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Blood, 15 February 2002, Vol. 99, No. 4, pp. 1273-1281
IMMUNOBIOLOGY
Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural
killer cell differentiation from progenitor cells
Matthew J. Loza,
Loris Zamai,
Livio Azzoni,
Emanuela Rosati, and
Bice Perussia
From The Kimmel Cancer Center, Jefferson Medical
College, Philadelphia, PA.
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Abstract |
To determine whether production of type 1 and type 2 cytokines
defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro
in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical
cord blood hematopoietic progenitors. Like peripheral NK cells, the
CD161+/CD56+ NK cells from these cultures
contained a tumor necrosis factor alpha
(TNF- )+/granulocyte macrophage-colony-stimulating
factor (GM-CSF)+ subset, an interferon gamma
(IFN- )+ subset, mostly included within the former, and
very few IFN- /IL-13+ cells. Instead, most
immature CD161+/CD56 NK cells, detectable
only in the cultures with IL-2, produced IL-13, TNF-, and GM-CSF,
but not IFN-, and contained an IL-5+ subset. In
short-term cultures with IL-12 and feeder cells, a proportion of the
immature cells acquired the ability to produce IFN-. Part of these
produced both IFN- and IL-13, irrespective of induced CD56
expression. These in vitro data indicate that ability to produce the
type 2 cytokines IL-13 and IL-5 defines CD161+ NK cells at
intermediate stages of differentiation, and is lost upon terminal
functional differentiation, concomitant with acquired ability to
produce IFN-.
(Blood. 2002;99:1273-1281)
© 2002 by The American Society of Hematology.
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Introduction |
Natural killer (NK) cells mediate the early,
nonadaptive, responses against virus-, intracellular bacteria-, and
parasite-infected cells,1 and modulate the activity of
other effector cells of the adaptive and innate systems of defense. In
the mouse, interleukin-12 (IL-12)-induced interferon gamma (IFN-)
production by mature NK cells2,3 directs the development of
Ag-specific cell-mediated responses to intracellular pathogens,
controlling Th1 cell differentiation4 (reviewed in
Trinchieri and Scott5 and Coffman et al6). NK
cells participate in the regulation of myeloid
hematopoiesis,7 and activation of myeloid8 and
monocytic cells (reviewed in Trinchieri et al9) via
production of granulocyte macrophage-colony-stimulating factor
(GM-CSF), IL-3, IFN- and tumor necrosis factor alpha
(TNF-).10-14 They have also been proposed to
participate in the regulation of humoral immune
responses,15,16 and to play a significant role in the
asthma-associated eosinophilia17,18 via IL-5 production, and possibly other factors. A minor subset or subsets of
IL-1319 and of IL-5 producing NK cells exists in adult
peripheral and umbilical cord blood.20,21 Whether
combinations of different cytokines are produced by NK cells at
distinct stages of differentiation or by distinct mature NK cell
subsets remains to be established.
NK cell differentiation is controlled by cytokines produced in an
intact bone marrow microenvironment22,23 (reviewed in Sivakumar et al24). In the murine system, these include
Flt-3 ligand1 (Flt3-L),25 c-kit
ligand (stem cell factor, SCF),25,26 and
IL-1525,27 that act, alone or together, on NK cells at different stages of differentiation.25 Flt3-L and IL-15
sustain differentiation of human CD34+ bone marrow cells to
cells functionally and phenotypically similar to mature peripheral
blood NK cells.28-30 Produced by stromal and monocytic/myeloid cells,31 they likely also act in vivo in
physiologic conditions (reviewed in Carson and Calgiuri32).
Possible differential effects of these cytokines on NK cell
differentiation have been analyzed only at very early developmental
stages,30,33,34 when NK lineage-specific markers are not
yet identifiable.
In rodents (mouse35,36 and rat37) and
humans38-42 IL-2 efficiently substitutes for IL-15 in vitro
to support NK cell differentiation from CD34+ or lineage
negative (Lin) hematopoietic progenitor cells. We have
reported an in vitro model of human NK cell differentiation involving
coculture of umbilical cord blood Lin hematopoietic
progenitor cells with IL-2 and a murine stromal cell line expressing
the membrane-bound form of SCF (mSCF).21 Using this
system, we established that expression of CD161 in the absence of other
mature NK cell markers defines NK cells at a relatively immature stage
of differentiation. CD161+/CD56 NK cells
mediate TRAIL-L-mediated, but not FasL-mediated or granule exocytosis-mediated cytotoxicity,43 and do not express
IFN- upon stimulation.21 However, in culture conditions
including IL-12 and feeder cells, a proportion of these cells is
induced to differentiate to mature cells expressing CD56 and,
constitutively, IFN- mRNA.21 It is currently not known
whether NK cells at this stage of differentiation produce IFN- or
other cytokines produced by mature NK cell subsets or if they can be
induced to produce them when cultured with the cytokines we have
previously shown able to induce expression of IFN- mRNA.
