (LCs, CD8α+ DCs and pDCs),as well as B cells and NK cells16,17,19,38, suggesting that these cells shareregulatory mechanisms of development. Targeted gene inactivation ofother transcription factors such as RelB and interferon consensussequence-binding protein (ICSBP) also affects DC development41–44.RelB–/– mice lack CD11c+CD11b+CD8α– DCs and have LCs41–43, where-as, as demonstrated here, Id2–/– mice lack LCs and are compromised inthe frequency of CD11c+CD11b–CD8α+ DCs. Thus, RelB–/– and Id2–/–

mice display defects in complementary DC subtypes, which may indi-cate that these factors act on independent pathways of DC develop-ment. ICSBP–/– mice are deficient in pDCs and CD8α+ DCs44. Given thefact that Id2–/– mice are deficient in two important DC subsets, it willnow be interesting to determine whether these mice are impaired intheir response to viral and bacterial infections or to tumors.

MethodsCells and cell culture. Human DCs were obtained by in vitro differentiation from CD34+

stem cells using a two-step amplification and differentiation protocol22. Briefly, CD34+ cellswere isolated from cord blood using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) and two cycles of immunomagnetic bead selection (purity of CD34+

cells was 83–90%). CD34+ cells (0.3–0.5 × 106 cells/ml) were cultured in StemSpan serum-free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml SCF, 50ng/ml FL (PeproTech, London, UK), 20 ng/ml thrombopoietin and 10 ng/ml recombinanthyper-IL-645.

After 10–15 d of culture, progenitor cells (0.5 × 106 cells/ml) were induced to differen-tiate into DCs in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mML-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO-BRL, Paisley, UK), 500 U/ml GM-CSF and 500 U/ml IL-4 for 6 d22,46. We added 10 ng/mlTNF-α at day 6 for an additional 24 h to induce DC maturation. TGF-β treatment of prog-enitors and DCs was for 4, 16 and 36 h with 10 ng/ml TGF-β (R&D Systems, Minneapolis,MN). Alternatively, cells were simultaneously treated with TGF-β1 (10 ng/ml) and cyclo-heximide (20 µg/ml, Sigma, St. Louis, MI) for 2 h.

For CD1a selection, differentiated DCs (day 6) were exposed to antibody to CD1a(NA1/34, DAKO, Glostrup, Denmark), and cells were then subjected to magnetic bead sep-aration as above. CD1a+ cell purity was 90–95%.

Mouse DCs were generated from mouse bone marrow as described46,47. Alternatively,bone marrow progenitors were first amplified in RPMI 1640 medium with 10% FCS, 100ng/ml SCF, 25 ng/ml FL, 40 ng/ml long-range insulin-like growth factor (IGF-1; Sigma), 10ng/ml hyper-IL-6, 20 U/ml of GM-CSF and 10–6 M dexamethasone (Sigma) for 7 d and thendifferentiated into DCs with 200 U/ml GM-CSF (T.H. and M.Z., unpublished data). SplenicDCs were prepared as described48. Briefly, spleens were treated with Liberase CI and DNaseI (both Roche Diagnostics, Mannheim, Germany), and CD11c+ cells were obtained byimmunomagnetic bead selection with CD11c-MACS beads (Miltenyi Biotech). Yields were3.6–5.4 × 106 CD11chi cells per spleen. For isolation of pDCs, single-cell suspensions ofspleens were depleted of CD3+, CD11b+ and CD19+ cells, and analyzed for CD11c, B220and Gr-1 expression by flow cytometry11,12. After depletion, 1.4–1.8 × 106 cells were rou-tinely obtained and analyzed. Id2–/– mice were on 129/Sv/NMRI background17. Proceduresinvolving animals were conducted according to the guidelines for animal treatment at thecentral animal facilities at MDC, Berlin, Germany, and the laboratory holds permission toperform the animal experiments described.

Flow cytometry and immunofluorescence analysis. Flow cytometry was carried out asdescribed46,49,50 with the following antibodies: human DCs, CD1a (NA1/34, DAKO), CD14(IOM2, clone RM052, Immunotech, Marseille, France), CD33 (WM53, CymbusBiotechnology, Chandlers Ford, UK), CD34 (Anti-HPCA-2, clone 8G12, BD Biosciences,San Jose, CA), CD80 (MAB104) and CD83 (HB15A, both Immunotech), CD86 (B70/B7-2,

clone 2331; BD Biosciences) and HLA-DR (clone CR3/43, DAKO); mouse DCs, CD11c-phycoerythrin (PE; clone HL3), CD4-biotin (bio; GK1.5), CD8α-fluorescein isothiocyanate(FITC; 53-6.7), CD11b-bio (M1/70), Gr-1-bio (RB6-8C5), B220-FITC (RA3-6B2) andrespective isotype controls (all BD Biosciences). Cells were then reacted with isotype-matched FITC-labeled secondary antibody (Sigma). Biotinylated monoclonal antibodieswere followed by Streptavidin–TRI-COLOR (Caltag Laboratories, Burlingame, CA). Cellswere analyzed by FACSCalibur flow cytometer and CELLQuest software (BD Biosciences).Cell sorting of CD11c+CD11b+ DCs from mouse spleen was done with CD11c–PE andCD11b-bio antibodies (and the respective secondary antibodies as above) and a FACSDivadevice (BD Biosciences).

