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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2625-2632
HEMATOPOIESIS
Differential requirement for the transcription factor PU.1 in the
generation of natural killer cells versus B and T cells
Francesco Colucci,
Sandrine
I. Samson,
Rodney P. DeKoter,
Olivier Lantz,
Harinder Singh, and
James P. Di Santo
From the Laboratory for Cytokines and Lymphoid
Development, Pasteur Institute, Paris, France; Howard Hughes Medical
Institute, The University of Chicago, Chicago, Illinois; and Institut
National de la Santé et de la Recherche Médicale U25,
Necker Hospital, Paris, France.
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Abstract |
PU.1 is a member of the Ets family of transcription factors
required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not
known. The study shows that PU.1 is expressed in NK cells and that, on
cell transfer into alymphoid Rag2/ c /
mice, hematopoietic progenitors of PU.1/
fetal liver cells could generate functional NK cells but not B or T
cells. Nevertheless, the numbers of bone marrow NK cell precursors and
splenic mature NK cells were reduced compared to controls. Moreover,
PU.1/ NK cells displayed reduced expression
of the receptors for stem cell factor and interleukin (IL)-7,
suggesting a nonredundant role for PU.1 in regulating the expression of
these cytokine receptor genes during NK cell development.
PU.1/ NK cells also showed defective
expression of inhibitory and activating members of the Ly49 family and
failed to proliferate in response to IL-2 and IL-12. Thus, despite the
less stringent requirement for PU.1 in NK cell development compared to
B and T cells, PU.1 regulates NK cell differentiation and homeostasis.
(Blood. 2001;97:2625-2632)
© 2001 by The American Society of Hematology.
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Introduction |
Natural killer (NK) cells are a distinct subset of
lymphocytes that mediate important functions in innate immunity being
able to eliminate tumor cells and to produce cytokines without prior sensitization (reviewed in Trinchieri1). NK cells can
distinguish cells with disparate levels of major histocompatibility
complex (MHC) class I expression, killing cells that, due to viral
infections or transformation, have low MHC expression, and sparing
those with normal expression (reviewed in Ljunggren and
Karre2). Such discrimination is mediated by self
MHC-specific inhibitory receptors on NK cells that belong to one of 3 groups: killer immunoglobulin-like receptors on human NK cells,
lectin-like Ly49 receptors on murine NK cells, and lectin-like
NKG2/CD94 cells that are found on both human and rodent NK cells
(reviewed in Lanier3). Despite our appreciation of these
different NK cell functions, the developmental relationship of NK cells
with other hematopoietic lineages is not clear. A rare population of
common lymphoid progenitors (CLPs) that can give rise to T, B, and NK
cells has been identified in mouse bone marrow (BM),4 and
NK and T cells have been suggested to derive from a common progenitor
during fetal life.5,6 NK and T cells also share expression
of several differentiation antigens and effector functions, yet the
great majority of T cells are generated in the thymus, whereas the
predominant site for NK cell development is the BM.
Part of the difficulty in understanding the developmental relationship
of NK cells with other hematopoietic lineages stems from our incomplete
knowledge of NK cell ontogeny. Notwithstanding, it is now clear that
development of NK cells is strictly dependent on cytokines that promote
survival, proliferation, and differentiation. Interleukin (IL)-15 plays
a pivotal role in NK cell differentiation, thus NK cells are extremely
reduced or absent in mice deficient for IL-157 or for any
of the IL-15 receptor subunits (IL-15R ,8
IL-2R ,9  ) or its downstream
signaling molecules Jak311 and Stat5.12
However, IL-15 intervenes in a rather late stage of development, when
the commitment to the NK cell lineage has already been made. Although
"early acting" cytokines, including IL-7, stem cell factor (SCF),
and Flk2L/Flt3L, are best candidates for driving the commitment to the
NK cell lineage (reviewed in Williams et al13), the
relative contribution of these growth factors is not fully appreciated.
Therefore, the molecular mechanisms marking NK cell specification are
not completely understood. It is conceivable that these cytokines
activate genetic programs that use multiple transcription factors to
silence or to activate lineage-specific genes. However, although the
transcriptional regulation of lineage commitment during lymphopoiesis
has been quite extensively studied in the context of B- and T-cell
development (reviewed in Glimcher and Singh14), the
transcription factors that control engagement to the NK cell lineage
have only recently started to be identified. Mice deficient for
interferon regulatory factor-1 fail to develop NK cells, due to
impaired transcriptional activation of the Il15
locus.15 The absence of CCAAT/enhancer binding protein (CREB-) impinges selectively on NK cell development and not on B or
T lymphopoiesis.16 Disruption of the Id2 gene results in the block of NK cell development, whereas B- and T-cell differentiation unfolds normally.17 The further
understanding of the transcriptional regulation of NK cells may help to
define stages in which lineage specification takes place, thereby
clarifying the developmental relationship of NK cells with B- and
T-cell lineages.