Here, we have analyzed cytokine production in human NK cells at
distinct stages of differentiation. Our data demonstrate that most
CD161+/CD56 immature NK cells produce IL-13,
TNF-, and GM-CSF and contain a minor fraction, undetectable in the
mature CD161+/CD56+ cells, that produces IL-5.
As previously suggested based on mRNA expression, the ability to
produce IFN- is acquired late, approximately at the same time as
acquisition of CD56 expression. Interestingly, late differentiation is
concurrent with decreased ability to produce IL-13 and/or IL-5 only,
and with the appearance of IFN-/IL-13 (or IL-5)
double-positive, CD56/dim cells. The data support
the conclusion that production of type 2 (IL-13 and IL-5) cytokines is
transient and defines an intermediate stage of NK cell differentiation
in this in vitro system. This is unlike production of TNF- and
GM-CSF, which are produced throughout differentiation.
| |
Materials and methods |
Monoclonal and polyclonal antibodies
Monoclonal antobodies (mAbs) to CD2 (B67.1, B67.6), CD4 (B66.6),
CD5 (B36.1), CD8 (B116.1), CD11b (B43.4), CD14 (B52.1), CD15 (B40.9),44 CD16 (3G8),45 CD56
(B159.5),44 CD161/NKR-P1A (B199.2),21 TNF-
(B154.1, B154.9, nonneutralizing; B154.2, B154.7,
neutralizing),10 and IFN- (B133.1, B133.3,
neutralizing)46 were previously characterized in our
laboratory. mAbs to CD3 (OKT3), CD21 (THB5), CD32 (IV.3), CD34 (My10),
CD64 (32.2), and the irrelevant P3 × 63-Ag8.653 Ig were produced
from cells obtained from the American Type Culture Collection (ATCC,
Rockville, MD). mAb to CD94/NKG2 (HP-3B1)47,48 was kindly
provided by Dr M. Lopez-Botet (Pompeu Fabra University, Barcelona,
Spain). When indicated, mAbs were labeled with biotin or fluorescein
isothiocyanate (FITC) according to standard procedures, after
purification on protein G-Sepharose (Pharmacia Fine Chemicals, Uppsala,
Sweden). Phycoerythrin (PE)-anti-CD56 (N901) and electron coupled dye
(ECD)-anti-CD3 (UCHT1) were from Coulter (Beckman Immunotec,
Marseille, France). FITC-anti-IFN- (B27),
FITC-anti-GM-CSF (BVD2-21C11), FITC-anti-CD3 (S4.1), FITC-control
mouse IgG1, and PE-anti-TNF- (MP9-20A4) were from Caltag
Laboratories (Burlingame, CA); PE-anti-IL-5 (TRFK5 or JES1-39D10),
PE-anti-IL-13 (JES10-5A2) (the only available reagents to these
cytokines), PE-rat IgG2a (R35-95) and PE-rat IgG1 (R3-34) were from
Pharmingen (San Diego, CA). FITC-goat F(ab')2-anti-mouse F(ab')2 (GaMIg) (Cappel Laboratories, Durham NC) was used
for indirect immunofluorescence, and the GaMIgs (produced in
our laboratory) used for panning were adsorbed on human Ig-purified and
affinity-purified mouse Ig-Sepharose before use. The rabbit IgG
anti-sheep erythrocytes (E) used to prepare immune complex
monolayers was from Organon-Teknika (Cappel).
Cell isolation
Lymphocytes were isolated from umbilical cord blood samples
(provided by Dr R. Wapner, Department of Obstetrics and Gynecology, Thomas Jefferson University Hospital, Philadelphia, PA) collected at
delivery from full-term pregnancies, and anticoagulated with heparin.
Lin cells were purified according to our previously
published protocol21 after sequential depletion of (1)
aminoethylisothiouronium bromide (AET)-E rosetting cells (AET, Sigma
Chemical); (2) FcR+ cells on rabbit IgG immune complex
monolayers; and (3) most other mature leukocytes following panning (30 minutes, 4°C) on GaMIg-treated dishes after sensitization of the
cells with the panel of mAbs to differentiation antigens on mature
hematopoietic cells listed above. The Lin cells were more
than 99%
CD3/CD161/CD16/CD56
in immunofluorescence analysis; they were not cytotoxic, and did not
express CD16 mRNA, as determined using reverse transcription polymerase
chain reaction (RT-PCR)21 (and data not shown). When indicated, CD34+ cells were positively selected from cells
at step (3) after sensitization with anti-CD34 (My10) mAb and panning
as above.
Homogeneous immature CD161+/CD56 NK cell
populations used in secondary cultures (see below) were purified from
30-day cultures of Lin cells with IL-2 depleting (panning
or fluorescence-activated cell sorting)
CD3+/CD5+ T cells (if needed),
CD32+/CD64+ myeloid cells, and
CD94+/CD56+ mature NK cells after sensitizing
the cells with a panel of mAbs to the indicated surface antigens. These
cell populations were more than 99%
CD3/CD161+/CD56 in direct
immunofluorescence. When indicated, total cell populations from primary
cultures, and homogeneous NK cell populations from 10-day cultures of
umbilical cord blood lymphocytes with 50 Gy irradiated RPMI-8866 cells
as described,44 were used as a source of NK cells. The
latter are referred to as 10-day NK cells.