Ear sheets of Id2–/– and Id2+/+ mice were prepared as described50 and subjected toimmunofluorescence analysis with specific antibodies for MHC class II (2G9, BDBiosciences), CD86 (RMMP-1, Immunotech) and langerin (CD207, clone HD24)51, fol-lowed by tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibody(Sigma). LC density was determined by fluorescence microscopy50.

Immunoblotting. Cells were lysed and subjected to blot analysis as described49. Briefly, fordetection of Id1 and Id2 proteins, polyclonal rabbit anti-mouse antisera (Santa CruzBiotechnology, Santa Cruz, CA) were used and developed with ECL reagents (ECL kit,Amersham Biosciences).

RNA isolation and hybridization. Total RNA was isolated with RNeasy Kits includingDNase digestion (Qiagen, Hilden, Germany). Total RNA (10 µg per lane) was loaded onto1.2% formaldehyde-agarose gels and hybridized following standard procedures. Human Id1and Id2 cDNAs were labeled with [32P]dCTP using the High Prime DNA Labeling Kit(Roche Diagnostics) and used for hybridization.

DNA microarray analysis. Total RNA (5 µg) was used to generate cDNA according to theExpression Analysis Technical Manual (Affymetrix, Santa Clara, CA). cRNA was generat-ed with the BioArray High-Yield Transcript Labeling kit (ENZO, Farmingdale, NY), and10 µg cRNA was hybridized to Affymetrix human HG-U95Av2 or mouse MG-U74Av2arrays at 45 °C for 16 h. The DNA chips were stained, washed and scanned according to themanufacturer’s protocol.

Bioinformatics. Scanned GeneChip DAT files were analyzed by the Gene Chip AnalysisSuite Software (Affymetrix) with global scaling to 1,000 as before52. Further analysis of dataoutput was carried out in Microsoft Excel. Genes were considered to be expressed if theirexpression values were above 300 and the expression call was ‘present’. Expression valuesbelow 300 were considered to be background, and for SOM analysis these values were set to300. Classification of genes into functional groups was done using information from variousdatabases including Entrez Pubmed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), theGeneCards database (http://bioinformatics.weizmann.ac.il/cards/) and the PROW databasefor CD molecules (http://www.ncbi.nlm.nih.gov/prow/).

Hierarchical clustering24 was done with GeneSpring software (Silicon Genetics, RedwoodCity, CA) using the Pearson correlation with a separation ratio of 0.5 and a minimum distanceof 0.001. Genes encoding CD molecules from one representative experiment were selected foranalysis. No additional filter was used. SOM analysis of transcription factors was done with theGenecluster program26 (http://www.genome.wi.mit.edu/MPR/GeneCluster/GeneCluster.html).Transcription factors that were consistently found to be up- or down-regulated, respectively, intwo independent experiments were used. Genes were filtered for Max/Min > 2 and normalizedfor a mean of 0 and a variance of 1 in the Genecluster software. Microarray data were submit-ted to ArrayExpress database (accession numbers E-MEXP-1 and E-MEXP-2).

Note: Supplementary information is available on the Nature Immunology website.

AcknowledgmentsWe thank F. Sablitzky for Id1 and Id2 cDNA; S. Saeland for the langerin antibody; D.Kobelt and A. Steinkasserer for HSV; H.D. Klenk for influenza virus;A. Mansouri and P.Gruss for sharing the Id2–/– mice; P. Grasshoff,T. Kaiser and K. Raba for cell sorting; S.S.Diebold and T. Blankenstein for discussions and support; S. Knespel and G. Blendinger fortechnical assistance; and P. Haink and P. Podlatis for secretarial assistance.This work wasfunded by grants of the Fonds der Chemischen Industrie, German Research Foundation(Ze 432/1 and Ze432/2) to M.Z. and a grant of the Edward Jenner Institute for VaccineResearch to M.Z. and T.C.G. X.-S.J. received a postdoctoral fellowship from the GermanAcademic Exchange Service.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 19 September 2002; accepted 3 February 2003.

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