The Ets (E26 transformation specific) family of oncogenic transcription
factors comprises more than 20 members that are conserved throughout
evolution, regulating cell fate of multiple cell types in worms, flies,
birds, and mammals (reviewed in Bassuk and Leiden18). Several mammalian Ets transcription factors are expressed in the hematopoietic system. Gene-targeting experiments have helped to define
the role of these transcription factors in mouse hematopoietic development. One Ets family member, PU.1 (purine rich box-1) or Spi-1
(spleen focus-forming virus integration site-1) is required for the
differentiation of multiple hematopoietic lineages.19,20 Although specification of erythrocytes and megakaryocytes can occur in
the absence of PU.1, monocytes, mature granulocytes, myeloid-derived
dendritic cells, and B lymphocytes fail to develop in
PU.1/ mice.19,20 T-cell
development is also strongly affected, with few or no T cells
developing in PU.1/ mice19,20
or in fetal thymic organ culture.21 In contrast, because
PU.1/ mice die at E17.5 during embryonic
life19 or just after birth,20 the role of PU.1
in NK cell differentiation has not been examined.
To overcome this limitation, we utilized a recently developed
complementation system in which fetal liver (FL) cells are used as a
source of hematopoietic stem cells (HSCs) to reconstitute alymphoid
Rag2/c/ mice. In these hematopoietic
chimeras, all lymphoid cells are donor derived.22-24 We
found that PU.1/
Rag2/c/ hematopoietic chimeras were
essentially devoid of B and T cells, whereas NK cells could be
generated. Thus, despite sharing a close developmental relationship, B,
T, and NK cells are differentially dependent on PU.1 for their generation.
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Materials and methods |
Mice and generation of FL hematopoietic chimeras
Mice with a null mutation in the common chain
() were from the fourth generation backcross to
the C57Bl/6 background. Rag2/ mice (10th
backcross to C57Bl/6) were bred with B10.BR (H-2k), and F1
progeny were intercrossed to generate RAG-2-deficient mice on the
H-2k background. These mice were then bred with
Rag2/c/ mice22 to generate
Rag2/c/ mice carrying the
H-2k haplotype. PU.1/+
mice19 (C57Bl/6 × 129, H-2b) were screened
by polymerase chain reaction (PCR), using genomic DNA and specific
primers for PU.1 and Neo gene (5'-CGG ATG TGC TTC CCT TAT
CAA AC-3', 5'-TGA CTT TCT TCA CCT CGC CTG TC-3', 5'-CAG AAA GCG AAG GAG
CAA AGC TG-3'). PU.1+/ mice were intercrossed
to generate PU.1/ and control
(PU.1+/+, or PU.1+/,
thereafter referred to as WT) embryos. The morning of the
vaginal plug discovery was designated as day 0.5 of gestation. FL cells were obtained from day 15.5 embryos by passage of the tissue through a
23-gauge needle, and the genotypes were determined by
fluorescence-activated cell sorter (FACS) analysis, using antibodies
specific for Mac-1 (macrophages fail to develop in
PU.1/ embryos).19
Rag2/c/ mice on H-2k
background (> 6 weeks of age) were irradiated with 300 rads from a
cobalt source and 2 hours later injected intravenously with 8, 25, or
75 × 105 FL cells as a source of hematopoietic
progenitors, including HSCs, which we will refer to as
FL-HSCs.
Flow cytometry analysis and cell sorting
Single cell suspensions were prepared from blood, thymus, BM,
spleen, and liver. Erythrocytes were lysed in ammonium chloride, and
cells were resuspended in phosphate-buffered saline with 1% bovine
serum albumin and 0.01% sodium azide. Cell viability was evaluated by
trypan blue exclusion. Monoclonal antibodies (mAbs) directly conjugated
to fluorescein isothiocyanate (FITC), phycoerythrin (PE), Tricolor
(TRI), allophycocyanin (APC), or biotin were used for
immunofluorescence analysis, including mAbs specific for immunoglobulin M (IgM), IgD, T-cell receptor (TCR), TCR , CD3, CD4,
CD8, CD11a (LFA-1), CD11b (Mac-1), CD19, CD45R (B220), CD90 (Thy-1.2), CD117 (c-kit), CD122 (IL-2R), CD161 (NK1.1), DX5, 2B4, Ly49A, Ly49C/I, Ly49D, Ly49G2, Gr-1, TER-119, and H-2k (all from
Pharmingen, San Diego, CA). Biotin-conjugated mAbs were revealed by
streptavidin-TRI (Caltag, Burlingame, CA). Cells (106) were
first incubated with anti-FcRII/III (hybridoma 2.4G2) for 20 minutes
on ice to avoid unspecific binding to low affinity FcRs. Thereafter,
cells were stained with a mixture of biotinylated and
fluorochrome-labeled mAbs at saturating concentrations, washed twice,
and finally incubated with streptavidin-TRI.
