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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-05-1501.
HEMATOPOIESIS
From the Cancer Immunology Immunotherapy
Center, Saint Savas Hospital, Athens; and the First Obstetrics and
Gynecology University Clinic, Alexandras Maternity Hospital, Athens,
Greece.
Natural killer (NK) cell differentiation from pluripotent
CD34+ human hematopoietic stem cells or oligopotent
lymphoid progenitors has already been reported. In the present
study, long-term cultures of the CD56 Natural killer (NK) cells are large granular
lymphocytes with innate immune function, playing a critical role in the
early host defense against viral, bacterial, and other infections, and against cancer. They exert their effector function, without prior sensitization, by killing virally infected cells and tumor cells and by
producing immunoregulatory cytokines and chemokines.1 Mature NK cells express CD56 alone or in combination with CD16. The
majority of adult peripheral blood (PB) NK cells are
CD56+16+, with a minor population of
CD56+16 Although there is considerable information about the phenotype,
function, and activation of mature human NK cells, comparatively less
information exists about their ontogenic development and differentiation/maturation. NK cells are known to develop from common
T/NK bipotent progenitors.9 The most primitive
human NK cell progenitors have been identified in the CD34+
HSC compartment, which gives rise to all hematopoietic lineages. Purified CD34+ cells from human adult bone marrow
(BM),10 umbilical cord blood (UCB), mobilized
PB,11 and fetal liver,12 cultured in the presence of flt 3 ligand (FL) or stem cell factor (SCF) and
interleukin-15 (IL-15), in the presence or absence of stromal
cells, differentiated into CD56+CD3 Intermediate differentiation stages of NK cells have also been
identified. NKR-P1A (CD161)+/CD56 Recent reports (for review see Graf19) suggest that the
model of gradual restriction in the differentiation potential of hematopoietic cells, does not always follow the pathway from
pluripotent (HSCs) to oligopotent (CLPs or CMPs) and finally to
monopotent progenitors. For example, CLPs can differentiate into
myeloid cells,20 early T progenitors can give rise to
myeloid cells,21,22 and B-cell progenitors can generate
macrophages.23 Katsura considers that T-cell and B-cell
progenitors are generated from a common myelolymphoid progenitor
(CMLP), based on his findings from the multilineage
progenitor assay of single cells from a progenitor-enriched population of mouse fetal liver.24
In the present study, we demonstrate that a human UCB-derived
population with phenotypical features of myeloid cells, characterized by the expression of CD14, CD11b, CD13, and CD33 surface antigens, differentiates into NK cells. FL in combination with IL-15 support differentiation of this myeloid-like cell population into
CD56+ functionally mature NK cells, capable of killing
NK- and lymphokine-activate killer (LAK)-sensitive
targets and producing cytokines. This is the first report demonstrating
differentiation of mature NK cells from
CD14+CD11b+CD13+CD33+
cell precursors and, as a consequence, the possible existence of a
human myeloid-lymphoid progenitor.
UCB samples and mononuclear cell purification
Cell lines
Cell cultures Depending on the cell number of the starting population (5 × 104 cells or 5 × 105 cells per culture), cultures were grown in 0.1 or 1.0 mL medium in 96- or 24-well culture plates, respectively. MyeloCult (Stem Cell Technologies, Vancouver, Canada) was used as a culture medium, supplemented with 10 6 M freshly dissolved hydrocortizone (Sigma, St
Louis, MO) and 50 µg/mL gentamicin. FL (R&D Systems,
Abington, United Kingdom) was added at 25 ng/mL and IL-15 (R&D
Systems) at 20 ng/mL. Every 3 to 4 days, half of the medium
was discarded and replenished by fresh medium containing freshly added
cytokines, whereby cell density was adjusted to
0.5 × 106 cells/mL.
