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Originally published online as doi:10.1189/jlb.0504278 on September 2, 2004

Published online before print September 2, 2004
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(Journal of Leukocyte Biology. 2004;76:1214-1219.)
© 2004 by Society for Leukocyte Biology

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte

Giuseppe Sconocchia1, Hiroshi Fujiwara, Katayoun Rezvani, Keyvan Keyvanfar, Frank El Ouriaghli, Matthias Grube, Jos Melenhorst, Nancy Hensel and A. John Barrett

Stem Cell Allotransplantation Section, Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

1 Correspondence: Hematology Branch, Hematopoietic Stem Cell Transplantation Section, National Heart Lung and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda MD 20892-0001. E-mail: giuseppe.sconocchia{at}roswellpark.org


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ABSTRACT
 
To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 ± 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56bright, mainly CD33– cells (7±10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5±2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-{gamma}. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.

Key Words: NK cells • monocytes/macrophages • cellular proliferation • cellular differentiation


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INTRODUCTION
 
Natural killer (NK) cells originate in the bone marrow from a CD34+Lin–CD45RA+CD10+ common lymphoid progenitor cell, which gives rise to CD56+ CD3– NK cells, B cells, and dendritic cells [1 ]. Initial studies showed that CD56+CD3– NK cell differentiation from CD34+ bone marrow cells required interleukin-2 (IL-2) and bone marrow stromal cells [2 3 4 ]. However, further studies showed that the stromal requirement for CD56+CD3– cell differentiation can be substituted with a combination of IL-2 and stem cell factor (SCF) [5 6 7 8 ]. Although IL-2 initiates CD56+CD3– cell differentiation from CD34+, in vitro bone marrow stromal cells do not secrete this cytokine. It is, therefore, not clear whether IL-2 is physiologically involved in NK differentiation. IL-15, which shares IL-2 {gamma} and ß chains with IL-2, may be a more physiologically relevant cytokine, as it is produced by bone marrow stroma and can induce CD34+ cells to generate CD56+CD3– cells [8 ]. It is remarkable that most studies of NK development have used IL-2 and IL-15 with CD34+ cells isolated from bone marrow [2 , 3 , 5 , 7 8 9 10 11 12 13 14 ] or cord blood [14 15 16 17 18 19 ]. With the exception of one study [20 ], little is known about the effects of IL-2 on the generation of CD56+CD3– cells from granulocyte-colony stimulating factor (G-CSF)-mobilized CD34+ cells. Considering that the use of G-CSF-mobilized CD34+ cell transplantation has become a common practice [21 ], it is important to investigate their ability to generate NK cells, as they may regulate graft-versus-host and graft-versus-leukemia effects following allogeneic stem cell transplantation. To further study the early stages and regulatory pathways of CD56+CD3– cell development from CD34 cells, we cultured purified, G-CSF-mobilized CD34 cells obtained from healthy stem cell donors in growth conditions favorable to CD56+CD3– cell growth and differentiation. Here, we describe two populations of CD56+ cells emerging after 3 weeks of culture, which can be separated into a CD56bright NK precursor and a novel CD56dim monocytic cell population.