To evaluate rare BM progenitor populations, 20 × 106
cells were incubated with purified rat mAbs for lineage (Lin) markers (CD11b, CD19, TER-119, and Gr-1) on ice for 20 minutes. After washing,
cells were incubated with goat antirat IgG coupled with magnetic beads
(Dynal, Oslo, Norway), and the majority of Lin+ cells were
removed on a magnet. Cells were then stained with H-2k (to
detect host-derived cells), washed, and incubated with mAbs specific
for rat IgG, CD3, CD4, CD8, and B220 (all coupled with TRI).
TRI+ cells were subsequently electronically gated, thereby
excluding Lin+ and host-derived precursors.
Analysis was performed on a FACScan or FACScalibur flow cytometer,
using the Cellquest software (Becton Dickinson, San Diego, CA). Dead
cells were excluded by means of their forward and side scatters, and an
electronic gate was set to acquire 104 lymphoid cells.
Cells derived from the host (H-2k) were excluded from the
analysis by electronic gating.
To purify splenic NK cells, cell suspensions were stained with mAbs
specific for NK1.1, and CD3 and NK cells
(CD3NK1.1+) were sorted using a FACStar+
(Becton Dickinson). The purity of the sorted populations was
reproducibility greater than 95%.
NK cell lytic activity
A standard 51Cr release assay was used to measure NK
lytic activity in vitro as described.22 YAC-1 cells (mouse
thymoma; H-2a) were used as target cells and were
maintained in complete medium (CM; RPMI-1640 with 10% fetal calf
serum, 105 M -ME, 100 mg/mL streptomycin, 100 U/mL
penicillin). Target cells were labeled with 100 µCi 51Cr
(ICN Pharmaceutical, Costa Mesa, CA), and 2.5 to 5 × 103
cells were incubated with graded numbers of effector cells in 200 µL
medium for 4 hours. Effector cells were either NK cells that were
isolated from splenocytes by cell sorting or IL-2-activated NK cell
cultures. The radioactivity released into the cell-free supernatant was
measured, and the percentage of specific lysis was calculated as
follows: 100 × (experimental release  spontaneous release)
/ (maximum release spontaneous release).
Reverse transcriptase-PCR and Western blotting
RNA was isolated from freshly sorted NK cell populations with
RNABle (EUROBIO, Les Ulis, France) according to the manufacturer's instructions. Complementary DNA was synthesized, using reverse transcriptase (RT) from avian myeloblastosis virus (PROMEGA, Madison, WI), hexanucleotides, and oligo-dT (Amersham Pharmacia, Uppsala, Sweden). PCR was performed, using Taq Platinum polymerase (GIBCO BRL).
Primer sequences were as follows: PU.1 forward 5'-GAG TTT GAG AAC TTC
CCT GAG-3' and reverse 5'-TGG TAG GTC ATC TTC TTG CGG-3'; Ets-1 forward
5'-CTA CGG TAT CGA GCA TGC TCA GTG-3' and reverse 5'-AAG GTG TCT GTC
TGG AGA GGG TCC-3'; Id2 forward 5'-TCT GAG CTT ATG TCG AAT GAT AGC-3'
and reverse 5'-CAC AGC ATT CAG TAG GCT CGT GTC-3'; IL-7R forward
5'-CTT TTA CGA GTG AAA TGC CTA ACT-3' and reverse 5'-CAG GTA TGA TTC
AAG AAT GCA ATA CA-3'; TCF-1 forward 5'-CTC TGC CTT CAA TCT GCT CAT-3'
and reverse 5'-TGG GTT CTG CCT GTG TTT TCA-3'; hypoxanthine phospho
ribosyl transferase (HPRT) forward 5'-CAC AGG ACT AGA ACA CCT GC-3' and
reverse 5'-GCT GGT GAA AAG GAC CTC T-3' (RNA control).