Limiting dilution analysis (LDA) For the LDA assay, highly purified UCB/CD14+ cells (> 99.5% purity) were cultured in U-bottomed 96-well plates (96 replicates per cell concentration), either alone at dilutions ranging from 105 to 102 cells/well or at 20, 50, and 100 cells/well on 5 × 104 irradiated (2000 cGy) CD14+ cells from the same UCB as feeders, in the presence of FL and IL-15. UCB/CD34+ cells were tested on only CD14+ feeders. Half of the medium was renewed every 3 to 4 days. After 30 days the wells containing viable cells were tested for CD56 expression by flow cytometry. All the wells containing viable cells were found positive for CD56 expression. For both conditions, the NK progenitor frequency was calculated as the reciprocal of the concentration of cells that resulted in 37% negative wells using Poisson statistics and the weighted mean method.25,26 No proliferation was observed in irradiated CD14+ feeders cultured as a control in parallel with the experimental groups under the same conditions (5 × 104 cells/well; 96 replicates for 30 days). Moreover, the same cultures did not give rise to any CD56+ cells.Monoclonal Abs and immunophenotyping mAb anti-CD34 conjugated with phycoerythrin (PE), anti-CD56, and anti-CD33 conjugated with fluorescein isothiocyanate (FITC) were obtained from Becton Dickinson (Mountain View, CA). Anti-CD8, -CD14, -CD56, and -CD161 mAbs conjugated with PE and anti-CD2, -CD3, -CD7, -CD19, -CD14, -CD16, -CD25, -CD158a, and -CD158b mAbs conjugated with FITC were purchased from PharMingen (San Diego, CA). FITC, PE-, or PE-cytochrome 5 (cy5)-conjugated anti-CD11b, -CD15, -CD56, -CD94, -CD117, -CD122 mAbs were obtained from Immunotech (Marseille, France). Anti-CD13/PE was purchased from DAKO A/S (Glostrup, Denmark). Cells were washed twice with ice-cold phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA), followed by incubation with saturating concentrations of the appropriate mAbs for 15 minutes at room temperature. Thereafter, cells were washed twice in ice-cold PBS/1% BSA and fixed with 1% paraformaldehyde in PBS. Samples were analyzed using FACSCalibur (Becton Dickinson) and CellQuest analysis software (Becton Dickinson, Mountain View, CA).Cluster determinant (CD) detection by reverse transcriptase-polymerase chain reaction (RT-PCR) To detect whether the purified CD14+ cells were contaminated by any NK/NK-progenitor cells, total RNA was extracted from cells isolated from samples of human UCB using the SV Total RNA Isolation System (Promega, Lyon, France), according to the manufacturer's protocol. First-strand cDNA synthesis was performed using approximately 1 µg total RNA, oligodT primers, and the SuperScript II RNase H (-) reverse transcriptase (Invitrogen, Paisley, Scotland). This cDNA material (2 µL) was used for PCR amplification with Taq Platinum (Invitrogen), using the appropriate set of primers and PCR conditions for each cluster determinant (CD56, CD161, CD7, and CD34).To determine the sensitivity of the applied technique for detecting the mRNAs mentioned in the previous paragraph, we prepared dilutions of purified CD56+ or CD34+ cells from UCB that were more than 98% positive for the expression of CD56, CD7, and CD161 or CD34, respectively, in the SP2/0-Ag14 myeloma mouse cell line (ATCC). We then proceeded to total RNA extraction and RT-PCR analysis in these samples, using the same sets of primers and PCR conditions. The sequences of the primers we used were as follows: CD56sense,
5'-GCTGGGACTTCAGGAGACTG; CD56antisense, 5'-CCCCTCAGCCTCTTCTTTCT; CD7sense, 5'-TTTACTACGAGGACGGGGTG; CD7antisense,
5'-AGGTGTAGGTGCCAGTGTCC; CD161sense, 5'-AGAATCCAGCCTGCTGCTTA;
CD161antisense, 5'-GAGCCGTTTATCCACTTCCA; CD34sense,
5'-CTTGGGCATCACTGGCTATT; CD34antisense1, 5'-AATTCGGTATCAGCCACCAC; CD34antisense2, 5'-CGTGTTGTCTTGCTGAATGG; The samples were denatured at 94°C for 5 minutes, followed by amplification rounds consisting of 94°C for 1 minute (denaturing), 60°C for 1 minute (annealing), and 72°C for 2 minutes (extension), for 39 cycles. Specifically for the detection of CD34 mRNA, seminested PCR was applied. In the first round of amplification CD34sense and antisense1 primers were used, and, subsequently, 2 µL of the products were used as template for the second round using the sense and antisense2 primers in the same conditions. All the amplified products were subjected to 2% agarose gel electrophoresis containing GelStar dye (FMC BioProducts, Rockland, ME) and visualized by ultraviolet (UV) light. Cytotoxicity assay Cytotoxic activity of cultured cells was determined in a standard 4-hour chromium 51 (51Cr)-release assay against the NK-sensitive cell line K562 and the NK-resistant cell line Daudi, as previously described.27 In brief, target cells were labeled with 100 µCi (3.7 MBq) sodium [51Cr] chromate (Radiochemical Centre, Amersham, Cardiff, United Kingdom) per 106 target cells for one hour. Effector cells were incubated with target cells at the indicated ratios. Spontaneous 51Cr release was measured by incubating target cells in the absence of effector cells. Maximum 51Cr release was determined by adding 1% Triton X-100 (Sigma). Spontaneous lysis did not exceed 10% of the maximum release. The amount of 51Cr released was measured in a -counter
(Packard, Downers Grove, IL), and the percent lysis was calculated as
follows: % specific lysis = [(experimental 51Cr
release  spontaneous 51Cr release)/(maximum
51Cr release spontaneous 51Cr
release)] ×100.
