Langerhans-Type Dendritic Cells in Vitro

Functional Involvement of E-Cadherin in TGF- β1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells This information is current as of September 29, 2021. Elisabeth Riedl, Johannes Stöckl, Otto Majdic, Clemens Scheinecker, Klemens Rappersberger, Walter Knapp and Herbert Strobl J Immunol 2000; 165:1381-1386; ; doi: 10.4049/jimmunol.165.3.1381 Downloaded from http://www.jimmunol.org/content/165/3/1381

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Functional Involvement of E-Cadherin in TGF-␤1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells1

Elisabeth Riedl,*§ Johannes Sto¨ckl,† Otto Majdic,† Clemens Scheinecker,‡ Klemens Rappersberger,§ Walter Knapp,† and Herbert Strobl2*

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-␤1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34؉ hemopoietic progenitor cells in response to TGF-␤1 costimulation (basic cytokine combination GM-CSF plus TNF-␣, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells.

This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is ﬁrst seen on day 4 after culture Downloaded from ,initiation of CD34؉ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway because parallel cultures set up in the absence of exogenous TGF-␤1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a؊, lysozyme؉, CD14؉). TGF-␤1 costimulation of CD34؉ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-␤1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, http://www.jimmunol.org/ associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34؉ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-␤1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells. The Journal of Immunology, 2000, 165: 1381–1386.

pidermal Langerhans cells (LC)3 are bone marrow-de- tion of epidermal LC, and lower expression levels of E-cadherin on rived immature dendritic cells (DC) that are highly spe- the surfaces of cultured LC correlate with decreased cell adhesive- cialized in Ag uptake and processing. LC form a three- ness (4, 7, 8). Apart from LC-type DC, most other hemopoietic E by guest on September 29, 2021 dimensional network in suprabasal epidermal layers and stay in the cells, with the exception of immature thymocytes (9, 10), murine epidermis for long periods of time, enabling them to fulfill a sen- dendritic epidermal T cells (11), and immature erythroid cells (12), tinel role in which they filter the surrounding tissue for foreign Ags lack E-cadherin expression. and pathogens (1–3). The cytokine TGF-␤1 plays a key role during LC development Differentiated epidermal LC share with epithelial cells of vari- and differentiation. Using an in vitro differentiation model of ous organs high cell surface expression levels of the homophilic CD34ϩ hemopoietic progenitor cells, we recently demonstrated calcium-dependent cytoadhesion molecule E-cadherin (4, 5). LC that development of LC from CD34ϩ progenitor cells in a serum- to keratinocyte adhesion (4) as well as homotypic adhesion of in free culture system is absolutely dependent on TGF-␤1 stimulation vitro generated murine LC (6) can be blocked by mAbs to E- (13). In vivo studies revealed that TGF-␤1-deficient mutant mice cadherin, suggesting an important role for E-cadherin in LC ad- selectively lack epidermal LC (14), but possess functional LC pre- hesion (reviewed in Ref. 2). Among DC, only differentiated LC cursors (15) and other myeloid cells and show normal bone mar- and a subpopulation of DC from skin-draining lymph nodes, which row morphology (16). Bone marrow cells from TGF-␤1-deficient may be derived from LC, express E-cadherin (7). E-cadherin ex- mice are capable, upon transplantation into normal mice, of dif- pression is markedly down-regulated upon migration and matura- ferentiating into LC, suggesting that paracrine TGF-␤1 stimulation of LC precursors in peripheral organs is sufficient for LC differentiation in vivo (15). Furthermore, similar numbers of bone mar- *Institute of Immunology, Vienna International Research Cooperation Center, No- Ϫ Ϫ vartis Research Institute, Vienna, Austria; and †Institute of Immunology, ‡Department row-derived LC were identified in TGF-␤1 / and control skin § of Internal Medicine III, Division of Rheumatology, and Department of Dermatology after engraftment onto nude mice. Additionally, epidermal-re- I, University of Vienna, Vienna, Austria stricted TGF-␤ type II receptor dominant negative transgenic mice Received for publication February 22, 2000. Accepted for publication May 23, 2000. contain normal numbers of LC (15). These observations suggest The costs of publication of this article were defrayed in part by the payment of page ␤ Ϫ/Ϫ charges. This article must therefore be hereby marked advertisement in accordance that the LC deficiency in TGF- 1 mice reflects a requirement with 18 U.S.C. Section 1734 solely to indicate this fact. of LC or precursor cells for TGF-␤1 and is obviously not due to 1 This work was supported by the Interdisziplina៮res Cooperations-Projekt program of modulation of the cutaneous microenvironment. This model seems the Austrian Ministry for Research and Transport. to be compatible with a critical role for endogenous TGF-␤1inLC 2 Address correspondence and reprint requests to Dr. Herbert Strobl, Institute of Im- development. In line with this possibility, LC-type DC are known munology, University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria. E-mail ␤ address: [email protected] to synthesize TGF- 1 abundantly (6, 17, 18), and a recent study ␤ 3 Abbreviations used in this paper: LC, Langerhans cells; SCF, stem cell factor; FL, showed that TGF- 1-transduced DC show increased survival and Flt3 ligand; DC, dendritic cells; CB, cord blood. persistence after transplantation into normal allogeneic mice (19).

