Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0206111 on September 7, 2006

Published online before print September 7, 2006
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(Journal of Leukocyte Biology. 2006;80:1337-1344.)
© 2006 by Society for Leukocyte Biology

A CD34+ human cell line model of myeloid dendritic cell differentiation: evidence for a CD14+CD11b+ Langerhans cell precursor

Saskia J. A. M. Santegoets*,1, Allan J. Masterson{dagger},1, Pieter C. van der Sluis*, Sinéad M. Lougheed{dagger}, Donna M. Fluitsma{ddagger}, Alfons J. M. van den Eertwegh{dagger}, Herbert M. Pinedo{dagger}, Rik J. Scheper*,2 and Tanja D. de Gruijl{dagger}

* Departments of Pathology,
{dagger} Medical Oncology, and
{ddagger} Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

2 Correspondence: Department of Pathology, VU University Medical Center, De Boelelaan 1117, Amsterdam 1081HV, The Netherlands. E-mail: rj.scheper{at}vumc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine-driven, transitional DC differentiation steps. The CD34+ acute myeloid leukemia cell line MUTZ-3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine-dependent manner. Phenotypic characterization revealed MUTZ-3 to consist of three distinct subpopulations. Small CD34+CD14–CD11b– progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34–CD14–CD11b+ stage to ultimately give rise to a morphologically large, nonproliferating CD14+CD11bhi progeny. These CD14+CD11bhi cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC-specific ICAM-grabbing nonintegrin, respectively. The identity of the MUTZ-3-derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ-3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.

Key Words: MUTZ-3 • progenitor • acute myeloid leukemia • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional APC and key regulators of the immune system, displaying an extraordinary capacity for T cell stimulation and the initiation of immune responses [1 , 2 ]. DC develop from CD34+ bone marrow-derived hematopoietic progenitor cells and are thought to undergo sequential differentiation through a number of intermediate precursor states, prior to populating the tissues as immature DC [3 ].

A number of specific markers have been proposed for the identification of CD34+ progenitors with the propensity to differentiate into myeloid DC, e.g., CD11c, CD14, CD33, CD40, and CD86 [3 4 5 6 ]. It is not known, however, whether these myeloid progenitors represent distinct lineages, giving rise to particular DC subsets within the peripheral tissues, or whether they represent consecutive precursor states along common differentiation pathways toward myeloid DC. Two major myeloid DC subsets have been identified so far, i.e., Langerhans cells (LC), populating epidermal surfaces in the body, and the dermal or interstitial DC (IDC). These subsets are thought to arise from one or more CD34-derived precursors, and TGF-β1 plays an obligatory role in LC differentiation [7 8 9 10 11 12 ]. However, the extremely low in vivo frequencies of distinct myeloid DC subsets and their precursors and the lack of homogeneous primary cell systems and sustainable, cytokine-dependent DC progenitor culture systems have hampered more extensive characterization of their precise inter-relationships [13 ].

We have demonstrated previously that the acute myeloid leukemia (AML)-derived human cell line MUTZ-3 can differentiate into functional DC in a cytokine-dependent manner with discrete immature and mature stages [14 ]. Here, we present data further characterizing MUTZ-3 as a DC progenitor line consisting of a proliferating pool of small CD34+ cells, which during cytokine-dependent culture, differentiate into large CD14+ cells. These early steps in differentiation are accompanied by a gradual shift in the expression of myeloid markers, costimulatory molecules, antigen-capture, and cytokine receptors, characteristic of more mature DC precursors. The resulting CD14+CD11bhi cells are shown to be immediate, common precursors of LC and IDC. These data clearly demonstrate the existence of a CD14+CD11b+ common IDC and LC precursor. We conclude that MUTZ-3 is unique in comparison with other myeloid leukemic cell lines [15 , 16 ] in that it allows the study of discrete transitional stages in myeloid DC development from CD34+ progenitor cells. The MUTZ-3 cell line thus represents a valuable and sustainable model system for the further elucidation of molecular mechanisms regulating early and late stages of human myeloid DC differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of MUTZ-3-derived IDC and LC
The human AML-derived cell line MUTZ-3 was obtained from the German collection of microorganisms and cell cultures (DSMZ, Braunschweig, Germany) [17 , 18 ]. MUTZ-3 was maintained in MEM-{alpha} with ribonucleosides and deoxyribonucleosides (Gibco, Grand Island, NY), supplemented with 10% conditioned medium from the human renal carcinoma cell line 5637 [18 ]. CD34+, CD14+, and CD34–CD14– MUTZ-3 cell fractions were isolated by MACS using anti-CD14 or anti-CD34-labeled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated cell fractions were >98% pure (as determined by flow cytometric analysis). During differentiation studies, 5637 conditioned media were replaced with defined cytokines as described previously [13 ]. For IDC generation, CD34+, CD14+, and CD 34–CD14– fractions were cultured in a cocktail of GM-CSF (100 ng/ml), IL-4 (1000 U/ml) and TNF-{alpha} (2.5 ng/ml) for 5 days. For the generation of LC, cell fractions were cultured with GM-CSF (100 ng/ml), TNF-{alpha} (2.5 ng/ml), and TGF-β1 (10 ng/ml) for 5 days. Differentiation of IDC or LC from unseparated MUTZ-3 cells was achieved with the above-listed cytokine cocktails over a period of 7 and 10 days, respectively. Maturation of MUTZ-3 IDC and LC was induced by adding monocyte-conditioned medium (MCM) mimic, a cytokine cocktail consisting of 50 ng/ml TNF-{alpha}, 100 ng/ml IL-6, 25 ng/ml IL-1β (Strathmann Biotec, Hamburg, Germany), and 1 µg/ml PGE2 (Sigma Chemical Co., St. Louis, MO) over a period of 3 days.

