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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3489-3497
IMMUNOBIOLOGY
Mice lacking flt3 ligand have deficient hematopoiesis affecting
hematopoietic progenitor cells, dendritic cells, and natural killer
cells
Hilary J. McKenna,
Kim L. Stocking,
Robert E. Miller,
Kenneth Brasel,
Thibaut De Smedt,
Eugene Maraskovsky,
Charles R. Maliszewski,
David
H. Lynch,
Jeffrey Smith,
Bali Pulendran,
Eileen R. Roux,
Mark Teepe,
Stewart D. Lyman, and
Jacques J. Peschon
From the Immunex Corporation, Seattle, WA.
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Abstract |
The ligand for the receptor tyrosine kinase fms-like tyrosine kinase
3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an
important role in hematopoiesis. The flt3 ligand (flt3L) is a growth
factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice
are treated with flt3L immature B cells, natural killer (NK) cells and
dendritic cells (DC) are expanded in vivo. To further elucidate the
role of flt3L in hematopoiesis, mice lacking flt3L (flt3L /) were
generated by targeted gene disruption. Leukocyte cellularity was
reduced in the bone marrow, peripheral blood, lymph nodes (LN), and
spleen. Thymic cellularity, blood hematocrit, and platelet numbers were
not affected. Significantly reduced numbers of myeloid and B-lymphoid
progenitors were noted in the BM of flt3L/ mice. In addition a
marked deficiency of NK cells in the spleen was noted. DC numbers were
also reduced in the spleen, LN, and thymus. Both myeloid-related
(CD11c++ CD8  ) and lymphoid-related
(CD11c++ CD8+) DC numbers were
affected. We conclude that flt3L has an important role in the expansion
of early hematopoietic progenitors and in the generation of mature
peripheral leukocytes.
(Blood. 2000;95:3489-3497)
© 2000 by The American Society of Hematology.
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Introduction |
The continual production of blood cells during an
individual's lifetime is mediated by the coordinated effects of a
number of cytokines acting on hematopoietic stem and progenitor cells in the bone marrow (BM).1,2 The fms-like tyrosine kinase 3 ligand (flt3L) is 1 of the cytokines that affects the development of
multiple hematopoietic lineages.3 These effects are
mediated through the binding of flt3L to the flt3 receptor, also
referred to as fetal liver kinase-2 (flk-2)4 and
STK-1,5 a receptor tyrosine kinase that is expressed on
hematopoietic stem and progenitor cells.4 Flt3L is
expressed as a membrane-bound protein on the surface of cells and can
be proteolytically cleaved to generate a soluble protein. Both the
membrane-bound and soluble forms are biologically active.6
Although a weak growth stimulator as a single factor in vitro, flt3L
synergizes with numerous hematopoietic growth factors to stimulate
myeloid progenitors to proliferate and differentiate.7-11
Flt3L synergizes with interleukin (IL)-7, c-kit ligand (KL),
IL-3, and IL-11 to stimulate B-cell lymphopoiesis in
vitro.12-15 Flt3L augments T-cell development from
BM-derived precursors cultured in the presence of thymic stroma plus
IL-1216 or BM stroma,17 and increases primitive
thymus-derived precursor expansion induced in vitro with IL-3 plus IL-6
plus IL-7.18 In this respect, flt3L shares many functional
properties with KL, augmenting the response of multipotent and
lineage-committed progenitor cells to a variety of cytokines. In
addition to the results obtained from in vitro experiments, studies of
hematopoietic disorders in humans support the concept that flt3L has an
important role in hematopoiesis. Healthy individuals have low levels of circulating flt3L in their plasma.19 However, in the case
of hematopoietic disorders that affect the stem cell compartment (pancytopenias), specifically Fanconi anemia and acquired aplastic anemia, soluble flt3L levels are highly elevated. Flt3L may be produced in an effort to compensate for the deficiency in stem cells in these anemias.19,20
When soluble flt3L is administered to mice, hematopoietic progenitors
in the BM and spleen are expanded, and potent mobilization of stem and
progenitor cells into the peripheral blood (PB) occurs.21 The mobilization includes cells with long-term, multilineage
reconstitution potential, demonstrating that pluripotent stem cells are
mobilized.22 In addition to the effect on stem and
progenitor cells of the myeloid lineage, expansion of immature B cells
is noted in the BM and spleen.21 Splenomegaly and
lymphadenopathy result, and examination of these tissues indicates
that administration of flt3L induces a large increase in the number of
dendritic cells (DC) in the spleen and lymph nodes (LN) as well as in
the PB, lungs, liver, Peyer patches, and thymus.23-25 The
flt3L-expanded DC can process and present antigen to naive T
lymphocytes in vitro and in vivo.23 Administration of flt3L
to tumor-bearing mice results in the generation of specific antitumor
T-cell responses, revealing a potent antitumor activity of the
protein.26-28 The role of the expanded DC in the generation
of this antitumor response is currently under investigation.