The mechanisms involved in TGF-␤1-dependent LC differentia- Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifu- ϩ tion, however, remained unclear. gation. CD34 cells were isolated from CB mononuclear cells using the Here we demonstrate that LC development from CD34ϩ hemo- MACS CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the instructions of the manufacturer. The ␤ ϩ poietic progenitor cells in response to TGF- 1 stimulation is as- purity of the CD34 population exceeded 90%. sociated with pronounced homotypic cell cluster formation of cultured cells. This cell-clustering phenomenon is mediated by TGF- ϩ ␤1-induced E-cadherin in cooperation with several constitutively Primary cultures of purified CD34 CB cells expressed adhesion molecules. Cluster inhibition studies suggest Primary cultures of purified CD34ϩ CB cells were performed in 24-well that TGF-␤1-dependent cellular interactions may be functionally plates (Costar, Cambridge, MA; 1 ϫ 104 cells in 1 ml/well) at 37°C in a involved in LC differentiation from their precursor cells. humidified atmosphere and in the presence of 5% CO2 as described previously (29). The serum-free medium X-VIVO 15 (BioWhittaker, Walk- ersville, MD) contained L-glutamine (2.5 mM), penicillin (125 U/ml), and Materials and Methods streptomycin (125 ␮g/ml). Cultures were supplemented with optimized Antibodies concentrations of the following human cytokines: Flt3 ligand (FL; 100 ng/ml; provided by Immunex, Seattle, WA), TGF-␤1 (0.5 ng/ml; purified The following murine mAbs were generated in our laboratory: VIAP (non- from platelets; British Biotechnology, Abington, U.K.), rTNF-␣ (50 U/ml; binding isotype control Ab, calf intestine alkaline phosphatase specific), Bender, Vienna, Austria), rGM-CSF (100 ng/ml; Novartis, Basel, Switzer- LZ-1 (human lysozyme), VIT6b (CD1a), 5E6 (CD11a), and 3B2/TA8 land), and recombinant human stem cell factor (SCF; 20 ng/ml; Amgen, (CD99). CD14 mAb MEM-18 was provided by An der Grub (Kaumberg, Thousand Oaks, CA). Austria); CD34 mAb HPCA2 was obtained from Becton Dickinson (San Jose, CA); CD18 mAb TS1/18 was purchased from American Type Cul- Downloaded from ture Collection (Manassas, VA); CD11b mAb 2LPM19C was obtained ϩ from Dako (Glostrup, Denmark). CD29 mAb 4B4, CD49d mAb HP2/1, Monoclonal Ab stimulation experiments of CD34 cells CD49e mAb SAM1, and CD103 mAb 2G5 were obtained from Beckman Primary cultures of CD34ϩ cells were set up exactly as described above. Coulter (Fullerton, CA). E-cadherin mAb HECD-1 was purchased from After 24 h cells were harvested and replated in duplicate in 96-well plates Zymed (San Francisco, CA); CD31 mAb CLB-HEC/75 was purchased containing identical growth media at a cell density of 1 ϫ 104 cells/well. from CLB (Amsterdam, The Netherlands). Cultures were supplemented with 20 ␮g/ml mAb when indicated, and The following above-mentioned Abs were used in cell culture experi- plates were incubated at 4°C to allow Ab binding. Afterward, cells were