Quantitation of cytokines in culture supernatants For cytokine production determinations, cells recovered from cultures with FL and IL-15 were washed twice with HBSS and incubated for 72 hours in fresh medium containing FL (25 ng/mL), IL-15 (20 ng/mL), IL-12 (2 ng/mL; R&D Systems), and IL-18 (100 ng/mL; R&D Systems). Supernatants were collected by centrifugation and stored at70°C until use. Cytokines (interferon- [IFN-],
granulocyte-macrophage colony-stimulating factor [GM-CSF], tumor
necrosis factor  [TNF], and IL-10) were quantitated
using commercially available enzyme-linked immunosorbent assay
(ELISA) kits (Diaclone Research, Besançon, France)
according to the manufacturer's instructions.
CD56+ cell differentiation from UCB-adherent
CD34 adherent progenitor cells under the influence of the
appropriate cytokine microenvironment. Based on this, we initially
asked whether such adherent CD34 progenitor cells could
give rise to mature NK cells when cultured in the presence of FL and
IL-15, which are known to promote NK differentiation from
CD34+ HSCs.10 If so, this would enable us to
make assumptions toward the identification of a new NK progenitor and
thus to better understand NK cell ontogeny.
At the initiation of our experiments, we studied the cell populations included in our total UCB preparations by forward scatter (FSC) and side scatter (SSC) analysis. In this way, we could distinguish 3 cell populations: a typical low SSC lymphoid population and 2 others scattered above this one, commonly characterized as myeloid (monocytic and polymorphonuclear) in adult PB (data not shown). We next attempted to study more closely the high SSC UCB populations by depleting lymphoid cells via a 2-step procedure: first, we sequentially eliminated CD34+, CD56+, CD3+, and CD19+ cells using immunomagnetic procedures, and then we allowed the recovered cells to adhere in order to remove residual lymphocytes included in the nonadherent cell fraction. The resulting ACF was devoid of cells expressing any of the lymphoid markers mentioned in this paragraph with a purity more than 99.5%, and when subjected to SSC/FSC analysis it showed a myeloid profile (data not shown). Phenotypic analysis of the ACF population revealed that 77.1 ± 11.4% of the cells expressed CD14; 76.4 ± 8.9%, CD11b; 70.6 ± 6.3%, CD13; 68.8 ± 4.2%, CD33; 100%, CD11c; and 87.8 ± 9.9%, CD4low (mean values ± SDs from 10 UCB samples). The ACF was overpopulated in cells coexpressing all of the cell markers mentioned in this paragraph (66.7 ± 4.6%, mean values ± SDs from the same UCB samples). The remaining cells were positive for at least one of the above-mentioned myeloid cell markers, although they did not coexpress all of them. During culture in the presence of FL and IL-15, there was a steady
increase in both the percentage and the absolute number of
CD56+ cells within the ACF (ACF/CD56+) (Figure
1; Table 1). The absolute number of
ACF/CD56+ cells increased
significantly by day 10 of culture, and by day 30, 100% of
the ACF expressed the CD56 marker. It is important to note that already
15 days after culture initiation 81 ± 7% of the ACF cell fraction
was CD56+ (Table 1), at which time point only 38 ± 5%
of UCB-derived CD34+ cells had developed into
CD56+ cells (Table 1).