Proteins were extracted from cell lysates derived from splenocytes,
thymocytes, pre-B cells (cell line 18.81), and purified IL-2-activated
NK cells. Proteins were then resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoreactive
PU.1 protein was revealed with an affinity-purified rabbit anti-PU.1
antibody as described.25
Cell-cycle analysis
Cell-cycle analysis was performed on in vitro IL-2-activated NK
cells and on circulating CD3NK1.1+ NK cells
from peripheral blood, using 7-aminoactinomycin-D (7-AAD) incorporation
into saponin-permeabilized cells as described.23 Sorted
splenic CD3NK1.1+ cells were plated at
104 cells/well in round-bottom microtitre plates in 200 µL CM and activated with 1000 U/mL huIL-2 (Peprotech, Rocky Hill, NJ)
for 7 days. To stimulate NK cell proliferation and to promote
subsequent activation-induced cell death, 2 ng/mL mIL-12 (Peprotech)
was added during the final 48 hours before the cell-cycle analysis.
Statistical analysis
Data were analyzed with the Microsoft Excel software, applying
the paired Student t test. The null hypothesis was rejected, and difference was assumed significant when P < .05.
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Results |
Expression of PU.1 in NK cells
Previous studies have assessed PU.1 expression in CLPs, immature
B- and T-cell precursors, and mature B cells.21,26,27 However, little is known about PU.1 expression in NK cells and its
putative role during NK cell differentiation. We, therefore, tested and
found PU.1 protein in cell lysates derived from sorted NK cells
expanded in IL-2 (Figure 1). As expected,
PU.1 was also found in control lysates derived from total splenocytes
and pre-B cells but not in thymocyte lysates (Figure 1). Thus, mature
NK cells, like B cells and unlike T cells, maintain PU.1 expression throughout development.
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| Figure 1.
NK cells express PU.1.
Cell lysates were generated from thymocytes, splenocytes, pre-B cells
(cell line 18.81), and purified splenic IL-2-activated NK cells.
Protein extracts were resolved by SDS-PAGE and probed by Western
blotting with an affinity-purified anti-PU.1 antibody. Blots were
stripped and reprobed with antitubulin antibody to control for
sample loading.
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Lymphoid cell development in the absence of PU.1
To address the role of PU.1 in NK cell development in vivo, we
generated hematopoietic chimeras by injecting
PU.1/ or WT FL-HSCs
(H-2b) into alymphoid Rag2/c/
(H-2k) mice. In this system,22-24 all lymphoid
cells are donor derived, and any host-derived cell can be identified by
virtue of their differential H-2 expression. Because
PU.1/ FL cells are known to contain fewer
hematopoietic progenitors than control cells,25 the
reconstitution capacity of 3 different doses (8 × 105,
25 × 105, or 75 × 105) of FL-HSCs from
PU.1/ embryos was analyzed and compared to
WT controls. Seven to 13 weeks after the transfer, chimeras were
killed, and the lymphoid cellularity in thymus, BM, spleen, and liver
was evaluated. Splenic T cells (CD3+NK1.1)
were virtually absent in PU.1/ chimeras
(Table 1), and the thymi of
PU.1/ chimeras were hypocellular, containing
almost exclusively early CD4CD8
double-negative thymocytes (Figure 2).
However, in very rare PU.1/ chimeras, we
could detect the 4 populations of CD4+,
CD4+CD8+, CD8+, and
CD4CD8 thymocytes (data not shown),
consistent with a variable penetrance of the T-cell-deficiency
phenotype.20,21 B220+IgM+ B cells
were absent in the spleen (Table 1) and BM of
PU.1/ chimeras (Figure 2), confirming the
essential role of PU.1 in B-cell differentiation.19,20
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| Figure 2.
NK cells but not B and T cells can be generated in the absence of PU.1.
Seven to 13 weeks after transfer of FL-HSCs, cells were isolated from
thymus, BM, and spleen and were stained respectively with mAbs specific
for CD8-FITC and CD4-TRI, IgM-PE and B220-TRI, as well as CD3-APC and
NK1.1-PE. An electronic gate based on morphologic criteria was set to
exclude most nonlymphoid cells. An example of the gating strategy for
lymphoid cell analysis in the spleen is shown. Lymphoid organs of
PU.1/ chimeras contained virtually no T or B
cells, whereas NK cells were present in all chimeras analyzed. Data are
from representative of 8 independent experiments.