Mice lacking the receptor for flt3L (flk-2/) exhibit
hematologic defects.29 Flk-2/ mice have
reduced numbers of B-cell precursors (pro-B cells) in the BM, though
normal numbers of functional B cells are present in the periphery.
Transplantation experiments have revealed a defect at the multipotent
stem cell level. In competitive repopulation experiments, stem cells
from flk-2/ mice transplanted to irradiated recipients do
not effectively reconstitute the hematopoietic system, especially the
T-lymphoid lineage.29 The interaction of KL with its
receptor c-kit is important for regulation of early events in
hematopoiesis. The complete lack of KL or c-kit expression in
mice is lethal, but some nonlethal mutations in this cytokine/receptor
combination have been described.30 These mice (eg,
W/Wv) have severe defects affecting hematopoietic
progenitors and erythropoiesis, in addition to nonhematopoietic
defects. Mice homozygous for mutations in both c-kit and flk-2
(W/Wv flk-2/ mice) display reduced
hematopoiesis and a life expectancy of 6 weeks.29 It was
concluded that flt3L and KL are both required for normal hematopoiesis.
In an effort to further elucidate the role of flt3L in hematopoiesis,
we have generated mice lacking flt3L and have found that
flt3L/ mice have a more profound hematologic phenotype than that noted for flk-2/ mice. Flt3L/
mice have significantly reduced cellularity in a number of
hematopoietic tissues, reduced numbers of myeloid and lymphoid
progenitors in the BM, and reduced numbers of DC and NK cells in the
lymphoid organs.
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Materials and methods |
Targeting of the flt3L gene by homologous recombination and
generation of flt3L-deficient mice
Genomic clones encoding murine flt3L were isolated from a C57BL/6
genomic library (Stratagene, La Jolla, CA) and mapped by a combination
of polymerase chain reaction (PCR) and restriction analyses. A
targeting vector was constructed by replacing a 2.8-kb NotI-SpeI
fragment encoding exons 1 to 5 (amino acids 1-164) with a PGK-neo
cassette. A thymidine kinase cassette (MC-TK) was inserted into the
3' end of the vector. C57BL/6-derived embryonic stem cells were
electroporated with the flt3L targeting construct and selected as
described previously.31 Approximately 1/80 G418 and
ganciclovir-resistant clones carried an flt3L allele disrupted by
homologous recombination as determined by both PCR and genomic Southern
blot analyses.
Flt3L-targeted embryonic stem cell clones were injected into day 3.5 BALB/c blastocysts and transferred to day 2.5 pseudopregnant Swiss
Webster recipients. Resulting male chimeras were bred to C57BL/6
females and offspring were analyzed for germline transmission of the
mutant alleles by PCR and genomic Southern blot analyses. C57BL/6 mice
heterozygous for the flt3L mutation (flt3L +/) were intercrossed
to yield mice homozygous for the mutation (flt3L/). The
lack of flt3L expression in flt3L/ mice was confirmed by PCR analysis of first strand complementary DNA (cDNA) synthesized according to manufacturer specifications (Amersham Pharmacia Biotech, Piscataway NJ) from spleen and BM total RNA and amplified using the
primers 5'-CACCTGACTGTTACTTCAGCC and 5'-CCTGGGCCGAGGCTCTGG, spanning the complete extracellular domain (481 nucleotides) of mature
flt3L.6 Amplified products were analyzed by Southern blot
analysis using a radiolabeled flt3L cDNA probe. Mouse  -actin reverse
transcriptase PCR (RT-PCR) analysis was performed as a control for
uniform RNA integrity and first strand synthesis using the primers
5'-AGGTAGTCCGTCAGGTCC and 5'-CGGAGTCCATCACAATGC.
Mice
Flt3L/ mice, maintained on a C57BL/6 background, were
bred at Immunex Corporation (Seattle, WA). C57BL/6 mice were purchased from Taconic Farms Inc (Germantown, NY), housed for a minimum of 1 week, and used as age- and sex-matched controls. DBA/2 mice were
purchased from Jackson Laboratory (Bar Harbor, ME) and Swiss Webster
mice were purchased from Taconic Farms. Data are presented from mice
aged between 5 and 14 weeks. Mice were housed under specific
pathogen-free conditions.