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MATERIALS AND METHODS
 
Antibodies and reagents
Fluorescein isothiocyanate (FITC)-conjugated anti-CD56, anti-Fc receptor for immunoglobulin G (IgG; Fc{gamma}R)III (CD16), anti-Fc{gamma}RII (CD32), anti-CD33, anti-CD3, anti-CD2, anti-CD11a, anti-CD94, anti-CD80, anti-CD44, anti-granzyme A, allophycocyanin (APC)-conjugated anti-CD56, anti-CD11c, anti-CD38, anti-CD69, anti-c-kit (CD117), phycoerythrin (PE)-conjugated anti-CD117, anti-Fc{gamma}RI (CD64), NKB1 (KIR3DL1), anti-CD3, anti-CD16, anti-CD56, anti-perforin, peridinin chlorophyl protein (PerCP)-conjugated anti-CD3, anti-CD69, anti-CD8, and matching isotype mouse monoclonal antibodies (mAb) were purchased from Becton Dickinson (San Jose, CA). PE-conjugated anti-CD34, P58.1 (KIR2DL1), P58.2 (KIR2DL2), NKG2A, and anti-CD14 were purchased from Immunotec (Marseille, France). Magnetic bead-conjugated anti-CD56 and mini-Macs magnet were purchased from Miltenyi Biotec (Auburn, CA). APC-conjugated anti-CD95 and PE-conjugated anti-CD95L were purchased from Caltag (Burlingame, CA). Hyaluronic acid (HA) and FITC albumin were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell isolation, activation, and expansion
Healthy stem cell donors gave permission for a portion of their CD34 cell donation to be used for research under Institutional Review Board-approved National Heart Lung and Blood Institute [NHLBI; National Institutes of Health (NIH), Bethesda, MD] stem cell allotransplantation protocols. CD34+ cells were positively selected from normal donor G-CSF-mobilized peripheral blood (PB) stem cells using an Isolex 300i cell separator. Residual lymphocytes were then removed by negative selection using a cocktail of C2, CD6, and CD7 mAb to produce a stem cell product with less than 0.001% contaminating lymphocytes [22 ]. CD34 cells were counted and frozen in liquid nitrogen until use. Peripheral blood mononuclear cells from healthy individuals were separated by Ficoll-Hypaque density separation. Cells were cultured in RPMI 1640 supplemented with 10% AB or 10% fetal calf serum glutamine (2 mM) gentamicin, hereafter referred to as complete medium (CM). CD34+ cells were cultured in CM in 96-well U-bottom plates (Costar, Corning, NY) for 14–21 days. Cells were stimulated every 5 days with SCF (50 ng/ml; Peprotech, Rocky Hill, NJ) with IL-2 200 U/ml. To obtain pure CD56+ cell populations, CD34+ cells stimulated for 15–21 days with IL-2 were stained with a PE-conjugated anti-CD56 and a FITC-conjugated anti-CD33 mAb, and CD56+ cells were isolated by electronic sorting using an EPICS ALTRA flow cytometer (Beckman Coulter, Miami, FL). In some experiments, immature CD56+ cells were stained with magnetic bead-conjugated anti-CD56 and passed through a magnetic column. A vigorous mechanic pressure eluted CD56+ cells retained in the column. PB NK cells were selected by magnetic sorting using a commercially available NK isolation kit (Miltenyi Biotech), which negatively selects NK cells by removing CD3+, CD4+, CD19+, and CD33+ cells.

Flow cytometric analysis
In some experiments, cells were stained with PE-conjugated anti-CD56 or anti-c-kit (one color). In other experiments, cells were incubated with FITC anti-CD56 and PE anti-c-kit (two colors) or a combination of PE, FITC, PerCP, and APC-conjugated antibodies specific for the desired molecules (four colors). In all cases, the cells were stained at 0°C for 30 min, washed twice, and fixed in 1% paraformaldehyde (PFA). For intracellular staining experiments, 106 cells were first stained with a PE-conjugated anti-CD56 for 15 min at room temperature (RT) in the dark, and then 2 ml fluorescein-activated cell sorter (FACS) lysing solution was added to the cell mixture. After 10 min incubation at RT, cells were washed and permeabilized with 0.5 ml FACS perm mix for 10 min at RT. Cells were than stained with a FITC-conjugated anti-granzyme A mAb or FITC-conjugated mouse control mAb isotypes for 30 min at RT, washed, and fixed in 200 µl 1% PFA. Cells were analyzed by a Becton Dickinson FACSCaliber© flow cytometer.

Microcytotoxicity assay
Effector cells were resuspended at the concentration of 8 x 105/ml in CM. Replicates of 20 µl (1.6x104) effector cells were incubated in a 60-well (40 µl depth) Terasaki plate for 30 min at RT. At the same time, 2 x 106 P815 cells were incubated in 1 ml CM supplemented with 10 µl calcein-AM (Molecular Probes, Junction City, OR) for 30 min at 37°C, washed four times, and diluted to 1 x 105/ml. After diluting the effector cells, 10 µl (1x103) target cells were added, and plates were centrifuged and incubated at 37°C for 4 h. A few minutes before scanning the plates using a fluorescent detector, 5 µl fluoro-quench was added to each well. The percent of lysis was calculated as follows: 1 – (mean test–mean blank)/(mean max–mean blank) x 100.

Proliferation assay
The proliferation of P815 cells was measured by tritiated thymidine (3H-TdR) incorporation. The first three U-wells of each horizontal row of 96-well plates were filled with 200 µl (1.6x104) of negatively selected NK cells or positively selected peripheral blood CD56+ cells, and then 100 µl cultured cells were serially diluted in the remaining wells previously filled with 100 µl CM. Later, 1 x 103 P815 cells were added to the cell cultures. After 2 days incubation, cells were pulsed with 1 µCi 3H-TdR per well (Amersham Biosciences, Piscataway, NJ). Eighteen hours later, 3H-TdR was measured using a ß scintillation counter.