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In contrast with the T- and B-cell deficiency,
CD3NK1.1+ NK cells were present in spleen,
liver, and BM of all PU.1/ chimeras analyzed
(n = 16), although they were reduced in absolute numbers
compared to controls (n = 11; Table 1). The kinetics of
peripheral NK cell generation in the absence of PU.1 was somewhat slower than controls (data not shown), a phenomenon already described for the in vivo generation of T cells in the viable
PU.1/ strain20 and the in vitro
generation of lymphoid-derived PU.1/
dendritic cells.28 The data in Table 1 demonstrate that by injecting more PU.1/ FL-HSCs, more NK cells
could be generated, but the NK cell numbers in
PU.1/ chimeras never reached those found in
WT chimeras. Thus, 10-fold less
PU.1/ NK cells were present in the spleens
of chimeras generated with 8 × 105 FL-HSCs, but only 3- to 4-fold less PU.1/ NK cells were present
in the spleens of PU.1/ chimeras generated
with 25 × 105 FL-HSCs. However, T- and B-cell numbers
did not significantly increase with higher doses of FL-HSCs and
remained 400- to 6000-fold reduced compared to controls. The numbers of
NK cells generated with even higher doses of FL-HSCs
(75 × 105) were not greater than those obtained by
injecting 25 × 105 FL-HSCs (Table 1 and data not shown),
suggesting that the reduction of NK cells in
PU.1/ chimeras is not only due to the
reduced frequency of progenitors in the FL of
PU.1/ embryo donors. These results clearly
demonstrate that NK cell development is permissive in the absence of
PU.1.
Reduced production of early NK cell precursors in the absence
of PU.1
HSCs of PU.1/ embryos fail to
express VLA-4/CD49d VLA-5/CD49e-CD11b integrins that has
been hypothesized to result in defective homing to the
BM.29 To directly test whether defects in engraftment of
PU.1/ FL-HSCs in the adult BM could explain
the lower numbers of NK cells in PU.1/
chimeras, we searched for donor-derived HSCs 8 weeks post-transfer. These cells are contained in a population of BM cells that is negative
for the host H-2k, does not express lineage-specific
markers (including CD19, B220, CD3, CD4, CD8, Gr-1, Mac-1, TER-119, and
NK1.1), and is positive for both Sca-1 and c-kit. We found HSCs
(Sca-1+, c-kit+, Lin, and
H-2k) in PU.1/ chimeras
(Figure 3A) and the presence of
donor-derived CD45+ cells at 16 weeks post-transfer (data
not shown). These results rule out a major defect in engraftment or
homing of FL-HSCs in PU.1/ chimeras.
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| Figure 3.
Reduced numbers of donor-derived NK cell precursors in
PU.1/ chimeras.
(A) BM cells of WT and PU.1/
chimeras were isolated, and lineage-positive cells were eliminated by a
combination of magnetic bead depletion and electronic gating. HSCs
(boxed cells) were identified by c-kit and Sca-1 expression as shown.
Percentages were calculated on total BM cells. Data are representative
of 4 independent experiments. (B) Lin-depleted BM cells were in
parallel stained with mAbs specific for DX5-FITC, NK1.1-PE, and
IL-2R-APC. Absolute numbers ± standard deviation of
Lin-depleted BM cells are indicated on top of the dot plots.
Lin-IL-2R+ cells were electronically gated, and their
percentages are indicated. (C) The percentages of gated
Lin-IL-2R+ cells that were positive or negative for
NK1.1 and DX5 are indicated. Data are representative of 3 independent
experiments, including 8 WT and 5 PU.1/ chimeras.
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Collectively, these observations suggest that PU.1 plays an intrinsic
and essential role in early NK cell differentiation. We have recently
identified a cell population in murine BM that appears to represent a
committed NK cell precursor (NKP), having lost any potential for B, T,
or myeloid differentiation (E. Rosmaraki et al, manuscript submitted).
NKPs are Lin and share IL-2R+ expression
with mature NK cells, but in contrast they do not express NK1.1 or DX5.
Eight to 10 weeks after the transfer, we enumerated these
Lin IL-2R+ NK1.1-DX5 NKPs in WT
and PU.1/ chimeras. NKPs were 10- to 12-fold
reduced in the BM of PU.1/ chimeras (Figure
3B,C). This observation suggests that PU.1/
hematopoietic progenitors can only poorly generate the early NK cell
compartment. Accordingly, the numbers of NK cells in older PU.1/ chimeras (> 30 weeks; n = 5)
decline with age to almost undetectable levels (data not shown).
Characterization of PU.1/ NK
cells
The development of hematopoietic progenitors is associated
with dynamic changes in the expression of a number of transcription factors.26,27 The transcription factors Ets-1 and Id2 have been shown to be crucial for NK cell development.17,30
Figure 4A shows that
PU.1/ NK cells express Id2 transcripts and,
interestingly, up-regulate expression of Ets-1 compared to control
NK cells.
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| Figure 4.