Progenitor cell cultures
For primary cultures, Lin or CD34+
cells, as indicated, were incubated (37°C, humidified 8%
CO2 atmosphere) in 24-well tissue culture plates
(2 × 105 cells per well/mL RPMI-1640 medium
[Biowhittaker, Walkersville, MD] supplemented with 10%
heat-inactivated fetal bovine serum [FBS; Sigma Chemical]). When
indicated, the murine bone marrow stromal cell line
Sl/Sl4hSCF,220 expressing human
mSCF49 (provided by Dr D. Williams, University of Indiana
School of Medicine, Indianapolis, IN) was used as feeder, after
irradiation (30 Gy). Alternatively, Flt-3/Flk-2 ligand (5 ng/mL; specific activity 3 × 106 U/mg; R&D Systems,
Minneapolis, MN) was used in a feeder cell-free system. rIL-2 (50 U/mL;
Hoffman-LaRoche, Nutley, NJ, obtained through the Biological Response
Modifiers Program, National Cancer Institute, Bethesda, MD) and rIL-15
(10 ng/mL; specific activity 2.95 × 108 U/mg protein;
provided by Immunex, Seattle, WA) and/or rIL-12 (2 ng/mL; specific
activity 4.5 × 106 U/mg protein in an IFN- induction
assay; provided by Dr S. Wolf, Genetics Institute, Andover, MA) were
added at the beginning of the cultures and every 3 to 4 days during a
20- to 30-day culture period. The culture medium was partially replaced
once a week, and nonadherent cells were subcultured when confluent.
For secondary cultures, 106 unseparated or
CD3/CD161+/CD56 NK cells from
30-day primary cultures of Lin or CD34+ cells
with IL-2 were cultured for 8 to 10 days in 24-well culture plates (2 mL medium/well). rIL-2, rIL-15, rIL-12 (concentrations indicated
above), or rIL-4 (10 ng/mL, specific activity  107
U/mg protein in a proliferation assay with CTLLhuIL-4R1.d cells; Genzyme, Cambridge, MA) and their combinations were added at the beginning of the culture and every 3 to 4 days. When indicated, 50 Gy-irradiated Daudi cells (5:1 lymphocyte-to-feeder cell ratio), anti-TNF- and/or anti-IFN- mAb (ascites, 1:500 final dilution) were added throughout primary or secondary culture.
Intracellular cytokine detection
Cells were incubated (5 × 106/mL, 6 hours,
37°C) in medium with or without phorbol myristate acetate (PMA)
(109 M) and Ca++ ionophore (A23187, 0.1 µg/mL) (all reagents from Sigma Chemical). Brefeldin A (10 µg/mL)
was added during the last 3 hours. A Fix/Perm cell permeabilization kit
(Caltag Laboratories, Burlingame, CA), or formaldehyde (3.7% in
phosphate buffered saline [PBS], 10 minutes, room temperature) and
18-hour incubation in PBS containing 0.5% saponin, 0.2% FBS, 0.005%
Tween 20, 0.01% NaN3, were used to fix and permeabilize
the cells for intracellular cytokine detection combined with surface
phenotyping as described in detail.50 Single-color and
multiple-color (up to 4) immunofluorescence analyses (flow cytometry)
were performed with the indicated FITC-, PE-, ECD-, or biotin-labeled
mAbs, as described.21 FITC-labeled (Vector Laboratories,
Burlingame, CA), PE-labeled (Becton Dickinson), R670-labeled (Gibco
BRL, Gaithersburg, MD), or CyChrome-labeled (CyC, Pharmingen)
streptavidin was used to detect biotin-labeled mAbs. Samples were
analyzed on an EPICS Elite, a Profile II, or an XL-MCL automated flow
cytofluorimeter (Beckman Coulter, Miami, FL). Listmode data were
analyzed with WinMDI Flow Cytometry Application (J. Trotter, the
Scripps Research Institute, La Jolla, CA,
http://www.facs.scripps.edu/). When 4-color analysis was performed, the
percentage of CD56 cells within the total
CD161+ NK cells (including
CD161+/CD56 and
CD161+/CD56+ cells) was calculated, within the
gated CD3 cells, as follows: (% CD161+
cells  % CD56+ cells)/CD161+ cells. The
proportion of cytokine-positive cells within these was calculated,
taking into account the fraction of cytokine-positive CD56+
cells within the total cytokine-positive CD161+ NK cells,
using the following formula: X = [A (B × C)]/(100 C) × 100, where X indicates
cytokine-positive cells within
CD3/CD161+/CD56 cells; A and B
indicate percentage of cytokine-positive cells within gated
CD3/CD161+ and
CD3/CD56+ cells, respectively, and C
indicates percentage of CD56+ cells within gated
CD3/CD161+ cells.
