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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 984-991
IMMUNOBIOLOGY
The receptor tyrosine kinase c-kit provides a critical signal for
survival, expansion, and maturation of mouse natural killer cells
Francesco Colucci and
James P. Di Santo
From the Institut National de la Santè et de la Recherche
Medicale (INSERM) U429, Hôpital Necker-Enfants
Malades, Paris, France.
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Abstract |
Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF)
have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the
contribution of signaling through the receptors for these growth
factors for in vivo NK cell development remains ill-defined. We have
analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver
(FL) HSCs of c-kit / (W/W) mice.
Although c-kit/NK cells were generated in
W/W chimeras, they were reduced in number, contained a lower
percentage of CD45R (B220)+ cells, and were poorly
cytolytic. In vitro experiments showed that generation of NK cells from
FL precursors was reduced in the absence of c-kit signaling and that
SCF promoted the survival of peripheral
c-kit+ NK cells. We conclude that c-kit/SCF
interactions in vivo are dispensable for the commitment of HSC to the
NK lineage, but they provide essential signals for generating normal
numbers of fully mature NK cells.
(Blood. 2000;95:984-991)
© 2000 by The American Society of Hematology.
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Introduction |
Natural killer (NK) cell differentiation is a multistep
process that involves the commitment of hematopoietic stem cells (HSCs) to the NK cell lineage followed by further differentiation to yield
functional mature NK cells. This process occurs primarily within the
bone marrow (BM) microenvironment and requires cell-to-cell interactions and soluble factors derived from BM stromal cells. Based
on a series of studies, models for NK cell development have been
proposed whereby purified HSC populations could be made to differentiate into cells that can express NK markers and are capable of
mediating natural cytotoxicity in vitro.1 William et
al1 suggest that distinct phases in NK cell differentiation
can be discerned. Early-acting cytokines, including interleukin-7
(IL-7), stem cell factor (SCF), and fetal liver kinase ligand
(flk2L/flt3L), act on HSCs to commit them to the NK
lineage with concomitant induction of the  subunit of the IL-15
receptor (IL2R chain) expression. Subsequently, triggering of the
IL-2R/ c (common gamma chain) complex with IL-15 (or
with high doses of IL-2) acts to further expand and differentiate these
cells to fully mature NK cells.2 Of the above-mentioned
cytokines or growth factors, none are capable of supporting NK cell
development by themselves.2-4 However, certain early acting
factors appear more potent in driving NK cell differentiation in
vitro.4-5 This model provides a useful starting point to
decipher NK cell development, although most of the data derive from in
vitro experiments using cytokines at concentrations that may have
little relevance to conditions normally encountered in vivo.
Administration of exogenous SCF, flk2L/flt3L, or IL-2 to humans or mice
results in an augmentation in NK cell number and lytic activity,6-9 further supporting a role for these factors in
promoting NK development in vivo. However, in these settings it is not
clear whether the injected cytokines directly influence NK cell
development or act indirectly by modifying the stromal cell microenvironment.
The analysis of natural mouse mutants and gene-targeted mice have made
an important impact on our understanding of the in vivo roles of
cytokines and their receptors in lymphoid development. IL-15 appears to
play a major role in NK cell development. Mice deficient of any chains
composing the receptor for IL-15, such as IL-15R , IL-2R, or
c, are devoid of mature NK cells.10-12 In
contrast, IL-2-deficient mice are capable of generating lytic NK
cells.13 Thus, while both IL-15 and high doses of IL-2 can support NK development in vitro, only IL-15 appears to subserve this
function in vivo. Targeted disruption of the interferon regulatory factor-1 causes a severe reduction of mature NK cells due to impaired IL-15 production within the BM microenvironment.14,15
Treatment of mice with the BM localizing isotope strontium
89 (89Sr) has been reported to deplete NK cells by a
similar mechanism.16 Thus blockade of IL-15 signaling in
vivo results in defective NK differentiation and confirms the initial
in vitro studies that suggest an important role for IL-15 in the
generation, proliferation, and activation of human and murine NK
cells.3,17,18
In contrast to IL-15, the role of flk2/flt3 and c-kit signaling for NK
cell development in vivo is less well-defined. Flk2/flt3 and c-kit
belong to a family of transmembrane receptor tyrosine kinases, which
also includes the receptors for platelet-derived growth factor (PDGF),
epidermal growth factor (EGF), insulin, insulin growth factor-1
(IGF-1), and colony stimulating factor-1 (CSF-1).19 Both
flk2/flt3 and c-kit are expressed by early multipotent HSCs and various
subsets of lineage-committed cells including B, T, NK, erythroid, and
mast cells.20-23 These 2 cytokines share some biological
activities, but they maintain distinct and unique functions in the
differentiation process of discrete cell lineages.24 For
instance, targeted deletion of flk2/flt3 leads to deficiencies in
primitive hematopoietic progenitors and early myeloid and B-lymphoid precursors.25 Preliminary observations in
flk2L/flt3L/ mice indicated
an absence of a subset of NK cells bearing high levels of the NK1.1
marker,26 although the nature of this defect has not been
defined. On the other hand, lack of c-kit/SCF interactions in mice
homozygous for a
c-kit/ null allele
(W/W) causes deficiencies in germ cells, melanocytes, HSCs,
early lymphoid progenitors, mast cells, T cells, and red blood cells,
which in turn can lead to severe anemia and death by day 10 of
postnatal life.27 The lethal nature of the W
mutation has impeded any in-depth analysis of the role of c-kit on NK
cell development, although mice with a viable
c-kit mutation (W/Wv) have
been shown to develop NK cells with reduced natural
killing.28 Still, the Wv mutant
receptor retains significant kinase activity (about 10% of wild type
levels29), which precludes a definitive conclusion on the
role of c-kit/SCF interactions in NK cell differentiation in vivo.