Characterization of
PU.1/ NK cells. (A) RT-PCR for
the indicated transcripts was performed, using RNA prepared from sorted
splenic NK cells (CD3NK1.1+) from
WT and PU.1/ chimeras. (B)
Splenic NK cells were stained with mAbs specific for the indicated
surface antigens. Histogram profiles are shown for
CD3NK1.1+ gated cells. Data are
representative of 8 independent experiments.
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We further analyzed the cell surface phenotype of the splenic NK cells
that developed in the absence of PU.1. A series of differentiation
antigens, including NK1.1, DX5, CD2, 2B4, Mac-1, and Thy-1, were
expressed at expected frequencies in PU.1/
NK cells and at levels comparable to those of controls (Figure 4B and
data not shown). The LFA-1 complex is expressed on all leukocytes and
is composed of CD11a and CD18 integrins. CD11a is a putative target
gene of PU.1,31,32 nevertheless
PU.1/ NK cells expressed normal levels of
LFA-1 (Figure 4B). Mature NK cells express inhibitory and activating
members of the Ly49 family of receptors that recognize MHC class I
antigens on the surface of target cells and play a critical role in
regulating NK cell cytotoxic activity (reviewed in
Raulet33). PU.1/ chimeras
contained normal percentages of splenic NK cells expressing the
inhibitory receptors Ly49C/I and Ly49G2 (Figure 4B). However, the NK
cell fractions expressing the inhibitory receptor Ly49A and the
activating receptor Ly49D were clearly underrepresented in
PU.1/ NK cells (Figure 4B). Along these
lines, the Ly49a locus has been shown to contain a consensus
PU.1 binding site (E. Hofer, 18th International NK Cell Workshop,
Marseille, France, May 2000). Reduced Ly49A expression has been
described in mice deficient for the high mobility group transcription
factor TCF-1.34 Figure 4A shows that TCF-1 transcripts
were normally detected in PU.1/ NK cells,
excluding the possibility that PU.1 is an essential regulator of TCF-1 expression.
Mature PU.1/ NK cells fail to
proliferate in response to IL-2 and IL-12
A survival and/or proliferation defect of mature NK cells could
contribute to the reduced absolute numbers of NK cells in PU.1/ chimeras. We, therefore, measured the
percentages of cycling cells and hypodiploid apoptotic cells among
circulating NK cells in PU.1/ chimeras.
Although there was no obvious increase in the proportion of
PU.1/ NK cells undergoing apoptosis (data not
shown), the fraction of NK cells in cycle was significantly lower in
PU.1/ NK cells, as compared with controls
(Figure 5A). IL-2 mediates survival,
activation, and expansion of NK cells in vitro,1 and
addition of IL-12 to IL-2-stimulated NK cells promotes their activation-induced cell death. Purified splenic NK cells from PU.1/ chimeras remained viable throughout
the culture period in IL-2 but did not appreciably expand (Figure 5B).
In addition, they proliferated little in IL-12, although a normal
apoptotic response to IL-2 + IL-12 was preserved (Figure 5C).
Finally, we failed to generate NK cells in vitro from
PU.1/ FL cells (data not shown), using a
combination of cytokines (SCF, Flk2L/Flt3L, IL-7, and IL-2) that drives
NK cell differentiation with high efficiency from WT FL
cells.13,23 Together, these results indicate that
PU.1/ NK cells have defective responses
to cytokines.
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| Figure 5.
Reduced proliferation in
PU.1/ NK cells. (A) Blood cells
were isolated from chimeras 4 weeks after the transfer of FL-HSCs. The
figure indicates the mean and standard deviation of cycling NK cells in
6 WT and 6 PU.1/ chimeras;
*P = .008. (B) Freshly sorted NK cells from spleen of
chimeras were plated at 2 × 104 cells/well and expanded
in IL-2 for 7 days, and thereafter counted. (C) The same cells were
replated at 105 cells/well and further stimulated with
IL-12 overnight. For detection of apoptotic and proliferating NK cells,
cells were stained with mAbs specific for NK1.1-PE and further stained
with 7-AAD to reveal the DNA content. Proliferating cells (in G2/M)
contain more DNA, whereas cells dying by apoptosis are
hypodiploid.
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Expression of growth factor receptors on
PU.1/ NK cells
PU.1 has been implicated in the regulation of cytokine receptor
genes.35 Developing NK cell precursors rely on
Flk2L/Flt3L, SCF, IL-7, and IL-15 to survive, proliferate, and
differentiate (reviewed in Williams et al13). The receptor
for IL-7 is expressed during the early lymphopoiesis, and transcripts
for the IL-7R chain are also found in mature NK cells (Figure
6A). However, PU.1/ NK cells failed to express IL-7R.