ments. They were selected based on previously published functional stud- transferred to 37°C and cultured for 7 days. On days 3 and 7, cultures were http://www.jimmunol.org/ ies: clone VIAP (control Ab, IgG1, purified from ascites using protein A analyzed for cell cluster formation by phase contrast microscopy using a chromatography) (20), clone 5E6 (CD11a, IgG1, purified from ascites fluid scoring system previously established in our laboratory (20) with slight using protein A chromatography, cell adhesion-blocking mAb) (20), clone modifications. Scores ranged from 0 to 4; 0 indicated that Ͻ10% of the 3B2/TA8 (CD99, IgG1, purified from ascites fluid using protein A chro- cells were in aggregates, 1 represented 10–50% aggregated cells, 2 indi- matography, mitogenic signaling mAb) (21), clone TS1/18 (CD18, IgG1, cated that about 50–75% of the cells were aggregated, 3 indicated 75- 90% purified from hybridoma supernatant using protein A chromatography, cell of the cells were aggregated, and 4 indicated that Ͼ90% of the cells were adhesion-blocking mAb) (22), clone 4B4 (CD29, IgG1 purified from as- found in large aggregates. On day 7 cells were harvested and analyzed by cites fluid using ion exchange chromatography, cell adhesion-blocking flow cytometry (see above). mAb) (23), clone HP2/1 (CD49d, IgG1, purified from hybridoma supernatant using affinity purification, cell adhesion-blocking mAb) (24), clone SAM1 (CD49e, IgG1, purified from hybridoma supernatant using affinity by guest on September 29, 2021 purification, cell adhesion-blocking mAb) (25), clone HECD-1 (E-cad- Results herin, IgG1, purified from ascites fluid using ammonium sulfate precipi- TGF-␤1-induced LC development from CD34ϩ progenitor cells tation, cell adhesion-blocking mAb) (26) and clone CLB-HEC/75 (CD31, is associated with cell cluster formation IgG1 purified from ascites fluid using affinity chromatography, cell adhesion-blocking mAb) (27). Before functional analysis, all Abs were dialyzed We recently established a serum-free culture system for the effi- against serum-free cell culture medium (see below). Abs were added to cient generation of Birbeck granule Ag Lagbright, CD1abright LC ␮ cultures at a final concentration of 20 g/ml. Ab preparations, which were from human CD34ϩ progenitors and observed that LC generation used in cell culture experiments, were tested for endotoxin (LPS). All the ␤ mAbs tested contained Ͻ0.1 ng/ml LPS, with the exception of mAb is strictly dependent on addition of TGF- 1 to the culture medium HEC/75 (CD31), which contained low levels of endotoxin (3.5 ng/ml). (29) containing the classical DC cytokines GM-CSF plus TNF-␣, Because all experiments were performed in serum-free medium, which SCF, and FL (30–32). does not contain significant amounts of LPS binding protein and soluble As shown in Fig. 1 (upper panel), TGF-␤1-dependent LC CD14, it is very unlikely that the observed functional effect of the mAb HEC/75 is due to LPS contamination. growth observed in this defined culture system is associated with the formation of large cell clusters. In the absence of TGF-␤1 (Fig. Immunofluorescence staining procedures 1, lower panel) cells do not form or form only few, very small, cell ϩ For membrane staining, 50 ␮l of cells (107/ml) were incubated for 15 min clusters and do not differentiate into CD1a LC (Fig. 1, lower at 4°C with 20 ␮l of conjugated mAb or unconjugated mAb. For staining panel). Ј using unconjugated mAb, FITC-conjugated F(ab )2 of sheep anti-mouse Ig Cell cluster formation in TGF-␤1-supplemented cultures starts Abs (An der Grub) were used as described previously (28). Cells were on day 4 of culture. By day 7 the vast majority of cultured cells in washed and analyzed by flow cytometry or were subjected to intracellular ␤ staining. the presence of TGF- 1 reside within large aggregates. These cell For suspension staining of the intracellular Ags, we used the commer- clusters can be easily disrupted by pipetting, but secondary cluster cially available reagent combination Fix&Perm (An der Grub) according to formation occurs within 24 h upon replating of generated cells in the manufacturer’s directions. Briefly, cells are fixed for 15 min at room identical growth medium at 37°C. Incubation of cells at 4°C or temperature (50 ␮l of cells plus 100 ␮l of formaldehyde-based Fixation Medium). After one washing with PBS, pH 7.2, cells are resuspended in 50 addition of EDTA (10 mM) to the cultures completely prevents ␮l of PBS and mixed with 100 ␮l of Permeabilization Medium plus 20 ␮l secondary cell cluster formation of LC (data not shown). of fluorochrome-labeled Ab. After a further incubation for 15 min at room In line with the selective generation of LC in the presence of temperature, cells are washed again and analyzed by flow cytometry. Flow TGF-␤1, only a minor portion of cultured cells show monocytic cytometric analyses were performed using a FACScan flow cytometer features (LZϩ, CD1aϪ; Fig. 1, upper right). Conversely, a large (Becton Dickinson). portion of cells generated in TGF-␤1-nonsupplemented cultures Cord blood (CB) cells develops into LZϩCD1aϪ monocytic cells (Fig. 1, lower right). CB samples were collected during normal full-term deliveries. Mononu- These data suggest an association of TGF-␤1-induced cluster for- clear cells were isolated within 10 h after collection using discontinuous mation and LC differentiation. The Journal of Immunology 1383