In this report, we address whether c-kit/SCF interactions are essential
for NK cell differentiation by analyzing hematopoietic chimeras made by
reconstituting alymphoid (T- B- NK-)
RAG2/c/ mice with W/W
c-kit/ fetal liver cells. This complementation
system is ideally suited for the analysis of genes involved in early
lymphoid and NK cell differentiation, since
RAG2/c/ mice (unlike
RAG2/ mice) are severely depleted
in lymphoid precursor cells and completely lack mature NK
cells.30 With the data discussed below, we can conclude
that signaling through c-kit is not required for commitment to the NK
lineage, although c-kit plays a nonredundant role in the expansion of
NK committed precursors and in the survival and final maturation of
murine NK cells.
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Materials and methods |
Mice and generation of fetal liver hematopoietic chimeras
Mice with a null mutation in the c
chain10 were from the fourth generation backcross to the
C57Bl/6 background. RAG2 mice31 were from the
10th generation backcross to C57Bl/6 (gift
of B. Rocha, INSERM U345, Paris, France). Doubly deficient mice
(RAG2/c/) were
obtained by intercrossing as described.30 We also used breeding stocks of WB-W/+ mice32
(gift of Dr H.-R. Rodewald, Basel, Switzerland). Mice were intercrossed
to generate the null allele W/W
(c-kit/) and the control
(c-kit+/+ or c-kit ±,
hereafter referred to as wt) day 13 embryos. The morning of the
vaginal plug discovery was designated as day 0. Fetal liver cell
suspensions were obtained by passage of the tissue through a 23-gauge
needle, and the genotypes were determined by fluorescence-activated cell sorter (FACS) analysis using an anti-c-kit monoclonal antibody (mAb). RAG2/c/ and
RAG2/ mice (more than 6 weeks of age) were
irradiated with 0.3 Gy (gray) from a cobalt source,
and 4 hours later they were injected intravenously with
4-8 × 105 FL cells as a source of HSCs. All mice
received tetracycline and bactrim in the drinking water for the period
following the FL cell transfer.
Flow cytometry
Single cell suspensions were prepared from spleen, thymus, liver,
and BM cells. Erythrocytes were lysed in ammonium chloride, and the
cells were resuspended in phosphate-buffered saline (PBS) with 3%
fetal calf serum (FCS) and 0.01% sodium azide. The following mAbs
(PharMingen, San Diego, CA) were directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), Tricolor (TRI), or biotin
(biot) and were used for immunofluorescence analysis: CD117 (c-kit),
CD122 (IL-2R), CD3, CD4, CD8, TCR, TCR ,
CD24 (HSA), NK1.1, B220, CD90 (thymine [Thy-1]), DX5, CD2, Ly49A,
Ly49C/I, Ly49D, CD43, immunoglobulin M (IgM), and IgD. Biotin
conjugates were revealed by streptavidin-TRI (Caltag,
San Francisco, CA). Cells (106) were first incubated with
anti-fragment(Fc)RII/III (hybridoma 2.4G2) and then stained with a
mixture of biotinylated and fluorochrome-labeled mAbs at saturating
concentrations, washed twice, and finally incubated with
streptavidin-TRI. Analysis was performed on a fluorescence-activated cell sorter (FACS) flow cytometer (CellQuest software;
Becton Dickinson, San Josè, CA). Dead cells were
excuded by means of their forward and side scatters, and an electronic
gate was set to acquire 5-10 × 103 lymphoid cells.