Cytotoxic potential of ACF/CD56+ cells CD56+ cells, differentiated from UCB/ACF after culture with FL and IL-15, were tested as effectors against NK- and LAK-sensitive targets, in parallel with CD56+ effectors differentiated from UCB-derived CD34+ cells and cultured under the same conditions. As shown in Figure 2, ACF/CD56+ cells, recovered from 15-day and 30-day cultures, exhibited significantly higher cytotoxicity against K562 (NK) targets than did autologous CD34-derived CD56+ effectors (60% and 76.3% compared with 10.9% and 46.3% at effector-target [E/T] ratio 2.5:1). An even more pronounced effect was observed when the same cells were tested for LAK cytotoxicity. As also shown in Figure 2, only marginal levels of killing against Daudi targets were observed when CD34-derived CD56+ cells were used as cytotoxic effectors. Even after 2 months in culture, CD34-derived CD56+ cells did not acquire significant LAK activity (data not shown). In marked contrast, ACF/CD56+ effectors efficiently lysed the Daudi targets (up to 80% cytotoxicity at E/T ratio 2.5:1; Figure 2). The levels of LAK cytotoxicity observed with ACF/CD56+ cells were comparable with those achieved with highly purified CD56+ effectors isolated from UCB and cultured under the same conditions (Figure 2).
Cytokine production by ACF/CD56+ cells We then examined cytokine production by the IL-15 plus FL-driven ACF/CD56+ cells, upon stimulation with IL-12 and IL-18. As shown in Figure 3, ACF/CD56+ cells were able to produce equally high levels of IFN-, GM-CSF, TNF, and IL-10 comparable with those achieved with
autologous UCB-isolated CD56+ cells. As with cytotoxic
responses, cytokine production by CD56+ cells
differentiated from UCB-isolated CD34+ cells was at
significantly lower levels compared with ACF/CD56+
cells.
Kinetics of CD14 expression in UCB-derived cell populations The UCB/ACF was cultured, as in Table 2, with FL and IL-15 to generate CD56+ NK cells. The CD56+ NK cells generated in vitro under these conditions (by day 15 almost 90% of the cells were CD56+; Table 2) expressed little (5% by day 15) or no (day 30) CD14 (Figure 4). As also mentioned in "Results," a fairly high percentage, up to 80% of freshly isolated UCB/ACF, expressed CD14 (day 0), which persisted at these levels until day 5 and thereafter declined rapidly (Figure 4A). Similar phenotypic profiles were obtained from UCB/CD14+ cells cultured under the same conditions (Figure 4A).
In contrast to ACF, UCB/CD34+ cells expressed no CD14 at
culture initiation. More than half (65%) of the cells were found to express CD14 by day 15 and thereafter this percentage progressively declined (Figure 4A). As also shown in Figure 4, UCB/CD56+
cells remained CD14 By gating on CD56+ cells, we could demonstrate that the vast majority of CD14+ cells were included in the CD56+ population that was generated from UCB/CD34+ cells. The CD14 marker was maintained on these cells until day 20 and thereafter declined sharply (Figure 4B,D). Approximately 60% of the CD56+ cells derived from the ACF or CD14+ cells also expressed CD14 by day 5 (Figure 4B-C). This percentage, in both cases, was strongly reduced within the next 5 days of culture (down to 5%). The similarities in the kinetics profile of CD14 expression on (1) the total ACF or the UCB/CD14+ cells (Figure 4A) and (2) the CD56+ cells differentiated from the total ACF or the UCB/CD14+ cells (Figure 4B) strongly suggested that CD14+ cells, representing the most dominant population among the UCB/ACF, are actually the cells that differentiate into CD56+ cells when cultured with FL and IL-15. To test this directly, we assessed the kinetics of FL- and IL-15-driven generation of CD56+ cells in parallel cultures with total ACF or highly purified CD14+ cells from UCB. There was an absolute overlap in the kinetics of appearance of CD56+ cells throughout the entire culture period (data not shown). To ensure that CD56+ cells generated from either total ACF
or UCB/CD14+ cells do not represent functionally distinct
populations reflecting different stages of maturity, we also assessed
kinetics of NK cytotoxicity and IFN- To exclude the possibility that UCB/CD14+ cell