Cytokine arrays
CD56+CD33+ cell supernatants cultured in the presence or absence of lipopolysaccharide (LPS) were assessed for cytokine content with a 42 human cytokine array system RayBioTM (RayBiotech, Norcross, GA), which detects the antibody-cytokine sandwich by chemiluminescence. Briefly, electronically sorted cells were cultured in duplicate with or without LPS in 96-well U-bottom plates, and the cumulative amount of supernatants was harvested and replaced with CM at 24, 48, and 72 h and stored frozen in a single pool until use. Antibody-coated membranes (duplicates) were incubated in 2 ml blocking buffer for 30 min. Cryopreserved supernatants were thawed, diluted 1:1 with blocking buffer, and added to the membranes. After 2 h incubation at RT after agitation, membranes were washed five times and incubated with a cocktail of biotin-conjugated antibodies. After 2 h, membranes were washed five times and incubated for 30 min with horseradish peroxidase-conjugated streptavidin. Membranes were incubated in the detection system for 5 min, wrapped in plastic wrap, placed in a Kodak cassette, and exposed to a X-OMAT for various periods of time.

IL-8 and IL-1ß measurement
Electronically sorted CD56+ or CD56– cells were cultured in CM. Supernatants were harvested at 72 h and stored frozen in a single pool. IL-8 content was measured by a standard immunoassay (Biosource, Camarillo, CA), IL-1ß was purchased from R&D Systems (Minneapolis, MN), and interferon-{alpha} (IFN-{alpha}) was purchased from PBL Biomedical Laboratories (Piscataway, NJ).


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RESULTS
 
Phenotype and morphology of cultured CD34 cells
A total number of 2.5 ± 2.8 x 106 (n=11) G-CSF-mobilized CD34 cells were cultured in the presence of IL-2 200 U/ml and SCF 50 ng/ml. After 2 weeks of culture, a fraction of 3.4 ± 2.4 x 106 nonadherent, hematopoietic cells was analyzed by flow cytometry. The majority of the cells in culture was CD33+ (Table 1 ). In 11 normal stem cell donors studied, there was a minor population of CD56+ cells segregating into CD56bright (7±10 range 0.2–34%) and CD56dim (2.5±2.0 range 0.38–5%). Flow-sorted CD56+ and CD56– cells stained with Giemsa showed that the CD56+ population was a heterogeneous mixture of large granular lymphocytes (LGL) and cells with myeloid or plasmacytoid appearance. The CD56– fraction was also heterogeneous but lacked LGL (Fig. 1A ). The phenotype of the CD56+ cells was examined further for NK cell markers and integrins (Table 1) . Some CD56bright cells expressed CD94 and NKG2A but lacked expression of killer inhibitory receptor (KIR) with Ig-like domain KIR2DL2, and CD56dim was negative for NK markers. Integrin expression further segregated CD56+ cells into CD11a-positive and -negative subsets with reciprocal expression of c-kit. Most of the CD56bright CD11a+ were c-kit- (74±6.7, n=4), and CD56bright CD11a– were c-kit+ (93±4, n=4). In contrast, the CD56dimCD11a+ and CD56dimCD11a– did not have a reciprocal c-kit expression.


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  		Table 1. Phenotype of CD34+ Cells at the Third Week Culture with IL-2 and SCF



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  		Figure 1. Characteristic of myeloid and lymphoid CD56+-cultured cells. (A) Morphology of immature CD56+ and CD56– cells. At the third week stimulation with SCF + IL-2 electronically sorted CD56+ and CD56– cells showing diverse size and mixed morphology of CD56+ cells. (B) Morphology of electronically sorted CD56low CD33+ cells showing large cytoplasm containing basophilic granules; morphology of electronically sorted CD56highCD33– cells showing some characteristic of LGL; flow cytometric segregation of CD56low+CD33+ population from a CD56highCD33– population after 3 weeks stimulation with SCF + IL-2 (representative of five experiments).

CD33 expression
CD56+ cells from 3-week CD34+CD33– cell cultures were sorted and resuspended in SCF and IL-2. In six individuals, CD56+ cells were CD56dimCD33+ (40.5±29) and had macrophage morphology, and the remaining cells CD56+CD33– resembled LGL (Fig. 1B) . Most individuals also had a minor population of CD56dim CD33– cells, and in five of 13 independent analyses of G-CSF-mobilized CD34 products derived from six individuals, we found a minor population of CD56brightCD33+ cells. CD56dimCD33– cells may represent precursors of CD56dimCD33+ cells, and CD56brightCD33+ cells may be CD33 cells that up-regulated CD56 with IL-2. CD56+CD33+ cells represented about one-third of the total CD56+ population.