RT-PCR analyses
These were performed as previously described,21
using total cellular RNA from cells (5 × 105/sample)
incubated (5 × 106/mL, 2 hours, 37°C) in medium with
or without combinations of IL-2, IL-12, and IL-15 (50 U/mL, 2 ng/mL,
and 10 ng/mL, respectively) for stimulation. The conditions and primers
used to detect  actin, IFN-, and TNF- mRNA (Clontech
Laboratories, Palo Alto, CA) have been reported.21 The
IL-5 and GM-CSF primer sequences used were those defined in a previous
report.51
Statistical analysis
Data were analyzed using the 2-tailed, paired Student
t test (Minitab statistical analysis software, State College, PA).
Values of P < .05 were considered significant.
| |
Results |
Kinetics of CD56+ NK cell generation from
Lin cell cultures
The number of CD3/CD56+ NK cells was
recorded at different time points during primary cultures of umbilical
cord blood Lin cells (including CD34+ and
CD34 cells) with feeder cells and IL-2 or IL-15, alone or
combined (Figure 1). Starting on the
third week of culture and up to 34 days, at the last time point
analyzed, the numbers of CD56+ NK cells generated from
cultures with IL-2 and IL-15, IL-2, or IL-15 alone at the doses used
were highest, intermediate, and lowest, respectively. The kinetics of
generation of NK cells in cultures of CD34+ cells with
Flt3-L and IL-2 were similar to those reported in Figure 1 (not
shown).
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| Figure 1.
CD56 expression to determine the kinetics of generation
of NK cells from Lin cells in cultures with IL-2 and
IL-15.
Lin cells (2 × 105/well) were cultured
with Sl/Sl4hSCF220 cells and rIL-2 (50 U/mL),
rIL-15 (10 ng/mL), or their combination, as described in "Materials
and methods." At the indicated times, the number of NK cells was
calculated as the product of the number of viable cells recovered by
the percentage of CD3/CD56+ cells in 2-color
immunofluorescence. Points are mean values from 3 to 20 experiments
performed for each time and condition. In 26- to 34-day culture: IL-2
and IL-15 versus IL-2 or IL-15 alone, P < .05.
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In agreement with our previous data52
CD3/CD161+/CD56+ cells were not
detected in cultures with IL-12 only, and NK cell generation in
cultures with IL-2 or IL-15 was inhibited by IL-12 regardless of the
presence of IFN- and/or TNF- neutralizing mAbs (not shown).
CD56 and CD161 expression in NK cells generated from progenitor
cell cultures with IL-2 and IL-15
Most CD3/CD161+ cells in primary
cultures of Lin cells with feeder cells and IL-15 were
CD56+ (Figure 2A). Like the
corresponding population in cultures with IL-2 and feeder
cells,21 or Flt3-L and IL-2, variable proportions of these
cells (lower than those in mature NK cells from the corresponding cord
blood samples) expressed all other mature NK cell markers (CD2, CD8,
CD16, CD94, and killer Ig-like receptors, not shown). All
CD3/CD56+ cells (herein referred to as
CD56+ NK cells) from cultures with IL-2 and Flt3-L, like
those from cultures with IL-2 and feeder cells (Figure 2B and Bennett
et al21), were included in the CD161+
population (Figure 2C), as confirmed independently in multiple-color and single-color immunofluorescence with anti-CD56 mAb alone or combined with anti-CD161 mAb. However, unlike the cultures with IL-15
and feeder cells, both cultures with IL-2 contained a significant proportion (up to 50%) of
CD3/CD161+/CD56 cells (herein
referred to as CD56 NK cells). Most of these immature NK
cells21 expressed CD7 (not shown) and CD161 at an average
density higher than that on their CD56+ counterpart
(Figure 2B-C).
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| Figure 2.
CD161 and CD56 expression on CD3 NK cells
from cultures of progenitor cells with feeder cells and IL-2 or IL-15,
or Flt3-L and IL-2, to determine the conditions that support
accumulation of immature CD56 NK cells.
(A, B) 2-color and (C) 3-color immunofluorescence was performed, with
the indicated FITC- or PE-, and biotin-labeled mAb detected with
streptavidin-PE or CyC, on cells generated from Lin (A,
B) or CD34+ cells (C) after 30-day culture with IL-15 (A)
or IL-2 (B) and the Sl/Sl4hSCF220 feeder cells,
or with Flt3-L and IL-2 (C), as described in "Materials and
methods." Correlate measurements of red and green fluorescence (x and
y axis, respectively, log10 scale) are displayed as
2-dimensional contour plots. In C, analysis was performed on gated
CD3 cells (CD3-FITC). The contours were divided into
quadrants in which less than 0.5% control cells (treated with
irrelevant isotype-matched mAbs) were included: top left, cells with
green fluorescence (binding FITC-labeled Ab only); top right, double
positive cells; bottom right, cells with red fluorescence (binding
PE-labeled Ab only); bottom left, double-negative cells. Histograms in
(C) are from samples treated with biotin-labeled anti-CD56 +/
anti-CD161 mAb detected with streptavidin-CyC on the same
CD3 cells (dotted line, negative control; solid line,
mAb+ cells; x axis, fluorescence intensity, y axis,
relative cell number). The experiment in A and B is representative of
10, and that in C is representative of 3 performed with similar
results.