cultures contain any NK/NK-progenitor contaminants that could give rise
to mature CD56+ cells, we tested UCB/CD14+ cell
preparations by RT-PCR for the presence of CD56-, CD161-, CD34-, or
CD7-specific transcripts, which characterize such cells. The
sensitivities of the applied technique were more than 10
Frequency of NK progenitors within the UCB/CD14+ cell fraction LDA of highly purified UCB/CD14+ cells, cultured alone for 30 days in the presence of FL and IL-15 at cell numbers ranging from 105 to 102, revealed an NK precursor frequency of 1 in 11 426 ± 992 cells (0.009%, n = 3). However, when highly purified UCB/CD14+ cells were cultured on feeders of CD14+ cells isolated from the same UCB sample, at cell numbers 100, 50, and 20 per culture, NK precursor frequency was significantly increased to 1 in 50.5 ± 1.8 cells (1.98%, n = 3). UCB/CD34+ cells cultured on CD14+ feeders, as UCB/CD14+ cells, revealed an NK precursor frequency of 1 in 35 ± 6 cells (2.86%, n = 3).Phenotypic characterization of CD56+ cells generated from UCB/ACF or UCB/CD14+ cells UCB/ACF or UCB/CD14+ cells were cultured in the presence of FL and IL-15 to generate CD56+ cells for phenotypic analysis (Table 2). CD56+ cells generated from UCB/ACF or UCB/CD14+ cells expressed CD2, CD7, CD8, CD16, and CD25 at moderate to high levels. The vast majority also expressed the C-type lectin-like receptor CD94/NKG2. The KIRs CD158 and/or
CD158b and the c-kit receptor (CD117) were expressed at low levels.
Early in culture (5-15 days) UCB/ACF stimulated with FL and IL-15
expressed CD56 at low density, whereas at later stages (> 15-day
cultures) these cells became CD56bright (Figure
6). Similar results were obtained also
with UCB/CD14+ cells (Figure 6). Finally, CD161 expression
was evident at culture initiation but thereafter was gradually lost
(Table 2). The phenotypic profile of ACF/CD56+ and of
CD56+ cells differentiated from UCB/CD14+ cells
was similar to that of UCB/CD56+ cells cultured under the
same conditions (Table 1). CD56+ cells differentiated from
UCB/CD34+ cells exhibited a more immature phenotype (Table
2). CD34-derived CD56+ cells expressed significantly lower
levels of CD94 and KIRs (CD158a and CD158b), as well as of CD2,
CD7, and CD8.
In the present study we identify for the first time, in human UCB,
a nonlymphoid adherent CD34 The studies presented here show that the CD34 So far, the most primitive human hematopoietic progenitors shown to
differentiate into NK cells are CD34+Lin Another interesting finding in this study is that mature NK cells
differentiated from UCB/CD14+ adherent cells behave
functionally differently from those differentiated from
UCB/CD34+ nonadherent cells, although the latter
transiently expressed CD14. UCB/CD14+-derived NK cells
produced high levels of cytokines (ie, IFN- Taken together, our data presented herein identify a new myeloid-like CD14+ NK cell progenitor fraction of UCB. When cultured in the presence of FL and IL-15, this progenitor, without acquiring the CD34+ marker, follows a differentiation pathway that leads to the generation of NK cells with enhanced maturational state compared with those derived from similarly activated UCB/CD34+ cells. This novel lineage for NK cell maturation, observed in vitro, points to the fact that NK cell development, and consequently lymphoid cell development, is likely to be substantially more complex in vivo.
Submitted May 22, 2002; accepted December 17, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-05-1501.
Supported by a grant from the Regional Operational Program Attika no. 20, MIS code 59605GR (M.P.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sonia A. Perez, Cancer Immunology Immunotherapy Center, Saint Savas Hospital, 171 Alexandras Ave, Athens 115 22, Greece; e-mail: cacenter{at}otenet.gr.
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Top
Abstract
Introduction
-ACTINsense,
5'-TGCTATCCCTGTACGCCTCT; 
), UCB/CD34+ (
), and UCB/CD56+
(
) after culture with FL and IL-15. Values represent the means ± SDs from 8 UCB samples.
), UCB/CD34+ (
) cells with FL and IL-15. Bars represent the
mean values ± SDs from 5 experiments.
), UCB/CD14+
(