Lymphokine-activated killer cell cultures and cytotoxic assay
A chromium 51 (51Cr) release assay was used to measure
NK lytic activity in vitro, as previously described.21 For
natural cytotoxicity, mice were injected intraperitoneally with 200 µg of poly (I:C), and 40 hours later, to deplete
macrophages, red cell-depleted splenocytes were incubated on plastic
for 1 hour at 37°C. YAC-1 and EL-4 thymoma cells
were maintained in complete medium (CM; RPMI-1640 with
10% FCS, 10-5 mol/L -ME, 100 µg/mL streptomycin, and
100 units/mL penicillin). Target cells were labeled with
3.7 MBq (100 µCi) 51Cr (ICN
Pharmaceutical, Costa Mesa, CA), and 2.5-5 × 103
cells were incubated with graded numbers of effector cells in 200 units/L (µl) of medium for 4 hours. In some
experiments, effector cells were CD122+
DX5+ NK cells isolated from splenocytes by cell sorting.
To generate adherent-lymphokine activated killer (A-LAK) cells, red
cell and macrophage-depleted splenocytes were cultured at
5 × 106 cells/mL in CM supplemented with 1000 units/mL of dihydrouridine IL-2 (huIL-2) (Peprotech,
Rocky Hill, NJ) or with 0.5 µg of huIL-15 (Immunex, R&D Systems,
Minneapolis, MN). After 3-4 days the nonadherent cells
were removed, and the A-LAK cells were refed every 2-3 days and
cultured until days 8-10. A-LAK cultures produced in this manner
routinely contained more than 80% NK1.1+
CD3cells. For antibody-dependent cell cytotoxicity
(ADCC), day 8-10 A-LAK cells were used as effectors, and target cells
were EL-4 cells coated with anti-Thy 1.1 mAb, as previously
described.21 Radioactivity released into the cell-free
supernatant was measured, and the percentage of specific lysis was
calculated as the following: 100 × (experimental release  spontaneous release) / (maximum release spontaneous release).
Generation of NK cells in vitro from FL cells
Day 13 FL cells prepared from normal C57Bl/6 timed pregnancies were
plated at 5 × 103 cells/mL in round-bottom
microtitre plates in CM supplemented with 50 ng/mL
muIL-7 (Peprotech), 100 ng/mL of huflk2L/flt3L
(Immunex), and 100 ng/mL rat SCF (Peprotech). After 8 days, the
cytokines were removed, and the cells were cultured with CM containing
huIL-2 (1000 units/mL) for an additional 8-10 days.
Effects of SCF on mature NK cells in vitro
Purified splenic NK1.1+ c-kit+ and NK1.1+ c-kit cells were
isolated from RAG2/mice by cell
sorting. Stringent sorting gates were applied to avoid
cross-contamination of the 2 sorted subsets. Sorted cells (greater than
97% pure) were plated at 104 cells/well in round-bottom
microtitre plates in 200 µL of CM supplemented with graded doses of
huIL-2, in the presence or absence of 100 ng/mL muSCF. Cell viability
was evaluated daily by trypan blue dye exclusion, and after 6 days of
culture, cells were analyzed for cell-surface phenotype, cell cycle
distribution, and cytotoxic activity. Cell- cycle analysis was
performed using 7-aminoactinomycin-D (7 AAD) incorporation into
saponin-permeabilized cells as described.32
Statistical analysis
Data were analyzed (Statview software, Version 4.5, SAS Institute, San Francisco, CA) applying the paired Student t
test. The null hypothesis was rejected, and the difference
was assumed significant when P < .05.
| |
Results |
c generation of FL  RAG/c hematopoietic
chimeras
We have recently developed a novel mouse strain harboring mutations
in both the RAG2 and common cytokine receptor
c genes.30 RAG2/c/mice are
alymphoid and have severely reduced numbers of BM and thymic lymphoid
precursors, thereby making them ideal hosts for genetic complementation
experiments analyzing lymphopoiesis. In order to define the role of the
c-kit receptor tyrosine kinase in NK cell development, we reconstituted
0.3 Gy irradiated
RAG2/c/ mice with
c-kit-deficient HSC precursors. W/W mice harbor a splice donor
site mutation that causes deletion of the transmembrane region, thereby
completely lacking cell-surface expression of c-kit molecules. The
absence of c-kit results in severe anemia in these mice and death by
day 10 after birth.27 In our experiments, c-kit-deficient embryos were identified by their extreme pallor, and
day 13 FL cells were used as a source of HSCs. The genotypes were
confirmed by flow cytometry using an anti-c-kit mAb (data not shown).