Flow cytometric analysis
For flow cytometric analysis, cells were incubated on ice for 30 min in PBS with 0.1% BSA and 0.01% NaN3, in the presence of appropriate dilutions of mouse isotype-matched control mAb or PE-labeled mAb against CD40, CD34, CD207, CD126, and fetal liver tyrosine kinase 3 (Coulter Immunotech, Marseilles, France); CD1a, CD11b, CD11c, CD36, CD91, and CD117 (PharMingen, San Diego, CA); CD86, CD45RA, CD4, CD123, and CD14 (Becton Dickinson, San Jose, CA); blood DC antigen 4 (BDCA4; Miltenyi Biotec); TNF-{alpha} receptor-II (TNF-{alpha}RII; R&D Systems, Oxon, UK); or FITC-labeled mAb against HLA-DR, CD14, and CD45RO (Becton Dickinson); CD116 and DC-specific ICAM-grabbing nonintegrin (SIGN; PharMingen); CD34 (Coulter Immunotech); BDCA1 and BDCA3 (Miltenyi Biotec); CD13 (Sigma Chemical Co.); conjugated linoleic acids (Becton Dickinson); and TNF-{alpha}RI (R&D Systems). The cells were subsequently analyzed, using a FACStar Plus and Cellquest FACS analysis software (Becton Dickinson).

Cell division studies
For CFSE labeling, cell fractions were washed with PBS, incubated with 10 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C, and washed twice with cold PBS. Labeled cells were cultured for up to 14 days in routine culture medium. At various time-points, CFSE dilution by cell division was determined by flow cytometry.

MLR
MLR was performed with immature and MCM mimic-matured IDC and LC. DC were added as stimulator cells to round-bottom, 96-well, tissue-culture plates (Costar, Corning, NY) at graded doses, reflecting the indicated responder:stimulator ratios. Allogeneic, plastic, nonadherent PBL were used as a source of responder cells, and 1 x 105 lymphocytes per well were added to the DC. Stimulations were performed in triplicate. The cells were cultured for 5 days in medium containing 10% FCS (Life Technologies, Paisley, Scotland). During the last 18 h of culture, [3H]TdR was added (0.4 µCi per well; Amersham, Aylesbury, UK), after which the cells were harvested onto fiberglass filters, and [3H]TdR incorporation was determined using a flatbed scintillation counter.

Electron microscopy (EM)
MUTZ-3-derived LC were prepared for EM according to standard procedures to check for the presence of Birbeck granules, based on their typical subcellular morphology. Cultured cells were fixed in 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, dehydrated in ethanol, infiltrated with propylene oxide, and embedded in Agar 100 resin (Agar Scientific, Stansted UK). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (Philips CM 100 Bio Twin).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MUTZ-3 represents ongoing, early-stage myeloid DC differentiation
We previously demonstrated the unique potential of the AML-derived MUTZ-3 cell line to differentiate into DC in a cytokine-dependent manner [14 ]. Here, we set out to further characterize the precursors from which the MUTZ-3-derived DC originate. Three phenotypically distinct populations were identified by flow cytometry within the MUTZ-3 cell line: CD34+CD14– (hereafter referred to as the CD34+ subpopulation), CD34–CD14– [hereafter referred to as the double-negative (DN) subpopulation], or CD34–CD14+ (hereafter referred to as the CD14+ subpopulation; see Fig. 1A ). Magnetic microbead-isolated subpopulations differed in cell size and morphology, and the larger CD14+ cell fraction exhibited a more differentiated, DC-like phenotype as compared with the smaller and more rounded CD34+ fraction (see Fig. 1A ). CFSE incorporation studies about the isolated subpopulations (>98% pure) demonstrated that the CD34+ cells underwent cell division, whereas the more differentiated CD14+ cells did not (Fig. 1A) . Routine maintenance culture of isolated CD34+ cells revealed an ongoing sequential differentiation process, resulting in the appearance of DN cells within 24 h, which in turn, gave rise to CD14+ cells by Day 5 (Fig. 1B) . These results identify the CD34+ fraction as the proliferating pool of progenitor cells from which the CD14+ progeny arise.