Granzyme A expression
Intracellular staining of cultured CD56+ cells from three individuals identified granzyme A in 21 ± 7.6% CD56high cells compared with 0.7 ± 0.1% in CD56dimCD33+cells (Fig. 2 ).



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  		Figure 2. Cytotoxic granule content of immature CD56+ cells. After 3 weeks of stimulation with IL-2 and SCF, cells were triple-stained with the indicated antibodies. The expression of CD33 on CD56bright and CD56dim was analyzed using the indicated electronic gates. (Upper panel) CD33 expression on immature PB CD56dim granzyme A– cells compared with immature PB CD56bright granzyme A+/–. Upper quadrant numbers represent the percentage of positive cells in the upper quadrants; histogram numbers are percentages of CD33+ cells in gated, immature PB CD56+ cells. (Lower panel) IL-2-activated PB CD56+ cells used as a positive control. The quadrant number is the percentage of PB CD56+ granzyme A+ cells (representative of three experiments).

Functional assays of CD56+ cells
CD56+ cells were flow-sorted into bright and dim populations and cultured with high doses of LPS. In three consecutive donors, CD56dimCD33+ cells promptly incorporated 3H-TdR and proliferated in culture, and CD56brightCD33– cells did not (Table 2 ). Monocyte-macrophages have antiproliferative properties but lacked perforin-dependent cytotoxicity [23 , 24 ]. CD56dimCD33+ cells targeted against the P815 cell line, as a classical NK cell target inhibited P815 proliferation but was not cytotoxic in a 4-h cytotoxicity assay, suggesting that CD56dimCD33+ cells behaved like monocyte-macrophages (Table 3 ).


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  		Table 2. Functional Features of Electronically Sorted CD56+ Cells


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  		Table 3. Antiproliferative Effects of CD34-Derived CD56dimCD33+ Cells

Cytokine production
CD34-derived NK cells generated in vitro produce minimal levels of IFN-{gamma}, tumor necrosis factor {alpha}, and granulocyte macrophage-CSF but can up-regulate these cytokines upon activation [8 ]. We therefore studied cytokine production in CD56+ cell CM in a cytokine array. Figure 3 shows that without further stimulation, CD56dimCD33+ cells constitutively produced IL-8 [relative cytokine units (RCU) 101±51, and CD56brightCD33– cells minimally secreted IL-8 (RCU 1.1±1.1)]. LPS stimulation of CD56dimCD33+ cells maintained IL-8 production (RCU 147±6) and induced IL-6, MCP-1 MDC, and IL-10. After LPS, CD56brightCD33– cells produced more IL-8 (RCU 74±1) and MCP-1 (RCU 27±12). To further validate the cytokine array data, we measured the IL-8 content of CD56dimCD33+ and CD56brightCD33– cell culture-conditioning medium by an immunoenzymatic assay. The IL-1ß content was used as a negative control. In unstimulated, CD56-derived CD34+ cells, the amount of IL-8 detected in CD56dimCD33+ cell culture CM (8.6±2.2 ng/ml) was tenfold higher than the IL-8 content measured in the CM of CD56brightCD33+ cells (0.9±1.2), and in both cases, no IL-1ß was detected. These results showed that the cytokine production in the two CD56 populations was quantitatively and qualitatively different.



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  		Figure 3. Cytokine production. G-CSF-mobilized CD34 from two donors (Donors 1 and 2) were cultured for 3 weeks with SCF + IL-2, and CD56dimCD33+ and CD56+CD33– were electronically sorted and cultured in the presence or absence of LPS. A cumulative amount of supernatants was harvested within 72 h and tested for cytokine content. 8, 6, and 10, IL-8, IL-6, and IL-10, respectively; MCP1, monocyte chemoattractant protein-1; MDC, macrophage-derived chemokine; GRO, growth-related oncogene; I309, CC chemokine.