Tissues
Peripheral blood was harvested by cardiac puncture and collected
into heparinized tubes (Venoject, Fischer Scientific, Pittsburgh, PA).
Hematocrits were performed as previously described.21 White blood cell (WBC) counts, blood differentials, and platelet counts were
analyzed using a Hemavet Multispecies Hematology Analyzer (CDC
Technologies, Oxford, CT). BM cell suspensions were prepared by
flushing cold phosphate-buffered saline (PBS) containing 5% fetal
bovine serum (FBS) (Intergen, Purchase, NY) through isolated femurs.
Cell suspensions were prepared from spleens, LN (2 inguinal plus 2 brachial plus 2 axillary), and thymi by gently breaking up the tissues
between frosted glass slides.
In vitro hematopoietic progenitor assays
Colony-forming units-granulocyte-macrophage (CFU-GM), burst-forming
units-erythroid (BFU-E), and B-cell colony-forming units (B-CFU) lymphoid assays were performed by plating BM cell
suspensions in methylcellulose (Stem Cell Technologies, Vancouver, BC,
Canada) supplemented with 30% FBS (Intergen) and 50 U/mL penicillin,
50 µg/mL streptomycin, and 2 mmol/L glutamine (JRH Biosciences,
Lenexa, KS). Methylcellulose for CFU-GM and BFU-E assays was
supplemented with 100 ng/mL recombinant murine (rmu) IL-3, 200 ng/mL
rmu KL, plus 2 U/mL recombinant human (rhu) erythropoietin. CFU-GM and BFU-E were scored in the same assay. Methylcellulose for CFU-B lymphoid
assays was supplemented with combinations of 50 ng/mL rhu IL-7, 100 ng/mL rhu flt3L, and 200 ng/mL rmu KL. All cytokines were produced and
purified at Immunex Corporation except erythropoietin (R & D Systems,
Minneapolis, MN). Cells were plated in quadruplicate wells in 24-well
plates (Costar, Cambridge, MA) at 0.5 mL/well. Plates were incubated at
37°C, 6.5% CO2. B-lymphoid colonies and myeloid
colonies containing more than 50 cells were scored after 7 and 9 days, respectively.
Colony-forming units-spleen13 (CFU-S13)
assays
The BM cells were isolated from the femurs of 4 to 5 mice per
experiment and pooled. Adult female C57BL/6 mice (9-20 weeks old)
served as recipients and were lethally irradiated (1000 rads) using a
137Cs source (Mark 1 irradiator, J. J. Shephard and
Associates, Glendale, CA) at a rate of 89 to 115 rads/min. BM cells
(2.5 × 104) were injected intravenously (IV) in a
volume of 0.2 mL PBS, 3 to 4 hours after irradiation (n = 10/group).
After 13 days, the mice were killed, the spleens isolated and fixed in
Tellyesniczky solution, and the macroscopic colonies were scored.
Monoclonal antibodies and flow cytometry
The following antibodies were used for flow cytometry: anti-CD3
(145-2C11), anti-NK1.1 (PK136), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), anti-CD11b (M1/70), anti-CD4 (RM4-5), anti-CD8 (53 6.7), anti-CD11c (HL3), anti-IAb (AF6-120.1), (Pharmingen, San
Diego, CA), polyclonal goat-anti-IgM, and anti-IgD (SBA 1) (Southern
Biotech, Birmingham, AL). Isotype-matched controls used included mouse
IgG2a, rat IgG2a, rat IgG2b, and hamster IgG labeled with the appropriate fluorochromes (Pharmingen). Up
to 1 × 106 cells per sample were first incubated in
FACS buffer (PBS containing 2% FBS and 0.02% sodium azide),
containing 2% mouse serum (Biocell, Rancho Dominguez, CA), 2% hamster
serum (Jackson Immunoresearch Laboratories, West Grove, PA) and 10 µg/mL anti-CD16/32 (2.4G2) (Immunex) to minimize nonspecific binding.
Cells were then labeled with the appropriate antibodies for 30 minutes
at 4°C in FACS buffer. Finally propidium iodide (2.5 µg/mL)
(Boehringer Mannheim, Indianapolis, IN) was added to exclude dead cells
from the analysis. Samples were analyzed on a FACScan (Becton
Dickinson, San Jose, CA), or FACSCalibur (Becton Dickinson). Between
20 000 and 100 000 events were collected for analysis. Data were
analyzed using Cellquest software (Becton Dickinson).