FIGURE 1. DC generated in the presence of TGF-␤1 in defined serum- free medium show specific features of epidermal LC and form large cell clusters. Purified CD34ϩ cells were cultured for 7 days in serum-free medium supplemented with the cytokines GM-CSF plus TNF-␣, SCF, and FL in the presence or the absence of TGF-␤1. Cells were harvested and analyzed for cell morphology (see Materials and Methods) and the expression Downloaded from of informative molecules. Typical microscopic appearance of generated cells in the presence or the absence of TGF-␤1(left) (original magnification, ϫ20). Diagrams show correlated expression of E-cadherin (center)or the monocyte marker molecule lysozyme (LZ) vs CD1a by generated cells in the presence (upper panel) or the absence (lower panel) of TGF-␤1 (right). http://www.jimmunol.org/

FIGURE 2. Analysis of cytoadhesion molecule expression by generated cells. Cells were generated in defined serum-free medium in the presence TGF-␤1 induces expression of the homophilic adhesion or the absence of TGF-␤1 (see Materials and Methods). On day 7 cells molecule E-cadherin were harvested and stained for expression of a panel of adhesion molecules. Overlay histograms show total generated cells analyzed for the ex- ␤ As described above, TGF- 1-induced LC differentiation is strictly pression of the adhesion molecules as indicated (open histograms) vs iso- associated with cell cluster formation, suggesting regulation of ad- tope-matched negative (filled histograms) controls. Data are representative hesion molecule expression by TGF-␤1. The homophilic epithelial of three experiments. adhesion molecule E-cadherin is known to be expressed in vivo by guest on September 29, 2021 not only by epithelial cells but also by LC (5), and mediates high affinity binding of LC to keratinocytes (4). As shown in Fig. 1, the cules and studied whether cell cluster inhibition may affect LC ϩ vast majority of CD1a cells in vitro generated in the presence of differentiation in TGF-␤1-supplemented LC generation cultures. TGF-␤1 strongly express E-cadherin. Cells generated in the ab- Addition of mAb 3B2/TA8 or TS1/18, specific for CD99 or the Ϫ sence of TGF-␤1 remain E-cadherin , except for a small common ␤-chain of ␤2 integrins (CD18), respectively, strongly ϩ Ϫ E-cadherin CD1a subset. inhibited LC cluster formation when added on day 1 to TGF-␤1- Fig. 2 shows representative expression profiles of a panel of supplemented LC generation cultures. As shown in Fig. 3, cultures cytoadhesion molecules by cells generated in the presence or the supplemented with mAbs TS1/18 or 3B2/TA8 contained no or ␤ ␣ absence of TGF- 1. As shown, CD103 (HML-1, integrin E only very small cell clusters. chain), which associates with the ␤ subunit of the heterodimer Phenotypic analysis of day 7 cells generated in the presence of ␣ ␤ integrin E 7, a heteroligand of E-cadherin (33), could not be de- the CD99 mAb 3B2/TA8 revealed that only very low percentages tected on the surface of generated cells. Most cells generated in the of cells express the LC marker molecule CD1a (5.0 Ϯ 3.5%; n ϭ ␤ ␤ presence or the absence of TGF- 1 express the 1 integrins 3), and that, in turn, high percentages of cells express the monocyte CD49d/CD29 (VLA-4) and CD49e/CD29 (VLA-5) as well as the marker molecule LZ (35) and lack CD1a (46.8 Ϯ 8.8%; n ϭ 3; p ϭ ␤ Ͻ ␤ ϩ 2 integrin CD11a/CD18 (LFA-1); 10% of cells express the 2 0.002 compared with control cultures: 13.0 Ϯ 4.1% LZ cells; Fig. integrin ␣-chain CD11b (data not shown). Additionally a subset of 4). Thus, mAb 3B2/TA8 binding to CD99 not only inhibits cluster generated cells expresses CD31 (PECAM-1), and most cells ex- formation, but the overall differentiation pattern of CD34ϩ pro- press CD99 (MIC-2/E2). All these latter molecules are already genitor cells switches from LC to monocytes. Very similar effects ϩ expressed at the CD34 progenitor cell stage (34), and their ex- on differentiation were observed in cultures containing the CD18 pression pattern is not significantly affected by TGF-␤1 stimula- mAb TS1/18 (14.7 Ϯ 2.9% of CD1aϩ cells and 55.6 Ϯ 8.8% of tion. In contrast, E-cadherin is clearly induced by TGF-␤1 stimu- LZϩ CD1aϪ cells; Fig. 4). A shift in the lineage differentiation ϩ lation and cannot be detected on the cell surface of CD34 pattern from LC to monocytic cells was confirmed by the obser- hemopoietic progenitors (12). vation that substantial percentages of cultured cells in the presence of mAbs 3B2/TA8 or TS1/18 express the monocyte marker mol- Inhibition of cell cluster formation by mAbs to CD99 or CD18 ecule CD14 in the absence of CD1a (33.7 Ϯ 9.1 and 24.5 Ϯ 8.8%, abrogates LC differentiation and cells develop into a monocytic respectively; n ϭ 3; see Fig. 4). The numbers of cells recovered on pathway day 7 from Ab-supplemented cultures initiated with 1 ϫ 104 To analyze whether TGF-␤1-induced LC differentiation and cell CD34ϩ cells on day 0 did not differ significantly (mAb 3B2/TA8, cluster formation are functionally linked, we performed cell cluster 20.8 Ϯ 4.7 ϫ 104; mAb TS1/18, 18.1 Ϯ 4.8 ϫ 104; control mAb inhibition experiments using blocking mAbs to adhesion mole- VIAP, 25.1 Ϯ 4.9 ϫ 104; n ϭ 4). The differentiation pattern and 1384 INVOLVEMENT OF E-CADHERIN IN LC PRECURSOR CELL CLUSTERING