Interactions of SCF with its c-kit receptor are essential for expansion
of NK cell precursors and full maturation of NK cells.23 A
small subset of mature NK cells express the c-kit receptor, but this
subset was 6- to 7-fold reduced in PU.1/ NK
cells (Figure 6B). NK cells derived from c-kit-deficient FL-HSCs also
express low levels of the activation marker B220.23
Consistent with the reduction in c-kit expression, the
B220+ NK cell fraction was clearly underrepresented in
PU.1/ chimeras (Figure 6B). Signaling
through the IL-15 receptor is crucial to drive NK cell
development.7-9 This tripartite receptor is composed of
the IL-15R, IL-2R, and c chains (the latter 2 are shared
with the IL-2 receptor and can signal in response to high doses of
IL-2). In line with the capacity of IL-2 to promote survival of
PU.1/ NK cells (Figure 5B), essentially all
of these cells expressed IL-2R and contained RNA transcripts for
IL-15R and c (Figure 6B and data not shown).
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| Figure 6.
Expression of growth factor receptors and activation
markers on
PU.1/ NK cells. (A) RT-PCR for
the indicated transcripts was performed, using RNA prepared from sorted
splenic NK cells (CD3NK1.1+) from
WT and PU.1/ chimeras. (B)
Splenic NK cells were stained with mAbs specific for the indicated
surface antigens. Histogram profiles are shown for
CD3NK1.1+ gated cells. Data are
representative of 8 independent experiments.
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Lytic activity of PU.1/ NK
cells
We have previously observed a correlation between reduced
expression of B220 and defective lytic activity in
c-kit/ NK cells.23 Because B220
and c-kit expression were reduced in PU.1/
NK cells, we assessed the capacity of freshly isolated splenic PU.1/ NK cells to lyse YAC-1 thymoma targets
in a standard 51Cr release assay. As shown in Figure
7, PU.1/ NK
cells were fully competent in lysing this NK-sensitive target. In
addition, IL-2-activated PU.1/ NK cells
could kill both YAC-1 and P815 targets (data not shown). These results
show that differentiation of the lytic machinery for natural cytolysis
does not require PU.1.
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| Figure 7.
PU.1/ NK cells are able to lyse tumor cells in vitro.
Splenic
CD3NK1.1+ NK cells were purified by sorting
and were used as effectors in a classical 51Cr release
assay versus YAC-1 thymoma cells. There was no significant difference
in the lytic capacity of splenic NK cells purified from
PU.1/ ( ) or WT ( ) chimeras.
Data from 2 separate experiments are shown.
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Discussion |
In this report, we demonstrate that NK cells like B but not
T lymphocytes express PU.1, but this transcription factor is not strictly required for the generation of functional NK cells in vivo.
Our studies made use of a novel alymphoid mouse strain, Rag2/c/ mice, which were reconstituted
with PU.1/ FL hematopoietic progenitors. In
this setting, B-cell, T-cell, and NK cell development is entirely donor
derived, and little or no competition with endogenous early lymphoid
precursors is observed.22-24 Moreover, the potentially
lethal effects associated with defective granulocyte and macrophage
development in the absence of PU.1 are avoided since these
hematopoietic lineages develop normally in
Rag2/c/ mice.36 This genetic
approach allowed us to assess the role of PU.1 in NK differentiation.
PU.1/ chimeras remain B and T cell deficient
but generate BM precursors and peripheral functional NK cells, although
both are reduced in numbers.
We show here that NK cells maintain expression of PU.1 throughout
differentiation. Consistent with this finding, mature
PU.1/ NK cells displayed phenotypical
abnormalities, such as reduced expression of certain surface antigens,
including the receptors for IL-7 and SCF, and defective proliferative
responses to potent NK cell mitogens, such as IL-2 and IL-12. However,
PU.1/ NK cells could mediate natural
cytotoxicity and survive in vitro on stimulation with IL-2. Taken
together, our results demonstrate a differential requirement of PU.1
for NK versus B- and T-cell lymphopoiesis.