Isolation of DC
The DC were enriched as previously described.32 Briefly,
spleens, thymi, and LN (2 inguinal plus 2 brachial plus 2 axillary nodes) were harvested. Spleens and thymi were infused with 100 U/mL of
type III collagenase (Worthington Biochemicals, Lakewood, NJ) and
incubated in 400 U/mL of type III collagenase for 30 minutes at
37°C. Intact LN were incubated in 400 U/mL of type III collagenase for 30 minutes at 37°C. Tissues were further dissociated in
Ca++-free media (Hanks balanced salt solution [HBSS])
(Life Technologies, Grand Island, NY) containing 10 mmol/L EDTA (Life
Technologies), and cells were separated into low- and high-density
fractions on a Nycodenz gradient (density 1.080 gm/mL) (Nycomed Pharma, Distributor in the United States, Life Technologies). The low-density cells, enriched for DC, were harvested from the interface. The cells
were incubated with anti-CD11c and anti-CD8 and the DC purified on
a FACSVantage (Becton Dickinson).
Mixed lymphocyte reaction
The CD4+ T lymphocytes from the LN and spleens of DBA/2
adult female mice were purified as previously described.23
One hundred thousand purified CD4+ T cells were incubated
in 10% CO2 in air at 37°C with serially diluted
purified DC in 96-well round-bottomed plates (ICN Biomedicals, Aurora,
OH) in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS (Life Technologies), 50 U/mL penicillin, 50 µg/mL streptomycin and 2 mmol/L L-glutamine (JRH
Biosciences), 5 × 105 mol/L 2-ME, 0.1 mmol/L
nonessential amino acids, and 1 mmol/L sodium pyruvate (Life
Technologies). On day 4, 0.5 µCi of tritiated thymidine was added to
the wells and after 5 hours the cells were harvested onto glass fiber
sheets for counting on a gas-phase counter.
Cytotoxicity assays
Mice were injected intraperitoneally with PBS or 100 µg of
polyinosinic-polycytidylic acid (poly-I:C) (Sigma, St Louis, MO) in PBS
and their spleens were isolated the following day. Cell suspensions
were prepared and cytotoxicity was measured in a standard 4-hour
51Cr-release assay. Yac-1 cells were labeled with
Na251CrO4 (100 µCi/3 × 106 cells) for 1 hour at 37°C. Serial
dilutions of effector cells were mixed with labeled targets
(5 × 104/well) in 96-well round-bottomed plates and
centrifuged at 400g for 3 minutes. Assays were performed in
RPMI media (Life Technologies) containing 10% FBS (Intergen), 50 U/mL
penicillin, 50 µg/mL streptomycin and 2 mmol/L
L-glutamine (JRH Biosciences),
5 × 105 mol/L 2-ME, 0.1 mmol/L nonessential
amino acids, and 1 mmol/L sodium pyruvate (Life
Technologies). In some experiments, labeled P815 target cells were
included as a control for NK-specific lysis. Cell-free supernatants
were harvested after 4 hours of incubation at 37°C, 5%
CO2 using a cell harvester (Skatron, Sterling, VA). Percent
specific lysis was calculated as ([experimental
release-spontaneous release]/[maximum release-spontaneous release]) × 100.
Statistical analyses
Data were analyzed using a 2-tailed Student t test.
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Results |
Generation of flt3L/ mice
Mice genetically deficient in flt3L (flt3L/) were
generated by gene targeting using a vector that deletes the majority of the flt3L extracellular domain (Figure 1A).
Southern blot analyses using diagnostic restriction digests in
conjunction with probes lying beyond the regions of homology contained
within the targeting vector confirmed that the flt3L gene was disrupted
by homologous recombination (Figure 1B), and a sensitive RT-PCR
analysis confirmed the lack of flt3L expression in flt3L/
mice (Figure 1C). Mice homozygous for the mutation were generated from
crosses of heterozygotes at the expected mendelian frequency, displayed
no overt phenotype, and bred normally. The flt3L/ mice
used throughout these studies were maintained on a C57BL/6 inbred
genetic background.
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| Fig 1.
Generation and molecular characterization of the flt3L
mutation.
(A) Schematic diagram of the wild-type flt3L gene and flt3L targeting
vector. Exons are depicted in filled boxes. Relevant restriction sites,
as well as the probe used for genomic Southern analysis, are shown. The
positions of the initiator methionine (ATG) and transmembrane (TM)
domain are depicted. (B) Genomic DNA from flt3L-targeted embryonic stem
cells as well as representative +/+, +/, and /
mice were digested with XbaI and subject to Southern blot analysis
using the depicted probe. (C) First strand cDNAs derived from either
bone marrow (B) or spleen (S) of +/+ and / adult mice and
a genomic DNA control (G) were amplified using flt3L specific primers
and subjected to Southern blot analysis using a flt3L cDNA probe.
