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IMMUNOBIOLOGY
From The Kimmel Cancer Center, Jefferson Medical
College, Philadelphia, PA.
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- 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- 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 Here, we have analyzed cytokine production in human NK cells at
distinct stages of differentiation. Our data demonstrate that most
CD161+/CD56 Monoclonal and polyclonal antibodies
Cell isolation
Homogeneous immature CD161+/CD56 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 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.
Kinetics of CD56+ NK cell generation from
Lin /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).
In agreement with our previous data52
CD3 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).
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.
Cytokine production by CD56 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).
Cytokine-induced differentiation of immature
CD161+/CD56 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.
Using 3 in vitro models of hematopoietic cell
differentiation of Lin 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 Whatever the reason for our observation, the results of the studies on
the CD161+/CD56 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 Our data indicate that most immature
CD161+/CD56 A major proportion of cells with phenotype (CD56 and other
differentiation antigens) and functions (IFN- Only a fraction of the CD56+ NK cells from the
primary cultures were induced to express IFN- 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- Our data support the conclusion that the ability to produce IFN-
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.
Submitted April 23, 2001; accepted October 10, 2001.
Supported, in part, by USPHS grants CA45284 (B.P.) and T32-CA09683 (M.J.L.); and by a grant from CNR (Italy), posizione 12115645, and the MURST Funds 1997, Project "Meccanismi immunologici di resistenza alle neoplasie" (E.R.).
M.J.L. and L.Z. contributed equally to this work.
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: Bice Perussia, Jefferson Medical College, Kimmel Cancer Center, BLSB Room 750, 233 S 10th St, Philadelphia, PA 19107; e-mail: bice.perussia{at}mail.tju.edu.
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L. Zamai, C. Ponti, P. Mirandola, G. Gobbi, S. Papa, L. Galeotti, L. Cocco, and M. Vitale NK Cells and Cancer J. Immunol., April 1, 2007; 178(7): 4011 - 4016. [Abstract] [Full Text] [PDF]
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P. Schierloh, N. Yokobori, M. Aleman, R. M. Musella, M. Beigier-Bompadre, M. A. Saab, L. Alves, E. Abbate, S. S. de la Barrera, and M. C. Sasiain Increased Susceptibility to Apoptosis of CD56dimCD16+ NK Cells Induces the Enrichment of IFN-{gamma}-Producing CD56bright Cells in Tuberculous Pleurisy J. Immunol., November 15, 2005; 175(10): 6852 - 6860. [Abstract] [Full Text] [PDF]
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C. A. J. Vosshenrich, T. Ranson, S. I. Samson, E. Corcuff, F. Colucci, E. E. Rosmaraki, and J. P. Di Santo Roles for Common Cytokine Receptor {gamma}-Chain-Dependent Cytokines in the Generation, Differentiation, and Maturation of NK Cell Precursors and Peripheral NK Cells in Vivo J. Immunol., February 1, 2005; 174(3): 1213 - 1221. [Abstract] [Full Text] [PDF]
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T. Katsumoto, M. Kimura, M. Yamashita, H. Hosokawa, K. Hashimoto, A. Hasegawa, M. Omori, T. Miyamoto, M. Taniguchi, and T. Nakayama STAT6-Dependent Differentiation and Production of IL-5 and IL-13 in Murine NK2 Cells J. Immunol., October 15, 2004; 173(8): 4967 - 4975. [Abstract] [Full Text] [PDF]
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A. Ahmad and F. Alvarez Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis J. Leukoc. Biol., October 1, 2004; 76(4): 743 - 759. [Abstract] [Full Text] [PDF]
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M. Terme, E. Tomasello, K. Maruyama, F. Crepineau, N. Chaput, C. Flament, J.-P. Marolleau, E. Angevin, E. F. Wagner, B. Salomon, et al. IL-4 Confers NK Stimulatory Capacity to Murine Dendritic Cells: A Signaling Pathway Involving KARAP/DAP12-Triggering Receptor Expressed on Myeloid Cell 2 Molecules J. Immunol., May 15, 2004; 172(10): 5957 - 5966. [Abstract] [Full Text] [PDF]
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J. Brady, Y. Hayakawa, M. J. Smyth, and S. L. Nutt IL-21 Induces the Functional Maturation of Murine NK Cells J. Immunol., February 15, 2004; 172(4): 2048 - 2058. [Abstract] [Full Text] [PDF]
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E. M. Schneider, I. Lorenz, M. Muller-Rosenberger, G. Steinbach, M. Kron, and G. E. Janka-Schaub Hemophagocytic lymphohistiocytosis is associated with deficiencies of cellular cytolysis but normal expression of transcripts relevant to killer-cell-induced apoptosis Blood, September 26, 2002; 100(8): 2891 - 2898. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
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-