Figure 1
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  		Figure 1. CD34+ cells are the proliferating fraction of the MUTZ-3 cell line and give rise to nonproliferating CD14+ precursors. (A) MUTZ-3 consists of three subpopulations, based on CD34 and CD14 expression: CD34+ cells, DN cells, and CD14+ cells. MUTZ-3 CD34+ and CD14+ subpopulations were separated by MACS isolation and examined for differences in morphology and proliferative capacity. (Upper panels) May-Günwald Giemsa staining of CD34+ cells and of CD14+ cells (original magnification, 400x). (Lower panels) CD34+ and CD14+ cell fractions were labeled with CFSE, cultured for 14 days, and analyzed for CFSE fluorescence by flow cytometry. Results are from one experiment representative of five. (B) CD34+ MUTZ-3 progenitor cells were purified by MACS (>98% purity) and cultured for 7 days in routine culture medium. At the indicated intervals, aliquots were assayed for the expression of CD34 and CD14. Results are from one experiment representative of three.

 
MUTZ-3 subpopulations were also examined for the expression of myeloid DC (precursor) markers and of cytokine receptors. Over the course of differentiation from the CD34+ progenitors through the DN fraction to the CD14+ precursors, a clear up-regulation of myeloid differentiation markers was observed (e.g., CD1c, CD11b, CD11c, CD13, and CD45RO; see Table 1 ). Expression of CD1c (i.e., BDCA1) preceding the expression of BDCA4 is in agreement with a previous report, showing expression of CD1c on blood DC precursors and BDCA4 expression only upon in vitro culture of DC precursors [19 ]. Receptors for cytokines, commonly associated with early DC precursor proliferation, were expressed selectively on the CD34+ cells (IL-3R, IL-6R, SCFR), whereas CD14+ cells preferentially expressed receptors for cytokines involved in DC differentiation (GM-CSFR, TNF-RI and-II; see Table 1 ) [20 ]. Similarly, increased expression of costimulatory molecules (CD40, CD86) and scavenger receptors (CD36 and CD91) on the CD14+ precursors is in keeping with their more mature differentiation state.


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  		Table 1. Phenotyping of MUTZ-3 Subpopulationsa

 
CD14+CD11bhi MUTZ-3 cells are common precursors of LC and IDC
By inducing MUTZ-3 DC differentiation with GM-CSF and TNF-{alpha} in the presence of TGF-β1 or IL-4, CD1a+Langerin+ LC or CD1a+DC-SIGN+ IDC were obtained, respectively (Fig. 2A ). Double-staining with CD1a revealed this DC-SIGN and Langerin expression on IDC and LC to be mutually exclusive (Fig. 2A) . This discrete, TGF-β1-dependent LC and IL-4-dependent IDC differentiation is entirely in keeping with observations from bone marrow-derived, primary CD34+ progenitor cells. However, the exact nature of the direct precursors of these DC subsets remains controversial. It was suggested recently that the CD14+CD11b+ phenotype denoted precursors that were committed exclusively to the IDC lineage [16 ]. CD11b expression was found to be absent from the CD34+ MUTZ-3 subpopulation, and intermediate and high expression levels were found on the DN and CD14+ subpopulations, respectively (see Table 1 and Fig. 2B ). To further delineate the ontogeny of the myeloid DC subsets, isolated CD34+CD11b–, DN/CD11b+, and CD14+CD11bhi MUTZ-3 subpopulations were cultured in the presence of GM-CSF and TNF-{alpha}, with or without IL-4 or TGF-β1. Upon 5 days of culture in the presence of IL-4, CD1a+DC-SIGN+ IDC were generated from the CD14+ precursors but not from the CD34+ or DN cells (Fig. 2B) . Similarly, CD1a+Langerin+ LC were generated in the presence of TGF-β1, only from the CD14+ precursors, which did not express Langerin prior to culture (Fig. 2B) . Differentiation of IDC and LC from the isolated CD14+CD34– precursors was accompanied by a loss of CD14 expression (not shown) and resulted in the following yields: 28.3 ± 12.7% for the IDC and 41.8 ± 18.6% for the LC. Thus, DC differentiation from this nonproliferating population was accompanied by considerable cell death.