While HSCs express c-kit, previous studies have shown that the content
of HSCs in day 13 FL from SCF-deficient mice is no different from
control mice.34 We therefore transferred equal numbers of
FL cells from W/W or wt day 13 embryos, and 8 weeks
posttransfer, analyzed thymic, splenic, and BM lymphoid populations in
the RAG2/c chimeras.
B- and T-cell development in hematopoietic chimeras
Consistent with previous reports,33 we found that B-cell
development was permissive in the absence of c-kit (Figures
1A-C). No obvious defects in BM B-cell development were
observed, and the absolute numbers of pre-B (B220+
IgM) and mature B cells (B220+
IgM+) were not significantly different between wt
(more than RAG2/c) and W/W (more than
RAG2/c) chimeras (Table
1, Figure 1B). In contrast, we found a 10- to 20-fold reduction in total thymocyte numbers in the absence of c-kit
(Table 1). The diminished thymopoiesis after transfer of
c-kit-deficient FL cells likely reflects the important role for
c-kit/SCF interactions in the expansion of early thymic
double-negative precursors, as described by Rodewald et
al.35 Once past this critical stage, intrathymic
development in the absence of c-kit appeared normal, and all mature
thymocyte subsets could be identified in W/W chimeras,
including single positive CD4 and CD8 T cells, T cells,
and NK T cells (Figure 1A; data not shown). We confirmed previous
results of Takeda et al33 demonstrating that
c-kit-deficient FL cells fail to reconstitute the thymus of irradiated
RAG2/ hosts (data not shown). The
ability of W/W HSCs to repopulate RAG2/c/ but not
RAG2/ hosts can be explained by the
severe reduction in both thymic and BM lymphoid precursors in
RAG2/c/
mice30 (Table 1).
RAG2/c/ mice thereby
offer a less competitive host microenvironment than RAG2/ mice, where mutant (eg,
c-kit/) HSCs can further
differentiate. These results attest to the utility of the
RAG2/c strain for reconstitution experiments and the analysis of genes affecting HSCs and their immediate progeny.
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| Fig 1.
T- and B-cell development in W/W chimeras.
Thymocytes and BM cells were depleted of red cells and stained with the
indicated antibodies. Percentages are indicated within the dot plots.
(A) Thymocyte numbers in W/W chimeras are much reduced,
although thymopoiesis appears rather normal in the absence of c-kit.
(B) BM pre-B (B220+ IgM) and B cells (B220+ IgM+) are
represented in normal percentages in W/W
chimeras. FACS profiles are representative of 4 independent
experiments. (C) Reconstitution of splenic lymphoid populations with
the presence of CD3+ T cells and B220+ B cells
in W/W chimeras.
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IgM+/IgD+ B cells and CD4+ and CD8+ T cells seed the peripheral
lymphoid organs in W/W chimeras more than
RAG2/c chimeras (Figure 1C; data not shown).
Total splenic lymphocyte numbers were slightly reduced in the absence
of c-kit, and this decrease was largely confined to the T-cell
compartment. Thus, splenic CD3+ cells were
6.5 ± 1.8 × 106 in wt (n = 7) and
4.6 ± 0.7 × 106 in W/W chimeras
(n = 7; P < .05) (Figure 1C). However, compared to the
dramatic decrease in thymopoiesis (5%-10% of normal), peripheral T-cell numbers were about 70% of controls, suggesting that peripheral T-cell expansion had occurred. Together these results suggest that T
lymphocytes do not require continual c-kit/SCF interactions for
peripheral homeostasis.
NK cell development in the absence of c-kit
Since all NK cells in RAG2/c chimeras are
donor-derived,30,36 any abnormalities in NK cell
differentiation in W/W chimeras result from cell intrinsic
defects due to lack of c-kit signaling. The ability of W/W FL
cells to fully replenish total BM lymphoid cellularity and BM and
splenic B-cell compartments suggests that HSC function in the absence
of c-kit was not a limiting factor.34 NK cells were
detected in the BM and spleens of W/W FL chimeras (Figure
2A and B), but absolute numbers of BM NK
cells were only 40% of normal in the absence of c-kit. The difference
was found to be statistically significant (P < .05). Thus,
c-kit/SCF interactions are dispensable for NK lineage commitment but
are required for normal NK cell differentiation (Table 1, Figure 2A).