FIGURE 5. Time kinetics of total cell numbers in LC generation cultures supplemented with mAbs to CD11a, CD31, or E-cadherin. CD34ϩ cells (1 ϫ 104) were cultured in deﬁned serum-free medium containing the

␤ ␣ Downloaded from FIGURE 3. Typical morphology of cells generated in the presence of cytokines TGF- 1 plus GM-CSF, TNF- , SCF, and FL. Parallel cultures TGF-␤1 and various mAbs to adhesion molecules. Cells were generated in were supplemented with mAbs to CD11a, CD31, or E-cadherin or negative cultures supplemented with TGF-␤1 plus GM-CSF, TNF-␣, SCF, and FL control Abs (see Materials and Methods). Cell numbers were determined for 7 days as described in Materials and Methods. On day 1 cultures were on days 3 and 7. Data are representative of three experiments. supplemented with mAbs of the following speciﬁcity: A, control mAb; B, CD99; C, CD18; and D, E-cadherin. The representative microscopic ap- ϫ ϭ

pearance of generated cells is shown (original magnification, 20; n 4). http://www.jimmunol.org/ and SCF-supplemented cultures of CD34ϩ cells (36). De- creased viability of LC precursors in the presence of these LC morphology of cells generated in these cultures resemble those of cluster-blocking mAbs may, therefore, reflect a role of cell clus- ␤ cells cultured in the absence of TGF- 1 (Fig. 1) (29). Thus, ad- tering in mediating an anti-apoptotic activity of TGF-␤1on ␤ ϩ dition of mAbs to these cytoadhesion molecules inhibits TGF- 1- cultured CD34 cells. induced cell cluster formation and LC differentiation. E-cadherin ligation allows LC generation in the absence of cell Inhibition of cell cluster formation by mAbs to CD31 or LFA-1 cluster formation abrogates LC differentiation and inhibits cell proliferation Addition of mAb HECD-1 specific for E-cadherin on day 1 to by guest on September 29, 2021 Addition of mAb CLB-HEC/75 or 5E6, specific for CD31 or TGF-␤1-supplemented cultures of CD34ϩ cells strongly inhibits ␤ LFA-1, respectively, also inhibited TGF- 1-induced cell cluster cell cluster formation (Fig. 3). Phenotypic analyses revealed that a formation when added to TGF-␤1-supplemented LC generation ϩ large percentage of cells generated after 7 days, however, express cultures of CD34 cells. This effect was associated with strong high levels of CD1a. As shown in the representative phenotypic inhibition of cell proliferation. As shown in Fig. 5 cell numbers analyses in Fig. 4, E-cadherin mAb-supplemented cultures con- of cultured cells continuously decreased from days 0 to 3 to 7 tained reproducibly even higher percentages of CD1aϩ cells than in cultures supplemented with either of these mAbs. We previ- control cultures (47.3 Ϯ 2.9 vs 41.8 Ϯ 2.1%; n ϭ 4). ously demonstrated that TGF-␤1 exerts an anti-apoptotic effect ␣ on LC progenitor cells when added to GM-CSF- plus TNF- Discussion In the present study we describe a tight association of homotypic cell cluster formation and LC differentiation of cultured hemopoietic progenitor cells in response to TGF-␤1 costimulation. CD34ϩ CB hemopoietic progenitor cells stimulated in serum-free cultures in the presence of the basic cytokine combination GM-CSF plus TNF-␣, SCF, and FL for 7 days fail to form cell clusters and predominantly develop into monocytic cells. Addition of TGF-␤1 to these short term cultures results in pronounced cell cluster formation and a concomitant shift in the differentiation pattern of stimulated CD34ϩ cells from monocytic progeny to LC-type DC. This LC differentiation-linked cell-clustering phenomenon starts on day 4 after culture initiation of purified CD34ϩ cells and thus obviously requires the pregeneration of a committed precursor cell FIGURE 4. Phenotype of cells generated in TGF-␤1-supplemented cul- ϩ population. These data suggest that cell clustering may mark an tures in the presence of mAbs to adhesion molecules. CD34 cells were early branching point at which monocyte/LC precursor