Duplicate samples were amplified using mouse -actin specific primers
and visualized by ethidium bromide staining.
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Flt3L/ mice have reduced numbers of leukocytes in their
hematopoietic tissues and PB
We examined leukocyte cellularity in the PB and a number of
hematopoietic tissues in the flt3L/ mice. Total WBC
numbers were reduced by 45% in the PB from a mean of
11.0 × 106 cells/mL in C57BL/6 mice (flt3L+/+) to
6.1 × 106 cells/mL in flt3L/ mice
(Figure 2A). The relative proportion of
lymphocytes, neutrophils, and monocytes was examined in the PB of
flt3L/ mice using a Hemavet blood analyzer (n = 8). The proportion of monocytes was unchanged, but the proportion of
lymphocytes was significantly reduced from a mean of 84.4% in the
flt3L+/+ mice to 75.8% in the flt3L/ mice. Conversely,
the proportion of neutrophils was significantly increased from 12.1%
in the flt3L+/+ mice to 20.3% in the flt3L/ mice (Figure
2B). This translates to an absolute lymphocyte count of
9.2 × 106/mL in flt3L+/+ PB compared to
4.6 × 106/mL in flt3L/ PB, and an
absolute neutrophil count of 1.4 × 106/mL in
flt3L+/+ PB compared to 1.2 × 106/mL in
flt3L/ PB. The hematocrit (percent packed cell volume) was examined and appeared normal in the flt3L/ mice
(Figure 2C). Platelets were also quantified using the Hemavet blood
analyzer. There was no difference in the mean number of platelets in
the PB of flt3L/ mice compared to flt3L+/+ mice (Figure
2D). Eosinophils in the PB were quantified and no difference was noted
between flt3L/ and flt3L+/+ mice (flt3L/,
4.1 ± 2.6 × 104/mL; flt3L+/+,
2.8 ± 2.5 × 104/ml; n = 8).
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| Fig 2.
Flt3L/ mice have reduced numbers of leukocytes in
the PB.
PB was obtained from flt3L+/+ and flt3L/ mice by cardiac
puncture. (A) WBC counts (n = 8). (B) Blood differentials (n = 8)
(L = lymphocytes, n = neutrophils, M = monocytes). (C) Hematocrit
(% packed cell volume) (n = 7). (D) Platelet counts (n = 8) were
performed as described in "Materials and methods." Data are
presented as the mean ± SD. *P < .0004 for WBC,
P = .0011 for percent lymphocytes, and P = .0026
for percent neutrophils.
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Cellularity of the BM was reduced by 27% in flt3L/ mice
(Figure 3). In flt3L+/+ mice, mean total
leukocyte cellularity from 2 femurs was 47.9 × 106
cells, compared to a mean of 34.8 × 106 cells from
the femurs of flt3L/ mice. Analysis by flow cytometry revealed that the reduction resulted from a decrease in the number of
immature B lymphocytes (B220+, IgM) and
immature myeloid cells (CD11b+, Gr-1+) (data
not shown). Spleen cellularity was reduced by 30% from a
mean of 140.0 × 106 cells/spleen to
98.0 × 106 cells/spleen in flt3L/
mice (Figure 3). LN cellularity was reduced by 35% in
flt3L/ mice, from a mean of
27.7 × 106 cells/6 nodes to
18.0 × 106 cells/6 nodes (Figure 3). Analysis of
spleen and LN cells by flow cytometry revealed that both T cells and B
cells were reduced in number, but that the ratio of T cells (both
CD4+ and CD8+) to B cells was unchanged in the
flt3L/ mice when compared to flt3L+/+ mice (data not
shown). In contrast flt3L/ thymus cellularity was not
significantly affected (Figure 3). Histochemical analyses of spleen,
thymi, and LN revealed no disruption to normal architecture in the
tissues of flt3L/ mice (n = 8, data not shown).
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| Fig 3.
Reduced cellularity in the BM, spleen, and LN of
flt3L/ mice.
BM was isolated from the 2 femurs of each mouse (n = 25 mice) and
cellularity determined. Spleens (n = 14), LN (n = 7), and thymi
(n = 11) were isolated, single-cell suspensions prepared, and cell
counts were performed. LN counts represent cellularity from 6 LN per
mouse (2 inguinal nodes plus 2 brachial nodes plus 2 axillary nodes).
Data are presented as the mean ± SD. *P < .0001 for
BM, P = .0046 for spleen, and P = .0053 for LN.