Total numbers of splenic NK cells were similarly decreased in
W/W chimeras (Figure 2B), which suggests that either peripheral
expansion is not a mechanism to regulate NK cell homeostasis or that
this process normally relies on c-kit signaling pathways. In agreement
with previous reports,1 we found that about 10% of
peripheral NK1.1+ CD3-NK cells from wt chimeras expressed
c-kit, while W/W NK cells were c-kit, as expected
(Figure 2C).
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| Fig 2.
NK cells in W/W chimeras.
BM (A) and spleen cells (B) were red-cell depleted and stained with a
combination of antibodies for IL-2RFITC,
NK1.1PE, TCRbiot, and
CD19biot. Absolute cell numbers (×106) are
indicated on top of each dot plot and refer to the mean ± SD of 7 chimeras per group. An electronic gate was set to exclude
TCR+ T cells and CD19+ B cells, and the
percentage of IL-2R+ NK1.1+ NK cells was
calculated. (C) Spleen cells were stained for DX5FITC,
TCRbiot, and c-kitPE. The percentages of
c-kit+ DX5+ TCR NK
cells are indicated. Results are representative of 5 independent
experiments.
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In light of the existence of a bipotent NK/T thymic precursor, the
development of thymic NK cells and NK-T cells in the absence of c-kit
was also evaluated. Both of these minor thymic lymphoid subsets could
be detected in W/W chimeras, although their absolute numbers
were clearly reduced compared to control chimeras (NK: wt = 5.95 ± 3.0 × 104 cells versus
W/W = 0.5 ± 0.3 × 104 cells; NK-T:
wt = 15.7 ± 5.6 × 104 cells versus
W/W = 3.4 ± 1.7 × 104 cells;
n = 4 for each genotype; P < .05). The reduction
in absolute cell numbers of thymic NK and NK-T cells paralleled the
overall reduction in thymic cellularity. These results suggest that
thymic T, NK, and NK-T cells develop via a c-kit-dependent early
thymic precursor, and the results argue against a major pathway of NK cells generated from a putative NK/T bipotent precursor.
Phenotype and function of c-kit/NK cells
The phenotypic maturation of splenic NK cells from wt and
W/W chimeras was characterized by flow cytometry using a panel
of cell-surface markers (Figure 3). NK
cells that were c-kit-deficient expressed normal levels of CD2,
IL-2R, NK1.1, DX5, and CD90 (Thy-1). Percentages and levels of Ly49
family members (including Ly49A, Ly49C/I, and Ly49D) were normal in
W/W chimeras, which suggests that NK cell "education"
with respect to inhibitory receptors for MHC class I molecules does not
critically involve c-kit. Interestingly, c-kit-deficient NK cells
showed markedly reduced expression of the B220 antigen (Figure 3).
Similar results were obtained during analysis of BM NK cells (data not
shown). As B220 expression increases on NK cells upon
activation,16 we further compared the functional capacity
of wt and c-kit-deficient W/W NK cells ex vivo.
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| Fig 3.
Phenotype of W/W NK cells.
Spleen cells were red-cell depleted and stained with the indicated
FITC-labeled antibodies in combination with NK1.1PE,
TCRbiot, and CD19biot. An electronic gate
was set to exclude TCR+ T cells and CD19+
B cells. The percentage of NK1.1+ NK cells positive for the
indicated marker (white histograms) was calculated over the background
of an isotype-matched control mAb (gray histograms). Results are
representative of 3 independent experiments comprising 6 wt and
6 W/W chimeras.
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Previous studies had shown that unfractionated splenocyte populations
from mice with a viable c-kit mutation
(W/Wv) had a decreased level of natural
killing.28 However, since absolute NK cell numbers were not
determined in those studies, it was not possible to precisely define
the nature of the functional defect. We found that total spleen cell
preparations from W/W chimeras demonstrated less than one-third
of the natural killing lytic activity as compared with wt
splenocytes at equivalent effector:target ratios (n = 5; data not
shown), confirming the initial data by Seaman and Talal.28
In order to determine the lytic ability of
c-kit/NK cells on a per-cell basis,
we purified wt and W/W splenic NK cells by cell
sorting. As shown in Figure 4,
c-kit-deficient W/W NK cells showed less than half of the
lytic capacity of wt NK cells against YAC-1 targets on a
per-cell basis, thus confirming an intrinsic defect in natural killing
of NK cells in the absence of c-kit.