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Cytokine production by NK cells derived from progenitor
cells
Cytokine expression and surface phenotype were analyzed
simultaneously at the single-cell level (multiple-color
immunofluorescence, flow cytometry) (Figure
3) in cells from primary cultures of
Lin cells with feeder cells and IL-2, and from parallel
cultures of cord blood lymphocytes with B-lymphoblastoid cell lines
(10-day NK cells). Intracellular cytokines were not detectable in
control, nonstimulated cells (not shown). Coexpression of IFN-,
TNF-, and GM-CSF was detected in approximately 75% of the umbilical cord blood CD3/CD56+ mature NK cells from
10-day cultures (Figure 3, top) within 6 hours of stimulation.
IL-5, when present, was detected in a minor (<1.0%) NK cell subset
that did not produce IFN- but contained cells expressing GM-CSF.
About 35% of the CD3/CD161+ (including
both CD56+ and CD56) NK cells from primary
cultures of Lin cells with IL-2 and feeder cells
expressed intracellular cytokines upon stimulation (Figure 3, bottom).
Most if not all IFN-- and GM-CSF-expressing cells were included
within those expressing TNF-. A minor (<5%) cell subset expressed
IL-5; this was significantly greater than that detectable in mature
lymphocytes, was distinct from that producing IFN-, and overlapped
only minimally with that producing GM-CSF.
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| Figure 3.
Intracellular cytokine accumulation in
CD161+ NK cells from cultures of Lin cells
with IL-2 to determine the proportion of cells capable of cytokine
production, and cytokine production by distinct subsets.
Umbilical cord blood 10-day NK cells (top), and
CD3/CD161+ cells from 30-day primary cultures
of Lin cells with IL-2 and
Sl/Sl4hSCF220 feeder cells (bottom) were
stimulated (6 hours, 37°C) with PMA and Ca++ ionophore
(see "Materials and methods"). Surface phenotype and expression of
the indicated cytokines were detected simultaneously (3-color
immunofluorescence) on gated CD161+ cells as described in
"Materials and methods," and analyzed as in Figure 2. Percent
positive cells is indicated in each quadrant. Experiment representative
of at least 4 performed with similar results with each Ab
combination.
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Cytokine production by CD56 and CD56+
NK cells
RT-PCR analysis on cells from cultures with IL-2 and feeder cells
revealed inducible expression of IFN- mRNA only in the CD56+ cells, and constitutive expression of GM-CSF,
TNF-, and IL-5 in both mature CD56+ and immature
CD56 cells21 (and data not shown). Analysis
of intracellular cytokine expression upon stimulation indicated that
about 30% of the NK cells from primary cultures with IL-2 and feeder
cells (both CD56+ and CD56) expressed TNF-
and GM-CSF (Figure 4). The levels of
GM-CSF on a per cell basis were lower in the CD56+ cells
than in the CD56 cells, as indicated by the mean
fluorescence intensity (MFI) values in the 2 populations. IL-5 was
detected in a small proportion of the CD56 cells. In NK
cells from all primary culture conditions, IFN-+ cells
were detected mostly, if not exclusively, in the CD56+
subset (Table 1 and Figure 4, IL-2 and
IL-15 with feeder cells; Table 2 and
Figure 5, IL-2 and Flt3-L). Similar to
the CD3/CD161+ NK cells from cultures with
feeder cells and IL-2, approximately 10% of those from cultures with
Flt3-L and IL-2 expressed IL-5, and up to approximately 40% of them
expressed IL-13 (Figure 5). The majority of these, like those of the
IL-5+ cells, were included in the CD56 cells,
and only a minor proportion of the CD56+ cells expressed
IL-13 (Table 2). The percentage of cytokine+ NK cells
detected with anti-IL-5 mAb added to the anti-IL-13 mAb was identical
to that of the cells positive with the anti-IL-13 mAb alone,
indicating that the IL-5-producing NK cells overlapped completely with
those producing IL-13 (not shown). Although IL-5 and IFN- were
produced independently, a minor proportion of
IFN-+/IL-13+ cells was reproducibly detected
in both the CD56+ and the CD56 cells
(Figure 5).
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| Figure 4.
Intracellular accumulation of IFN-, IL-5, TNF-,
and GM-CSF in CD56+ and CD56 NK cells from
cultures of Lin cells to determine the sequence with
which the ability to produce type 1 and type 2 cytokines is acquired
during differentiation.
Cells from primary cultures of Lin cells with
Sl/Sl4hSCF220 feeder cells and IL-2 (left
panels) or IL-15 (right panels) were stimulated as in Figure 3.