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We conclude that targeted disruption of the flt3L gene results in
reduced leukocyte cellularity in primary hematopoietic tissue, secondary lymphoid organs, and the PB. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Although leukocyte cellularity was significantly reduced in the flt3L/ mice,
these animals do not appear to suffer an increased frequency of
infection, and their lifespan in a specific pathogen-free animal
facility appears to be equivalent to flt3L+/+ mice (data not shown).
Reduced numbers of hematopoietic myeloid and lymphoid
progenitors in the BM of flt3L/ mice
We hypothesized that the reduced numbers of leukocytes observed in
the periphery of flt3L/ mice resulted from reduced
numbers of lymphoid and myeloid hematopoietic progenitors in the BM.
The frequency and numbers of myeloid precursors were examined in vitro and in vivo. Although the frequency of precursors for the
myeloid lineage (CFU-GM) in the BM was not significantly
different (Figure 4A), the absolute number
of CFU-GM was reduced from a mean of 80.8 × 103/2 femurs in the flt3L+/+ mice to a mean
of 47.2 × 103/2 femurs in the flt3L/
mice (Figure 4B).
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| Fig 4.
Reduced numbers of CFU-GM precursors in the BM of
flt3L/ mice.
BM cells were cultured in methylcellulose in the presence of rmu IL-3
plus rmu KL plus rhu EPO (see "Materials and methods"). (A)
Frequency of clonal CFU-GM progenitors. (B) Absolute number of CFU-GM
per 2 femurs (n = 16 mice). Data were pooled from 4 experiments and
are presented as the mean ± SD. *P = .0012.
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In addition to CFU-GM, the frequency and total number of multipotent
CFU-S13 progenitors in the femurs were determined. BM cells
from flt3L+/+ and flt3L/ mice were injected IV into
lethally irradiated C57BL/6 recipients, and after 13 days, the spleens were isolated, fixed in Tellyesniczky solution, and the macroscopic spleen colonies were scored (Table 1). The
frequency of CFU-S13 progenitors in the BM was not
significantly different in the flt3L+/+ and the flt3L/
mice; however, the absolute number of CFU-S13 progenitors
in the BM of flt3L/ mice was reduced 39% from a mean of
9777 CFU-S13 progenitors per 2 femurs in the flt3L+/+ mice
to a mean of 5951 CFU-S13 progenitors per 2 femurs in the flt3L/ mice.
The numbers of BFU-E were scored as a measure of the number of
erythroid progenitors in the BM. The number of BFU-E per 2 femurs was
slightly reduced in the BM of flt3L/ mice but did not
reach significance (5.8 ± 3.0 × 103 in
flt3L+/+ BM compared to 4.3 ± 2.8 × 103 in
flt3L/ BM; n = 12).
The B-lymphocyte precursors (B-CFU) present in the BM were also
examined in vitro. B-cell precursors responsive to IL-7 alone were
reduced 90% from 13.5 × 103/2 femurs in flt3L+/+
mice to 1.4 × 103/2 femurs in flt3L/
mice (Figure 5). The frequency of
IL-7-responsive precursors was reduced from
28/1 × 105 BM cells in flt3L+/+ mice to
4/1 × 105 BM cells in flt3L/ mice.
B-lymphocyte precursors responsive to the cytokine combinations IL-7
plus KL or IL-7 plus flt3L were reduced 81% and 75%, respectively
(Figure 5). Unlike the myeloid precursors, which were reduced in
absolute number in the BM, but not in frequency, the absolute numbers
and the frequency of B-cell precursors were significantly reduced in
the BM of flt3L/ mice.
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| Fig 5.
Reduced numbers of B-lymphoid progenitors in the BM of
flt3L/ mice.
BM was isolated from the femurs of flt3L+/+ and flt3L/
mice and cultured in methylcellulose supplemented with rhu IL-7 alone
or combined with rmu KL or rhu flt3L to determine the frequency of
CFU-B progenitors. Data are presented as the absolute number of
progenitors per 2 femurs (n = 8 mice). Data were pooled from 2 experiments and are presented as the mean ± SD.
*P < .0001.
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Because the number of B-cell precursors in the BM was severely reduced,
we examined the peripheral B-lymphocyte populations to determine
whether these were affected qualitatively or quantitatively. B cells
present in the spleen and LN expressed normal levels of surface IgM and
IgD (data not shown), and normal levels of circulating IgA,
IgG1, IgG2a, IgG2b,
IgG3, and IgE were present in the serum (n = 7 mice, data
not shown). However, significantly increased levels of IgM
were noted in the serum of flt3L/ mice. The mean concentration of serum IgM in flt3L+/+ mice was 63 ±12 µg/mL, as
compared to 178 ± 66 µg/mL in flt3L/ mice.