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| Fig 4.
Cytotoxic activity of purified W/W NK cells.
Spleen cells were depleted of red cells, B cells, and T cells and
stained with IL-2R and DX5. Double-positive NK cells were sorted and
plated at the indicated E/T ratios with 2500-5000 YAC-1
target cells. All assays were done in duplicate or triplicate. Results
are representative of 2 independent experiments. The spontaneous
release was less than 10% of the maximal release.
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Restoration of NK cell differentiation in vitro
Combined signaling through c-kit and IL-2/IL-15 receptors has been
shown to synergistically enhance NK cell expansion and lytic activity
both in vitro and in vivo.8 We therefore tested whether
exposure of c-kit-deficient NK cells to IL-2 or IL-15 in vitro
(thereby generating A-LAKs) could correct their functional and
phenotypic defects. In contrast to freshly isolated W/W NK cells, A-LAK cells from W/W chimeras lysed YAC-1 targets in a fashion indistinguishable from controls (Figure
5A). A-LAK cells that were c-kit-deficient
also mediated normal levels of ADCC. In parallel with the restoration
of natural killing, A-LAK cells from W/W chimeras also
upregulated B220 surface expression (Figure 5B). Similar results were
obtained with IL-15 (data not shown). Taken together, these results
suggest that the abnormal differentiation of c-kit-deficient NK cells
in vivo can be corrected in vitro by signaling through the IL-2/IL-15
receptor.
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| Fig 5.
Cytotoxic activity and phenotype of IL-2-activated
W/W NK cells.
(A) Spleen cells were depleted of red cells, B cells, and T cells, and
the remaining cells were used to generate A-LAK cells. Day 7-10 A-LAK
cells were used as effectors against YAC-1 cells ( ) or
EL-4 cells in the presence ( ) or absence of -CD90
antibodies ( ). All assays were done in duplicate or triplicate. (B)
Day 7-10 A-LAK cells were stained with antibodies for
CD3FITC, NK1.1PE, B220TRIC, and the
expression of B220 on NK1.1+ CD3NK cells
was calculated. Data are representative of 3 independent experiments.
Similar data were obtained with IL-15 (n = 2; data not
shown).
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SCF provides an essential signal for expansion of NK cell
precursors
Our observations in W/W chimeras suggest that c-kit/SCF
interactions in vivo are essential for generation of normal numbers of NK cells and for full differentiation of this lymphoid subset. To
gain more insight into the stages at which c-kit signaling operates in
NK cell development, the 2-step culture system described by Williams
and colleagues2 was used to compare the cytokine requirements for differentiating NK cells from wt and
W/W day 13 FL cells. When cultured in a mixture of IL-7, SCF,
and flk2L/flt3L followed by high-dose IL-2, W/W FL cells
developed similar frequencies of IL2R+ NK1.1 and IL2R+
NK1.1+ cells as compared with wt FL cells (Table
2, Figure 6).
However, total cell numbers were 3- to 4-fold reduced in W/W
cultures before the addition of IL-2 (data not shown). At the
end of the culture period, c-kit/W/W
NK cells were found to be less numerous than wt NK cells (Table 2). These results confirm that SCF/c-kit interactions are not required
for NK commitment, but they show that SCF provides an essential signal
for the expansion of NK precursors, which possibly occurs during the
acquisition of IL-15 responsiveness (up-regulation of IL2R). NK
cells generated from W/W FL cultures were as cytolytic as
wt-derived NK cells (Table 2), which confirms that stimulation with high doses of IL-2 (or IL-15) in vitro can compensate for the lack
of c-kit signaling and suggests that SCF may synergize with IL-15 in
vivo to induce full lytic competence.
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| Fig 6.
Surface marker expression of in vitro-generated NK
cells.
Day 13 FL cells were cultured in the presence of flk2L /flt3L, SCF, and
IL-7 for a week followed by culture in IL-2 (1000 units/mL) alone, for
an additional week. Representative of 2 independent experiments.
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Characterization of c-kit/SCF interactions in mature NK cells
Evidence for direct effects of SCF on mature murine NK cells has not
been provided to date. Previous studies1,21 have shown that
a subset of mature murine and human NK cells express c-kit, and human
purified c-kit+ NK cells have been reported to show a lower cytotoxic
activity, which suggests that this subset is somewhat less
mature.21 In line with these studies, we found that freshly
isolated c-kit+ murine NK cells have lower cytotoxic activity
(70%-80%) than the c-kit-NK subset (data not shown). Studies by
Caligiuri and colleagues8 have demonstrated that in vivo
injection of SCF can enhance IL-2-mediated expansion of NK cells,
which presumably acted at the level of NK cell precursors.