cells start cultured in defined serum-free medium containing the cytokines TGF-␤1 ␣ to differentiate into LC. In support of this assumption, we previ- plus GM-CSF, TNF- , SCF, and FL. Parallel cultures were supplemented ϩ ␤ with mAbs to E-cadherin, CD18, or CD99 or with negative control Abs; ously observed that CD1a LC in TGF- 1-supplemented cultures (see Materials and Methods). Dot plots show LZ vs CD1a expression or seem to develop from a day 4 generated early monopoietic pre- CD14 vs CD1a expression, respectively, of total day 7 generated cells in cursor cell population (13) identifiable by the expression of ly- the presence of mAbs as indicated. Markers were set according to negative sozyme in the absence of CD14 (35). Furthermore, recent studies ϩ control staining. Data are representative of four experiments. demonstrated that even later stage CD14 precursor cells can be The Journal of Immunology 1385 induced to differentiate into LC in response to TGF-␤1 costimu- Interestingly, mAb HEC/75 or 5E6 (directed against CD31 or lation (37–39), thus supporting this possibility. CD11a, respectively) blocked TGF-␤1-induced LC precursor cell Homophilic E-cadherin interactions between developing LC clustering of CD34ϩ cells, and this effect was associated with in- precursor cells seem to be critically involved in the TGF-␤1-inhibition of cell proliferation early during culture. It is possible that duced, early appearing, cell-clustering phenomenon of developing CD31 and/or CD11a interactions are involved in initial TGF-␤1- LC precursor cells described herein. First, large proportions of induced cell cluster formation of CD34ϩ cells and may be func- cells from TGF-␤1-supplemented cultures express E-cadherin at tionally involved in mediating an anti-apoptotic (36) or (pro-) pro- high levels, whereas most cells from TGF-␤1-nonsupplemented liferative effect (39) of TGF-␤1 on LC precursor cells. Consistent parallel cultures lack E-cadherin expression (with the exception of with this possibility, both CD11a and CD31 molecules have pre- a small E-cadherindim subset). Second, physical separation of clus- viously been implicated in cell adhesion of hemopoietic progenitor tered cells from TGF-␤1-supplemented LC generation cultures (by cells. We previously demonstrated that mAb 5E6 (specific for 1 ϫ g sedimentation) results in strong enrichment of E-cadherinϩ CD11a; used in the present study) blocks CD34 ligation-induced CD1aϩ cells (data not shown). Third, addition of a blocking anti- homotypic cell clustering of the hemopoietic progenitor cell line E-cadherin mAb on day 1 to the cultures inhibits TGF-␤1-induced KG1a (20). Lastres et al. (27) observed that CD31 mAb HEC/75 cell cluster formation. Furthermore, cells generated in TGF-␤1- blocks TGF-␤1-induced cell clustering of the promonocytic cell supplemented LC generation cultures lack expression of CD103, a line U-937. In line with our observations, CD31 is expressed at previously characterized heterophilic E-cadherin ligand (33). high levels by virtually all CD34ϩ cells, and transmigration of Thus, the cell cluster phenomenon described herein obviously in- CD34ϩ cells (41) or a subset of CD34ϩ DC precursors (42) volves homotypic LC precursor cell interactions, which are medi- through vascular endothelial layers is dependent on CD31 Downloaded from ated by E-cadherin adhesion. Given the fact that E-cadherin has adhesion. previously been described as a reliable surface marker molecule As mentioned above, mAb HECD-1 directed against E-cadherin for developing LC-type DC in cultures of CD34ϩ cells (30), these inhibited TGF-␤1-induced LC precursor cell cluster formation, but observations seem to support above-mentioned tight linkage of cell in contrast to the above cell cluster-blocking mAbs, outgrowth of cluster formation and LC differentiation in our culture model. CD1aϩ LC was not impaired. These observations thus seem to