Figure 2
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  		Figure 2. MUTZ-3 CD14+CD11bhi cells are direct LC and IDC precursors. (A) Unseparated MUTZ-3, cultured in the presence of GM-CSF, TNF-{alpha}, and IL-4 or TGF-β1, turns into Langerin–DC-SIGN+ IDC (left panel) or Langerin+DC-SIGN– LC (right panel) in, respectively, 7 and 10 days. (B) The three MUTZ-3 subpopulations (CD34+CD14–, CD34–CD14–, and CD34–CD14+) were isolated by sequential anti-CD34 and anti-CD14 magnetic microbead separation, analyzed for CD11b expression, cultured for 5 days in the presence of GM-CSF, TNF-{alpha}, and IL-4 or TGF-β1 (as indicated), and examined for the expression of CD1a and DC-SIGN or CD1a and Langerin. Data are from one experiment representative of five.

 
MUTZ-3-derived IDC and LC could be matured further under the influence of a cytokine cocktail consisting of IL-1β, IL-6, TNF-{alpha}, and PGE2, as demonstrated by the up-regulation of the DC maturation markers CD86 and CD40 and de novo expression of CD83 on both subsets (Fig. 3A ). This maturation induction also increased the T cell stimulatory capacity of IDC and LC, as demonstrated in an allogeneic MLR (Fig. 3B) . Of note, expression levels of costimulatory molecules were generally higher on the LC subset (Fig. 3A) , which translated into stronger T cell proliferation elicited in the allo-MLR (Fig. 3B) .


Figure 3
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  		Figure 3. Phenotypic and functional maturation induction in MUTZ-3-derived IDC and LC, generated from unseparated MUTZ-3 precursors. (A) MUTZ-3-derived IDC and LC can be matured under the influence of a cytokine cocktail consisting of IL-1β, IL-6, TNF-{alpha}, and PGE2, as demonstrated by the up-regulation of the DC maturation markers CD86, CD40, and CD83, after 3 days of maturation induction. The markers in the FACS histograms denote fluorescence intensities obtained with isotype control antibodies. (B) Maturation induction results in higher T cell responsiveness to IDC and LC in an allogeneic MLR. Data shown are representative of at least three experiments.

 
These data thus clearly demonstrate the CD14+CD11bhi cells, derived from CD34+ progenitors, to be the immediate, common precursors of fully functional IDC and LC and refute the assertion that these cells would be strict IDC precursors. To further confirm the identity of the MUTZ-3-derived LC, along with the coexpression at the cell surface of CD1a, Langerin, and E-cadherin, the absence of DC-SIGN was shown as well as the down-regulation of CD11b (Fig. 4A ) and the presence of characteristic Birbeck granules, as established by EM (Fig. 4B) . From our extensive experience, we find the fraction of the CD1a+ LC, which are positive by flow cytometry for Langerin at their cell surface, to vary between 40% and 80%. Further, EM immunostaining revealed intracellular Langerin to localize to the Birbeck granules (data not shown; L. de Witte, Y. van Kooyk, T.B. Geijtenbeek, manuscript in preparation). Birbeck granules in the LC were found to be quite numerous and were observed in virtually every section plane by EM. Altogether, these hallmarks firmly establish these MUTZ-3-derived DC as bona fide LC.


Figure 4
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  		Figure 4. DC derived from unseparated MUTZ-3 precursors through culture in the presence of TGF-β1 display typical LC phenotype and morphology. (A) TGF-β1-induced CD1a+ MUTZ3-DC display a typical LC phenotype, i.e., positive for Langerin and E-cadherin but negative for the IDC markers DC-SIGN and CD11b. (B) EM reveals the presence of cytoplasmic Birbeck granules, typical for the LC lineage. Inset shows a close-up. Results shown are representative of at least three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although intensively studied, the delineation of human myeloid DC differentiation is hampered by low frequencies and the heterogeneity of primary DC progenitors and precursors. Platzer et al. [16 ] recently commented on the need for more homogeneous, primary cell systems, which selectively mimic the cytokine-driven transitional DC differentiation steps, as these are not displayed by expanded CD34+ cells or myelomonocytic cell line models. We believe that the MUTZ-3 cell line may offer an ideal model system to study these processes, as in contrast to other myelomonocytic cell lines, it does display these transitional, differentiation steps. As outlined in this paper, routinely maintained MUTZ-3 cells undergo cytokine-driven, continuous differentation from a proliferative CD34+ progenitor into a nonproliferative CD14+ DC precursor. These findings are in keeping with previous reports of small, proliferating progenitors, which spontaneously gave rise to progeny resembling DC, without the addition of exogenous cytokines, in long-term cultures of mouse thymic stroma [21 22 23 24 ]. Stromal cells at sites of DC differentiation (most notably, the bone marrow) are a source of a number of cytokines, including GM-CSF, which are also contained in the 5637-conditioned medium used in the routine maintenance culture of MUTZ-3 [23 24 25 ]. The 5637 supernatants may thus mimic the influence of stromal cells in the bone marrow on such processes as progenitor proliferation and early-stage differentiation.