To characterize the potential mechanisms by which c-kit/SCF
interactions act during NK cell differentiation, we analyzed the ability of SCF to promote survival, proliferation, and cytolytic activity of ex vivo purified c-kit+ and c-kit NK1.1+
cells from the pool of splenic NK cells. When c-kit+ NK cells were
incubated with high doses of huIL-2 (1000 units/mL), the addition of
SCF resulted in a modest (1.5- to 2-fold) increase in cell yield
(Figure 7, Table
3), although the cell viability (greater
than 90%) and the percentage of cycling cells (about 30%) of c-kit+
cells cultured in high doses of IL-2 were not significantly different
in the presence or absence of SCF (Table 3). In addition, the maximal
lytic activity was comparable between c-kit+ and c-kit NK cells
cultured under conditions of high IL-2 concentrations (Table 3). These
results are consistent with the ability of high doses of IL-2 to
stimulate normal lytic activity in W/W NK cells.
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| Fig 7.
Proliferation and cell survival of purified
c-kit and c-kit+ NK cells.
Splenic NK cells were sorted by FACS in c-kit+ () and
c-kit ( ) subsets, plated at 104 cells/well in CM
supplemented with the indicated doses of IL-2 in the presence () or
absence (, ) of SCF (100 ng/mL). Plots indicate the numbers and
percentages of live cells evaluated by trypan blue exclusion.
Representative of 2 independent experiments.
|
|
In contrast, when c-kit+ or c-kit NK cells were cultured in a
suboptimal concentration of IL-2 (100 units/mL), SCF had a clear effect
on survival, cell cycle progression, and lytic activity of c-kit+ NK
cells (Table 3). Survival in the presence of SCF was 70%, while in the
absence of SCF, few c-kit+ (23%) or c-kit (18%) NK cells were
viable after the 6-day culture period. In the absence of SCF, fewer
c-kit+ NK cells were cycling (11%), and more cells were apoptotic
(12%), based on their DNA content. In contrast, a normal rate of
cycling cells (25%) and apoptotic cells (4%) were detected in c-kit+
NK cells cultured in the presence of SCF. This results in the highest
absolute cell yield (Table 3, Figure 7). Taken together, the results
suggest that SCF can provide survival signals to c-kit+ mature NK cells.
| |
Discussion |
A number of soluble factors that play important roles in the
generation of NK cells from hematopoietic precursors have been identified.1 It is now generally accepted that IL-15 is a
dominant cytokine for NK cell development. Triggering of the
IL-2R/c receptor complex by IL-15 (or mimicking this
signal using high doses of IL-2) is required in vitro for NK cell
differentiation from HSCs, while mice lacking IL-15 receptor components
are NK deficient.3,10-12,14-16,18 Still, IL-15 alone only
poorly promotes in vitro NK cell development,2,3 and
additional factors may intervene in this process. The ligands for the
receptor tyrosine kinases c-kit and flk2/flt3 (SCF and flk2L/flt3L,
respectively) are 2 growth factors that play a role in the
differentiation of multiple hematopoietic cell
lineages.23,37 In vitro studies have shown
that both SCF and flk2L/flt3L can significantly enhance the expansion
of NK cells from HSCs in combination with IL-2 or IL-15.3,4
Williams et al2 and Yu et al5 have demonstrated that flk2L/flt3L induce expression of the IL-2R chain in murine and
human stem cell precursors. Together, these results suggest a 2-stage
model for NK cell differentiation, requiring first, c-kit and flk2/flt3
signaling, and second, IL-15 receptor signaling.1
The ability of either flk2L/flt3L or SCF to promote NK cell development
in concert with IL-15 would be in agreement with presumed redundant
roles for these 2 receptor tyrosine kinases in NK cell differentiation.
However, while the in vitro data are compelling, little is known about
the essential roles of these 2 pathways for NK development in vivo.