Intracellular cytokines and surface phenotype were analyzed
simultaneously (3-color immunofluorescence) on gated CD161+
cells within purified CD3 cells using FITC-, PE-, or
biotin-labeled mAbs to the indicated molecules and streptavidin-R670.
Quadrants were set to distinguish CD56+ and
CD56 cells. Percent positive cells is indicated in each
quadrant. Experiment representative of 4 performed with similar
results.
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Table 1.
Cytokine expression in CD56+ and
CD56, CD161+ natural killer cells generated
in primary cultures of Lin cells
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| Figure 5.
Analysis of intracellular accumulation of IL-5 and IL-13
in CD161+ and CD56+ NK cells during
differentiation to determine the stage(s) at which IL-13 is produced
and whether the same or distinct NK cell subsets produce the 2 cytokines.
Cells from primary cultures of CD34+ cells with Flt3-L and
IL-2 were stimulated as in Figure 3. Intracellular cytokines and
surface phenotype were analyzed simultaneously in 4-color
immunofluorescence with ECD-anti-CD3, biotin-labeled anti-CD56 alone
(bottom) or combined with anti-CD161 (top) detected with
streptavidin-CyC, FITC-anti-IFN-, and PE-anti-IL-5 or PE-IL-13
mAbs, as indicated. Analysis was performed, as indicated, on gated
CD3/CD56+ or
CD3/(CD161/CD56)+ cells (referred to as
CD161+ NK cells in the text). Percent positive cells is
indicated in each quadrant. Percentages in the left-hand plots are
percent gated cells in the total population. Experiment representative
of 3 performed with similar results.
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Cytokine-induced differentiation of immature
CD161+/CD56 NK cells
Cells from primary cultures of CD34+ cells with Flt3-L
and IL-2 (either unseparated or depleted of mature CD56+ NK
cells) were cultured with IL-2, IL-12, and Daudi feeder cells, conditions we previously demonstrated capable of supporting functional NK cell maturation, as indicated by the acquisition of IFN- mRNA expression.21 Intracellular cytokines and surface
phenotype were analyzed simultaneously after stimulation (3- or 4-color immunofluorescence) in gated CD3/CD161+
(total) and CD3/CD56+ (mostly mature) NK
cells. The initial cell number was maintained throughout the culture.
Within NK cells from nonseparated cultures (Table
3, donors 1 and 2) there was a variable
(range 2- to 20-fold) but consistent increase in the percentages of
IFN-+ cells. These were detected both among the
CD56+ and the CD56 subsets. Instead, the
percentage of NK cells capable of producing IL-13 or IL-5 did not
change, but a significant percentage of IFN-/IL-13 double-positive
cells was detected in both CD56+ and CD56
cells. These results suggested that cells originally capable of
producing only IL-13 had become capable of also producing IFN-, and
that differentiation to mature IFN-+ cells involves an
intermediate double-positive stage. To further analyze this,
CD3/CD161+/CD56 NK cells
(purified to homogeneity from primary cultures and containing only
approximately 0.1% and 52% of cells producing, respectively, IFN- and IL-13 exclusive of each other), were cultured with IL-2 and
IL-12 with or without irradiated Daudi cells as feeder (Table 3, donor
3). After secondary culture, approximately 50% of the cells expressed
CD56 and the proportion of IFN-+ cells increased to
7.6%. Whereas approximately 85% of the IFN-+ cells in
the population that were still CD56 also produced IL-13,
only about 50% of the IFN-+ CD56+ cells
were capable of doing so. As in the cultures using total cells, the
majority of the IL-13+ cells did not produce IFN-. In
cultures with IL-2 and IL-12, alone or in combination, without feeder
cells CD56 and IFN- expression was induced only in a minor
proportion of the cells. Similarly, secondary cultures of the
CD56 NK cells with IL-15 alone or with added IL-2, IL-4,
and/or IL-12 induced minimal to no CD56 and IFN- expression (not
shown). The data support the conclusion that IL-12, but none of the
other cytokines tested, is needed, together with other cellular or
soluble factors, to induce differentiation of
CD161+/CD56 cells to cells capable of
producing IFN-, and that this occurs gradually, concurrent with
decreased ability to produce IL-13 and expression of CD56.