Ab blockage experiments revealed that in addition to E-cad- argue against a functional involvement of cell clustering in LC http://www.jimmunol.org/ herin, several constitutively expressed adhesion molecules are differentiation. We cannot, however, rule out the possibility that functionally involved in TGF-␤1-induced homotypic LC precursor E-cadherin ligation by mAb may functionally alter the differenti- cell clustering. We demonstrate that addition of mAbs to CD11a ation pattern of cultured cells, eventually leading to enhancement (LFA-1), CD18, CD31, or CD99 to day 1 cultures of CD34ϩ cells of LC generation. Consistent with this possibility, E-cadherin has inhibits TGF-␤1-induced LC precursor cell clustering. Interest- previously been implicated in the differentiation processes of ep- ingly, in contrast to E-cadherin, all these latter adhesion molecules ithelial (43) and hemopoietic progenitor cells (10, 12). Addition- were previously shown to be constitutively expressed by freshly ally, mAb ligation of E-cadherin on the surface of epithelial cell isolated CD34ϩ cells (reviewed in Ref. 34), and we observed that lines was previously shown to mimic homophilic E-cadherin liga- their expression intensities by cultured cells were only slightly tion in inducing transmembrane signaling in epithelial cell lines by guest on September 29, 2021 modulated by TGF-␤1 costimulation. (44–46). The identiﬁcation of several cell cluster-blocking mAbs allowed In conclusion, cell cluster formation represents a regular re- us to study a possible functional linkage of cell cluster formation sponse of LC precursor cells to TGF-␤1 stimulation, and TGF- and LC differentiation. Ab addition experiments indeed seem to ␤1-dependent cellular interactions may modulate LC development argue in favor of this possibility. We observed that inhibition of and differentiation. This differentiation model is optimally suited LC precursor cell clustering by addition of certain mAbs to TGF- for studying the possible functional involvement of E-cadherin in ␤1-supplemented progenitor cell cultures selectively inhibits LC LC differentiation using gene transfer experiments. generation. For example, addition of mAb TS1/18 (CD18) or the mAb 3B2/TA8 (CD99) inhibited LC development, and cells de- Acknowledgments veloped into LZϩCD14ϩCD1aϪ monocytic cells. From this dif- We thank A. Renner for his invaluable contribution in cell separation. We ferentiation pattern these cells strikingly resembled cells generated are indebted to all the collaborating nurses and doctors of the gynecology ␤ in parallel cultures in the absence of TGF- 1 (Fig. 1). Thus, in- departments at Sozialmedizinisches Zentrum Ost and Kaiser Franz Josef hibition of cell cluster formation by mAbs to these adhesion mol- Spital (Vienna, Austria) for providing cord blood samples. 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