Inclusion of IL-4 or TGF-β1 in DC differentiation cultures from primary CD34+ precursors has been shown to result in the respective preferential differentiation of DC-SIGN+ IDC [26 ] or Langerin+ LC [10 ]. The MUTZ-3 model shows the exact same cytokine dependence for the differentiation of these specific myeloid DC lineages. Phenotypic characterization further confirmed the MUTZ-3-derived IDC and LC to be identical to their primary counterparts. Indeed, the presence of Birbeck granules establishes Langerin+ MUTZ-3 DC, generated in the presence of TGF-β1, as bona fide LC. IDC and LC could be matured further to end-stage differentiation, as evidenced by de novo expression of the characteristic maturation marker CD83 and up-regulation of costimulatory markers. This phenotypic maturation was accompanied by an increased T cell stimulatory capacity of both DC subsets in an allo-MLR, demonstrating their functionality. Further functional analyses (i.e., cytotoxic T cell-priming capacity, DC migration, and cytokine release) also point to characteristics of the MUTZ3-IDC and -LC, which are in line with previous reports about primary IDC and LC (S. J. A. M. Santegoets, A. G. Stam, R. J. Scheper, T. D. de Gruijl, submitted). In aggregate, these observations support the validity of the immortalized MUTZ-3 cell line, despite its leukemic origins, as a model for primary myeloid human DC differentiation. This was confirmed recently by Larsson et al. [27 ], who demonstrated through transcriptional profiling that MUTZ-3, in contrast to the more commonly used myeloid leukemic cell lines KG-1 and THP-1, closely resembled primary DC.

Two recent studies showed the inhibition of CD14+CD11b+ monocytopoiesis from CD34+ progenitors to arrest macrophage and IDC differentiation while leaving LC differentiation unaffected [16 , 28 ]. These results led the authors to conclude that LC differentiate from an early CD11b– precursor, which may or may not transit through a CD14+ stage. This is in line with earlier findings by Jaksits et al. [11 ], who reported CD34+ progenitor-derived CD14+CD11b– precursors to differentiate into LC in a TGFβ1-dependent manner. Whereas these reports suggest that CD14+CD11b+ monocytic cells are strict IDC precursors, previous studies clearly demonstrated the ability of peripheral blood-derived monocytes to adopt phenotypic LC traits upon culture in TGF-β1 or IL-15, respectively [29 , 30 ]. Although the physiological relevance of these in vitro models remains unclear, Larregina et al. [12 ] demonstrated the existence of CD14+CD11b+ precursors in the dermis of human skin with the ability to differentiate into CD1a+E-cadherin+Langerin+ LC in the presence of GM-CSF and TGF-β1. These results lend strong support to a physiological role for CD14+CD11b+ myelomonocytic precursors in LC differentiation but leave their connection to CD34+ progenitors unclear. The study of CD11b expression on the three MUTZ-3 subpopulations revealed it to be absent from the CD34+ progenitors, and intermediate and high expression levels were found on their DN and CD14+ progeny, respectively. We clearly showed the more mature, monocyte-like CD14+CD11bhi precursor cells to be immediate precursors of IDC and LC. Our data thus demonstrate irrevocably the existence of a CD34+ progenitor-derived CD14+CD11bhi LC precursor and refute the suggestion that these cells would be strict IDC precursors.

In conclusion, our data clearly demonstrate the existence of a CD34+ progenitor-derived CD14+CD11b+ LC precursor with an equal IDC differentiation capacity and serve as a case in point to demonstrate the suitability of the human MUTZ-3 cell line model for studies about cytokine-driven, regulatory mechanisms in the development of distinct myeloid DC lineages.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received February 22, 2006; revised June 22, 2006; accepted July 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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