| |
Discussion |
Using 3 in vitro models of hematopoietic cell
differentiation of Lin or CD34+ cells to
analyze cytokine production at distinct stages of human NK cell
differentiation and its regulation by IL-15 and IL-12, we present
evidence that (1) like in cord blood, most CD56+ NK cells
generated in these cultures produce TNF- and GM-CSF, contain a
subset of cells producing exclusively IFN-, a small proportion of
cells producing IL-13 only, and none producing IL-5 at detectable
levels; (2) IL-13 and IL-5 production, evident in immature
CD161+/CD56 cells unable to produce IFN-,
is lost gradually upon differentiation to phenotypically mature
IFN-+ cells, indicating that IFN- and IL-13 and/or
IL-5 production, characterize, respectively, final and intermediate
stages of functional NK cell differentiation; (3) IL-12, likely
together with other factors, supports terminal functional maturation of
the IFN- CD56 NK cells, as indicated by
the appearance of a significant proportion of both
IFN-+/IL-13+/IL-5+ cells and
cells producing only IFN- in cultures of the immature NK cells with
this cytokine and feeder cells; and (4) IL-2, IL-4, and IL-15 alone
support survival/proliferation, but not differentiation, of the
CD56 NK cells, as indicated by the lack of significant
changes in cytokine production in secondary cultures with these
cytokines without (this report) or with feeder
cells.21
NK cell differentiation in vivo is mediated, in part, by IL-15,
produced by stromal/myeloid cells,53 and NK cells are not generated in mice lacking IRF-1,22,23 essential for
induced expression of IL-15. In agreement with previous reports using different culture systems and bone marrow-derived or umbilical cord
blood-derived progenitor cells,27,28,30 IL-15 supports the generation of phenotypically mature NK cells in all 3 systems used,
and our data show that the relative proportion and phenotype of the
cytokine-producing NK cell subsets in either condition are similar to
those identified in peripheral blood. However, immature
CD161+/CD56 NK cells are not generated in
cultures with IL-15. This may depend on the inability of IL-15 to
induce their proliferation, and consequent detectable accumulation,
whereas IL-2 may be directly or indirectly mitogenic for the same
cells. Supporting this possibility, IL-15 maintains the
CD161+/CD56 cells without inducing their
proliferation in secondary cultures. However, we cannot exclude that
IL-15 has a more pronounced differentiation-inducing effect than IL-2,
resulting in faster differentiation and consequent difficulty in
experimentally identifying cells at intermediate stages of
differentiation. Alternatively, progenitor cells at different stages of
maturation may be differentially susceptible to the 2 cytokines, like
in the murine system,25,54 where distinct growth factors
act on cells at different stages of maturation. Undefined cytokines
contributing to differentiation of earlier NK cell progenitors may be
produced endogenously in response to IL-2 by other (eg, myeloid) cells
and be depleted at later times during culture, and/or receptors for
IL-15 may not be expressed on these cells. Finally, the accumulation of
immature NK cells in the cultures with IL-2 may be only apparent and
due to susceptibility of the mature cells to death induced by
membrane-bound or soluble factors expressed by the NK or other cells in
the cultures in response to IL-2 but not, or at lower and ineffective
levels, to IL-15.
Whatever the reason for our observation, the results of the studies on
the CD161+/CD56 NK cells generated
necessarily from cultures with IL-2 indicate that in vitro systems
containing this cytokine are appropriate for studying the origin and
significance of NK cell subsets in the peripheral blood. Likely because
of the sharing of the and c signal transduction
subunits of its receptor,53,55 IL-2 substitutes for IL-15
in vitro to study NK cell differentiation in the murine
system.54 A CD161+/CD56 NK cell
subset and IL-13+ NK cells have been detected, although in
minor proportions, in peripheral adult and neonatal
blood19,21; and NK cells producing high levels of IL-13 and
IL-5, with correspondingly lower IFN- levels, can be
expanded/induced from peripheral NK cells.56 Finally,
although IL-2 is unlikely to be a growth factor for NK cells in
physiologic conditions, it may influence differentiation of NK cells
from their progenitors in the bone marrow under pathologic conditions
in which IL-2-producing activated T cells are present, or in the
peripheral blood during infection, once specific activated T cells have
been generated.
As previously reported,52 IL-12 alone does not support NK
cell differentiation although, in concert with other
growth/differentiation factors, it supports differentiation of
hematopoietic progenitors (preferentially myeloid) both in
vitro57-60 and in vivo,61 and affects later
stages of differentiation of the CD56 NK cells generated
in cultures with IL-2.21,52 The inability of IL-12 to
support IL-2 and IL-15 in allowing NK cell differentiation in vitro,
and the observation that NK cells are generated, although functionally
impaired, in IL-12 p40/ mice,62
adds to the contention that this cytokine does not participate in early
steps of NK cell differentiation. The inhibitory effect of IL-12
indicates a negative regulatory effect of this cytokine. This may
depend on preferential induction of differentiation or proliferation of
immature myeloid cells, and may be mediated, in part, via induced
TNF- production and/or TNF-R1 expression.63-65 However,
neutralization of TNF- or IFN-, and addition of either cytokine
to IL-2 or IL-15 (not shown) had no significant effects on NK cell
differentiation, and direct or indirect inhibitory effects of IL-12
(eg, induction of Fas/FasL and consequent apoptosis) on progenitors or
differentiating NK cells cannot be excluded.
Our data indicate that most immature
CD161+/CD56 cells produce IL-13, and a
discrete subset of them coexpresses IL-5, in the absence of IFN-.