Flt3L/ mice were immunized with trinitrophenol-conjugated
keyhole limpet hemocyanin (TNP-KLH) in alum, and 3 weeks later
rechallenged with TNP-KLH. Concentrations of various immunoglobulin
isotypes were measured in the serum after the primary and secondary
challenge. No differences in the concentration of TNP-specific IgA,
IgG1, IgG2a, IgG2b, IgG3, IgE, or IgM were noted between flt3L/
and flt3L+/+ mice (data not shown). In addition, the spleens of
immunized animals were examined for the presence of peanut
agglutinin-positive germinal centers and no differences were noted in
the number formed in the spleens of flt3L/ mice compared
to flt3L+/+ mice (data not shown). These results indicate that although
the numbers of B-cell precursors present in the BM of
flt3L/ mice are severely reduced, peripheral B cells are
present and appear to function normally in response to immunization
with a T-cell-dependent immunization protocol.
Flt3L/ mice have reduced numbers of DC
Administration of flt3L to mice for 9 to 10 days induces a
dramatic expansion of DC in the PB as well as in multiple
tissues23 suggesting a role for flt3L in the development of
DC. We examined the spleens, thymi, and LN of flt3L/ mice
for the presence of DC. To enrich for DC, the tissues were treated with
collagenase and the resulting cell suspension was centrifuged
over Nycodenz (1.080 g/mL) to enrich for low-density cells. Cells were
incubated with antibodies to CD11c and CD8. Myeloid-related DC were
identified as low-density cells expressing high levels of
CD11c and lacking expression of CD8, whereas lymphoid-related DC
were identified as cells expressing high levels of CD11c and CD8.
All CD11cbright cells expressed high levels of major
histocompatibility complex class II (IAb) (data not shown).
Representative examples from the spleen, thymus, and LN are presented
in Figure 6. The absolute numbers of
CD8 and CD8+ DC in each tissue are
presented in Table 2. The numbers of
myeloid-related and lymphoid-related DC were reduced in the spleen,
thymus, and LN of flt3L/ mice. In some experiments the
number of DC present in the LN of flt3L/ mice was below
the level of detection (0.1 × 104/6 LN) (Table 2).
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| Fig 6.
Reduced numbers of DC present in the lymphoid tissues of
flt3L/ mice.
DC were enriched from the spleen, thymus, and LN (n = 6/mouse) by
first treating the tissues with collagenase followed by centrifugation
over Nycodenz to enrich for low-density cells. Cells were incubated
with antibodies against CD11c and CD8. A representative example of 3 to 5 separate experiments is shown in which tissues from 3 to 8 mice
were pooled (see Table 2).
|
|
The CD8+ DC and the CD8 DC
from the spleens of flt3L/ and flt3L+/+ mice
were purified by cell sorting, and their ability to stimulate
proliferation of allogeneic naive T cells was examined in a mixed
lymphocyte reaction (MLR) (Figure 7). The
CD8 DC from the flt3L/ mice were
equivalent to those isolated from flt3L+/+ mice at stimulating
allogeneic T-cell proliferation (Figure 7). The CD8+ DC
were less efficient than the CD8 DC at
stimulating allogeneic T-cell proliferation; however, no difference
between the CD8+ DC from the flt3L+/+ and the
flt3L/ mice was noted. We conclude that the number of
splenic DC is markedly reduced in flt3L/ mice, but the DC
that are present are able to efficiently stimulate naive allogeneic
T-cell proliferation in vitro.
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| Fig 7.
Splenic CD8+ DC and
CD8 DC from flt3L/ mice stimulate allogeneic
T-cell proliferation in an MLR.
Splenic CD8+ DC and CD8 DC from
flt3L+/+ and flt3L/ mice were purified by flow cytometry
(see "Materials and methods"), and cultured in titrating numbers
with 1 × 105 purified allogeneic naive
CD4+ T cells isolated from the spleen and LN of DBA/2 mice.
T-cell proliferation as measured by uptake of tritiated thymidine was
determined on day 4;  indicates flt3L+/+ CD8
DC;  , flt3L/ CD8 DC;  ,
flt3L+/+ CD8+ DC;  , flt3L/
CD8+ DC. Data are presented as the mean ± SD of
triplicate counts. The experiment is representative of 3 separate
experiments.
|
|
Flt3L/ mice are deficient in NK cells
The presence of NK cells (NK1.1high,
CD3) in the spleen was examined by flow cytometry
and yielded an unexpected finding. The number of NK cells in the
spleens of flt3L/ mice was dramatically reduced from a
mean of 4.14 × 106 cells in the spleens of flt3L+/+
mice (range 2.31-6.37 × 106) to a mean of
0.78 × 106 (range
0.39-1.64 × 106) in the spleens of
flt3L/ mice (Figure 8A). The
number of natural T (NT) cells (NK1.1low,
CD3low) present in the spleens of flt3L/ mice
did not appear to be affected (data not shown). Administration of
poly-I:C to mice induces quiescent NK cells to become activated, which
can be quantified by their ability to lyse Yac-1 lymphoma cells.