CD56+ cell generation is inhibited by IL-4 and HA acid
As IL-4 and HA are produced by marrow stromal cells and regulate hematopoiesis [25 , 26 ], we studied their effect on CD56+ cell development. IL-4 enhanced CD34+ cell proliferation in SCF + IL-2, and HA had an antiproliferative effect. Three-week cultures of CD34 cells in IL-4 and HA inhibited CD56+ cell development. Without HA and IL-4, 32% of CD34+ cells were CD56+, and only 9% and 1.5% were CD56+ in HA and IL-4, respectively. Cultures remained >95% viable by trypan blue (Fig. 4 ).



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  		Figure 4. Inhibition of CD56+ cell development by HA and IL-4. (A) After 3 days culture of CD34+ cells incubated in various cytokine mixtures, cells were labeled with 3H-TdR overnight, harvested, and analyzed in a ß-counter. (B) HA and IL-4 inhibit CD56+ cell generation from CD34+ cells after 3 weeks culture in HA or IL-4. Solid lines, No HA or IL-4; dashed lines, 300 µg/ml or 400 U/ml IL-4; dotted line, IgG1 isotype control (representative of three experiments).


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DISCUSSION
 
Several recent studies, investigating the in vitro growth of CD34 cells in IL-2 in the absence of marrow stroma, have described the induction of CD3– CD56+ functionally competent NK cells as well as a minor population of CD16+ cells [2 , 5 , 9 ]. To further study in vitro generation of NK cells from G-CSF-mobilized CD34+ cells derived from normal donors, we cultured them in combinations of SCF and IL-2. As CD56 (a 120- to 180-KD N-linked, glycosylated isoform of the neural cell adhesion molecule) [27 ] is a useful marker for NK cells and a subset of dendritic cells, we studied the emergence of CD56+ cells in culture. It is surprising that we found that after 2- to 3-week cultures, only 8.7 ± 8.8% of cultured cells expressed CD56, about eightfold less than described previously [2 , 5 , 9 ]. The majority of cells in our cultures was of myeloid morphology and CD33+, Fc{gamma}RI+, or Fc{gamma}RII+. Here, we describe the phenotype and function of cultured CD56+ cells, which include a novel myeloid subset of CD56dim CD33+ cells. They have the appearance of macrophages, express integrins, but lack NK cell markers such as KIR and granzyme A. Functionally, they are unable to kill the NK-resistant line P815 but exert a cytostatic effect on it. Finally, they constitutively produced IL-8 and up-regulated MCP-1 on stimulation with LPS. We also found a CD56bright subset, which appears to be immature NK precursors. They possess granzyme A but not granzyme B and do not express KIR or activation markers. Prolongation of some cultures to 70 days ultimately generated small numbers of NK cells with a more mature phenotype as described by others [18 ] (data not shown).

It is not clear why our results differ from the earlier descriptions of rapid NK cell induction from CD34 cells using the same cytokines or from a recently described CD34 cell-derived NK cell from cord blood [19 ]. One possibility is that a different source of CD34+ cells, including bone marrow, peripheral blood, and umbilical cord blood, may have different frequencies of hematopoietic precursors. Alternatively, the use of G-CSF-mobilized CD34 cells may have resulted in the induction of a precursor not present in bone marrow. It is also possible that the absence of marrow stromal cells in culture may have biased the maturation toward CD56+CD33+ cells, as suggested by the inhibitory effects of HA and IL-4 on CD56+ cell development.

The physiological significance of these cells is not known. However, it is likely that the cultured CD33+CD56+ cells are the in vitro counterparts of low frequency, circulating CD56lowCD33+ monocytes, which we recently identified (manuscript submitted). It is interesting that these cells share features of the rare CD56+CD33+ hybrid NK/myeloid leukemia. This aggressive leukemia is represented by two phenotypes: HLA-DR– CD16– and HLA-DR+ CD34–CD7+ [28 , 29 ]. The cells we identified appear to be distinct from the plasmacytoid DC (DC2), which is CD56+ CD33– CD11b– [30 31 32 ].

In conclusion, we have shown that six G-CSF-mobilized peripheral blood CD34+ cell products, exposed to lymphocyte growth factors and SCF in the absence of marrow stroma, generate a hierarchy of CD56+ cells with NK and myeloid characteristics. These results indicate a hitherto unsuspected diversity of CD34-derived CD56+ cells.


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ACKNOWLEDGEMENTS
 
We thank Charles Carter (Cell Processing Section, Clinical Center, NIH) for providing CD34+ cells. We also thank Philip McCoy (NHLBI, Hematology Branch, NIH), who kindly provided us technical assistance for electronic cell sorting.

Received May 6, 2004; revised June 11, 2004; accepted August 10, 2004.


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