Interestingly, it has been recently reported that an
asialoGM-1+, DX5+, non-T, non-NK/T, nonmature
NK cell type found in association with immature B cells in the bone
marrow66 is responsible for protecting these cells from
antigen-induced, apoptosis-mediated deletion. Given the phenotype
reminiscent of the human immature IL-13+ NK cells we report
here, and the antiapoptotic effects of IL-13 on B cells,67
it will be important to determine whether immature bone
marrow-resident NK cells play a role to control B-cell selection. Additionally, from our data, the suggestion can be made that resting IL-5+ or IL-13+ peripheral blood NK cells may
represent NK cells that exited the bone marrow at relatively late,
although not final, stages of differentiation.
A major proportion of cells with phenotype (CD56 and other
differentiation antigens) and functions (IFN-, but not IL-13
production) of mature NK cells and, importantly,
CD56/dim/IL-13+/IFN-+ NK cells
with intermediate phenotype, are generated from the CD161+/CD56 cells derived from both in vitro
culture models containing IL-2 after switch to culture conditions
including IL-12 and B lymphoblastoid cells as feeder. This occurs both
using total NK or purified CD161+/CD56 NK
cells. Also, similar numbers of cells were recovered from the cultures
without feeder cells (not shown), and cytokines alone did not support
significant changes in cytokine production. Thus, it is unlikely that
the cells with mature or intermediate phenotype derive from expansion
of minor contaminants in the original population. We favor the
interpretation that type 2 and type 1 cytokine production is a
functional characteristic of NK cells at distinct stages, rather than
of distinct subsets of cells at an identical stage of differentiation.
Unlike IL-221 (and IL-15, reported here), IL-12 in the
presence of feeder cells promotes terminal functional differentiation
of the immature CD161+/CD56 NK cells, as
previously proposed based on induced expression of IFN- mRNA and
CD56.21 Unlike IFN- and type 2 cytokine production, that of GM-CSF and TNF- does not define distinct stages of
differentiation, although the lower levels of GM-CSF detected on a per
cell basis in the CD56+ cells suggest its decreased
production during differentiation.
Only a fraction of the CD56+ NK cells from the
primary cultures were induced to express IFN-, similar to what is
observed, with interdonor variability, in peripheral blood NK cells.
The observation that only a proportion of the
CD161+/CD56 cells differentiate under the
appropriate conditions most likely depends on lack of additional
factors. NK cell functions are impaired in vivo (decreased cytotoxicity
and lower IFN- production by either T or NK cells) in mice with a
disrupted IL-12R 1 chain gene68 and in those lacking
IL-18.69 Further functional impairment is observed in the
NK1.1 (NKRP-1A, CD161)+ NK cells from IL-12
p40//IL-18/ mice,69
paralleling our in vitro data. This suggests that together with IL-12,
IL-18 (and possibly other factors) may be involved in the functional
final maturation of NK cells to IFN- production. It will be
important to determine whether peripheral NK cells in the IL-12
p40//IL-18/ mice are arrested at a
stage of differentiation equivalent to that of the
CD161+/CD56 NK cells reported here.
IL-12 with IL-2 or IL-15 alone (the latter 2 likely needed to support
survival/proliferation) induce CD56 expression at low density and
IFN- in a minor proportion of the cells, without concomitant
decrease in the proportion of IL-5+ and IL-13+
cells. The observation that most IL-13+ cells under these
conditions are still CD56 supports the hypothesis that
cytokines alone are incompletely effective to support differentiation
of the CD161+ NK cells. No significant changes were
detected, at the times analyzed, in the proportion of
IL-13+ and IL-5+ cells in cultures with IL-4
(not shown). In keeping with the data of Warren et al in mature NK
cells,20 IL-4 may support survival and/or proliferation,
rather than differentiation, of the relatively immature
CD161+/CD56 NK cells.
Our data support the conclusion that the ability to produce IFN- is
acquired by NK cells at intermediate to late stages of differentiation,
concomitant with the shut-off of the type 2 cytokines IL-5 and IL-13
specifically and irreversibly induced by IL-12 and possibly other
cytokines. We have recently demonstrated that, like in the 2 culture
systems that recapitulate and mimic the bone marrow environment
(including culture of progenitor cells with IL-2), the
CD161+/CD56 NK cells detectable in peripheral
blood produce IL-13, but not IFN-. These cells undergo
IL-12-induced differentiation to
IFN-+/IL-13/CD56+ NK cells,
transiting through an IL-13+/IFN-+ stage at
which the cells start expressing CD56.70 Thus, the 2 in
vitro systems dissected here will be invaluable in future studies of
the cytokine-mediated and molecular regulation of the differentiation
and functions of NK cells at stages preceding or following the
intermediate CD161+/IL-13+ stage.
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Acknowledgments |
We thank Dr R. Wapner and the staff in the Obstetrics and
Gynecology Department of Thomas Jefferson University Hospital for providing the umbilical cord blood samples, Mr B. Abebe for technical assistance, and Mr D. Dicker and P. Hallberg for assistance with flow cytometry.
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Abstract
Introduction