Preliminary observations in mice with a null mutation in the
flk2L/flt3L indicated an absence of NK1.1high
cells,26 although the nature of this defect has not
yet been fully explored. The NK cell deficiency in
flk2L/flt3L/mice would suggest,
however, that c-kit and flk2/flt3 signaling pathways are not entirely
redundant in this regard. Seaman and Talal analyzed NK cells in viable
(W/Wv) c-kit mutant mice and suggested that c-kit
was involved in the acquisition of natural cytotoxicity.28
However, these studies raised the question of whether c-kit was
essential for NK cell development, since the Wv
mutation retains significant kinase activity.29 We report
here that c-kit/W/W FL cells generate
reduced numbers of NK cells in vitro and only partially repopulate the
NK cell pool in alymphoid RAG2/c hosts. In
addition, the W/W NK cells generated in vivo show defective killing. These results provide clear evidence for a nonredundant role
of c-kit in the in vivo differentiation of murine NK cells. Together,
the available data support the notion of unique (and perhaps
additive4) roles for c-kit and flk2/flt3 signaling in NK
cell differentiation.
While the precise action of SCF on developing NK precursors remains to
be defined, a major role for c-kit/SCF interactions in the induction of
IL-15 responsiveness appears unlikely, as IL2R expression was
comparable on wt and W/W NK cells both in vivo and in
vitro (Figures 3 and 6). Assuming that this event restricts cells to
the NK cell lineage, we can conclude that c-kit/SCF interactions are
dispensable for NK cell commitment. Consequently, flk2L/flt3L might act
as the dominant factor in NK cell determination in vivo. In contrast,
c-kit signaling is required for generating normal numbers of NK cells.
We would propose that c-kit/SCF interactions augment absolute numbers
of BM and splenic NK cells by acting as a survival factor or (less
likely) by inducing proliferation at the level of NK cell precursors.
In this way, triggering of flk2/flt3 and c-kit on early lymphoid
progenitors would provide distinct yet synergistic signals for NK
cell differentiation.
Previous studies from Fehniger and colleagues8 suggested
that SCF could synergize with IL-2 to promote proliferation of mature
c-kit+ human NK cell subsets, although it did not appear that SCF had a
direct effect on NK cell proliferation. The same group showed that in
vivo SCF administration in mice also resulted in higher yields of NK
cells, but studies done on bulk splenic populations did not show any
direct effect of SCF on NK cell survival or proliferation.8
Our in vitro data demonstrate that under conditions where other growth
factors may be limiting, SCF can provide an essential signal for
survival and cell cycle progression of the c-kit+ cells among the pool
of mature NK cells. These c-kit/SCF interactions appear to act in an
anti-apoptotic fashion and provide protection from cell death in part
through the up-regulation of Bcl-2 levels.38 Taken
together, these studies support a role for c-kit/SCF interactions in
the survival of a subset of mature NK cells and their precursors in
both mice and man.
The reduction in peripheral NK cell function in the absence of c-kit is
noteworthy. Both lytic activity of freshly isolated c-kit/ W/W NK cells and
their activation status (as evidenced by B220 expression) were reduced.
These results would suggest that c-kit signals are required for the
final maturation of NK cells, and they are in line with previous
observations showing that human c-kit+ NK cells appear to be less
mature.21 The functional defects in
c-kit/NK cells could be reversed by
exposure to IL-2 in vitro, which suggests that this phenotype could be
overcome by compensatory pathways. These results are consistent with
previous reports which have demonstrated that IL-2 and SCF can act
synergistically to enhance NK cell expansion and lytic activity both in
vitro and in vivo.8 We would propose that SCF not only
expands the number of NK cell precursors, but that it also acts in
synergy with IL-15 to drive full maturation of NK cells in vivo.
In summary, c-kit/SCF interactions are essential for the normal
expansion of NK cell precursors and for the full maturation and
survival of mature NK cells. In view of a role for c-kit/SCF interactions in generation of NK cell mediated functions, we are currently investigating the role of c-kit-deficient NK cells during in
vivo responses to infectious pathogens and during tumor rejection.
| |
Acknowledgments |
We would like to thank Dr Hans Reimer Rodewald (Basel, Switzerland) for
discussions and for kindly providing WBW/+ mice; Drs Ana
Cumano (Paris, France) and Emma Colucci-Guyon (Paris, France) for
critically reviewing the manuscript; and the anonymous reviewers for
their helpful comments.
| |
Footnotes |
Submitted June 25, 1999; accepted September 30, 1999.
Supported by grants from INSERM (Paris, France), Ligue Contre le Cancer
(Paris, France), and Association pour la Recherche contre la Cancer
(Paris, France) and by a fellowship from INSERM (F. Colucci).
Reprints: James P. Di Santo, Laboratory for Cytokines and
Lymphoid Development, Institut Pasteur, 25, rue Dr. Roux, 75015 Paris,
France; e-mail: disanto{at}pasteur.fr.
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.
| |
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Abstract
Introduction