How do we explain the partial effects of PU.1 deficiency on NK
cell differentiation in the context of the known roles that this
transcription factor plays during hematopoiesis? PU.1 expression appears to be regulated in a complex and dynamic fashion throughout the
hematopoietic system. PU.1 is expressed in HSCs and in common myeloid
progenitors and in CLPs.26 Although the precise effects of
PU.1 deficiency on these hematopoietic subsets remain to be determined,
PU.1 is not required for the generation of HSCs, since this cell subset
could be detected after cell transfer of
PU.1/ FL precursors into
Rag2/c/ recipients. PU.1 is essential for
myeloid differentiation,19,20 yet its absence causes
divergent consequences in distinct cell subsets. Monocytes and
macrophages strictly depend on PU.1 for development,37
whereas neutrophils can develop in PU.1/
mice, although they show defective effector functions.38
Myeloid-derived dendritic cells also fail to develop in the absence of
PU.1, whereas the requirement for PU.1 in development of
lymphoid-derived dendritic cells is controversial, and the expression
of PU.1 in this subset has not been documented.28,39 PU.1
expression is maintained as CLPs differentiate toward the B-cell
lineage but is turned off early in T-cell
development.21,27
The requirement of PU.1 for B-cell development appears absolute, as
shown by the complete absence of fetal and BM-derived B lineage cells
in PU.1/ mice,19,20,25 which we
confirm in this report. PU.1 regulates the expression of the
IL-7R gene during fetal hematopoiesis.14,40 PU.1/ fetal hematopoietic progenitors do not
express transcripts for the B lineage-specific transcription factors
EBF and Pax-5.14,25 Thus, the profound block to B-cell
development caused by the PU.1 mutation may be due to
defects in both IL-7-induced proliferation as well as EBF and
Pax-5-mediated differentiation of B lineage progenitors. PU.1 may
directly regulate the expression of the immunoglobulin
loci.41-43 Interestingly, low levels of PU.1 are essential
for B-cell development, whereas high levels inhibit B-cell
differentiation and instead promote macrophage
development.44
In contrast, PU.1 expression is restricted to a discrete stage of T
lineage differentiation. Only the earliest intrathymic progenitors
(CD44+CD25) express PU.1, which is rapidly
extinguished as T cells mature.21,27 Still, T-cell
development is profoundly impaired in PU.1/
mice.19,20 However, studies using fetal thymic organ
culture have shown that the few thymocytes in
PU.1/ mice that can bypass this
developmental block develop into mature T cells that have normal
function,21 thus PU.1 expression in T-cell differentiation
is essential only during early thymopoiesis. CD44+CD25 cells of fetal thymi contain a
bipotent precursor endowed with both NK and T potential,45
and PU.1 deficiency may block development of these 2 closely related
cell lineages. However, this bipotent precursor may not represent the
main pool of NK progenitors in adult life, when NK lymphopoiesis occurs
in the BM. We show here a reduction of NKP in
PU.1/ BM, whereas their frequency of HSCs
was normal. This suggests a NK lineage-specific role for PU.1, which
may be independent of its role in early T-cell development.
The mechanisms by which the absence of PU.1 disrupts hematopoietic
development are being clarified. A major function of PU.1 is to control
the transcription of growth factor receptor genes in developing blood
cells.14 In the context of myeloid development, evidence
supports a model in which PU.1 is required for expression of the
granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and c-fms
receptors on early myeloid progenitors (see Held et al34 and references therein). However, retroviral infection with c-fms could
only restore the proliferation but not differentiation defect in
PU.1-deficient myeloid precursors, suggesting additional roles for PU.1
in macrophage development.40 Thus, the absence of
expression of certain cytokine receptors during early myeloid and
B-cell development in PU.1 mutants can partly explain the developmental blocks.
In line with this, PU.1 deficiency impaired the expression of some
cytokine and growth factor receptors on developing NK cells. Previous
studies have identified 4 ligand/receptor systems that play an
important role in the generation of NK cells from hematopoietic precursors: Flk2/Flt3, c-kit, IL-7, and IL-15 (reviewed in Williams et
al13). Of these, we found that
PU.1/ NK cells express IL-2R and
transcripts for IL-15R and c. In contrast, IL-7R was absent,
and c-kit expression was reduced on PU.1/ NK
cells. The reduction in numbers of NK precursors seen in
PU.1/ BM may well reflect a synergistic
effect of the defective IL-7R and c-kit expression. In the absence
of c-kit and IL-7R signaling, NK cell development may be sustained by
Flk2L/Flt3L, IL-15, or other cytokines. A similar cumulative defect in
these cytokine receptors may also explain the severe block in early T
lymphopoiesis in PU.1/ mice.46
Thus, the requirement for IL-7 and c-kit in early NK lymphopoiesis may
be less strict than for early thymopoiesis. Mice deficient in both IL-7
and c-kit will help to test this hypothesis.
T-cell progenitors lose the expression of PU.1 as they commit to the T
lineage (CD44+CD25+), whereas NK cells express
it throughout development. In line with this, mature
PU.1/ NK cells are less in cycle, fail to
proliferate in response to mitogens, and do not express a normal
pattern of surface antigens (including Ly49A, Ly49D, and B220) as they
differentiate. Yet they are competent for natural cytolysis of
lymphomas, suggesting </ |
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Abstract
Introduction