Flt3L+/+ and flt3L/ mice were injected intraperitoneally
with poly-I:C and the following day their spleens were isolated. NK
cytotoxicity was measured by culturing splenocytes with
51Cr-labeled Yac-1 cells. Poly-I:C treatment stimulated NK
cell activity in flt3L+/+ mice (Figure 8B). Poly-I:C treatment of
flt3L/ mice stimulated a small increase in the percent
specific lysis of Yac-1 cells, compared to the activity observed from
flt3L/ splenocytes from mice treated with PBS. However
the level of killing was below the level of background killing observed
with splenocytes from flt3L+/+ mice treated with PBS. An example of the
levels of specific lysis observed is presented in Figure 8B. We
conclude that targeted disruption of the flt3L gene affects
development of NK cells.
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[in a new window]
| Fig 8.
NK cells are deficient in the spleens of flt3L/
mice.
(A) The number of NK1.1++ CD3 NK cells
present in the spleens of flt3L+/+ and flt3L/ mice
(n = 14) was calculated from the percentage of NK cells detected by
flow cytometry combined with splenic cellularity. Data are presented as
the mean ± SD. *P < .0001. (B) Mice were injected
intraperitoneally with either PBS or 100 µg poly-I:C. The following
day spleens were harvested and a cell suspension prepared (effector
cells). Serial dilutions of effector cells were cultured for 4 hours
with 51Cr-labeled Yac-1 lymphoma cells (targets). Percent
specific lysis of the target cells was estimated as described in
"Materials and methods"; indicates flt3L+/+, poly-I:C
treatment; , flt3L+/+, PBS treatment; , flt3L/,
poly-I:C treatment; , flt3L/, PBS treatment. Data
shown are representative of data generated from 18 mice.
|
|
| |
Discussion |
We have generated mice deficient for the gene encoding the
hematopoietic growth factor flt3L. Although these mice are viable, breed normally, and appear healthy, at least when maintained in a
specific pathogen-free facility, we have observed a number of deficiencies in hematopoietic lineages. Specifically, leukocyte cellularity was significantly reduced in the PB, BM, spleen, and LN and
the numbers of DC and NK cells were dramatically reduced.
Examination of the PB revealed a decreased proportion of lymphocytes
and a corresponding increase in the proportion of neutrophils. The
proportion of monocytes was not changed (Figure 2B). Conversely, erythrocyte and platelet production was not affected in the
flt3L/ mice (Figure 2C and D). In addition to reduced
leukocyte cellularity, decreased numbers of hematopoietic progenitors
were present in the BM (Figures 4 and 5, Table 1). The absolute numbers
of myeloid progenitors were reduced, but the frequency of these cells
was not different from that observed in flt3L+/+ mice (Figure 4, Table 1). Similar observations have been made in W/Wv
flk-2/ mice.29 These mice, which lack flk-2
and have a mutated c-kit receptor, have severely reduced
hematopoiesis, including reduced absolute numbers of progenitors in the
BM, but the frequency of the myeloid progenitors is normal. This
implies that a mechanism to maintain normal myeloid progenitor
frequencies in vivo exists, which does not involve flt3L or KL. In
contrast to the myeloid lineage, both the absolute number and the
frequency of B lymphoid progenitors in the BM were reduced in the BM of
flt3L/ mice, implicating a critical role for flt3L in B
lymphopoiesis (Figure 5). The reduced numbers of lymphoid and myeloid
progenitors in the BM of flt3L/ mice are consistent with
the numerous observations that flt3L synergizes with myeloid and
lymphoid cytokines to augment expansion of these
progenitors.3 Interestingly, mice with mutations in KL
(Sl/Sld) have reduced numbers of hematopoietic progenitors
in their BM, along with reduced BM cellularity, as described here for
the flt3L/ mice, but, unlike flt3L/ mice,
peripheral leukocyte numbers are close to normal.30
The reduced numbers of DC noted in the spleens, LN, and thymus of
flt3L/ mice (Figure 6, Table 2) support our hypothesis that flt3L is an important cytokine in the generation of DC. This hypothesis was based on our previous observations that flt3L
administration to mice for 9 to 10 days induces a profound expansion of
DC in multiple tissues.23 Analysis of DC from the spleens,
LN, and thymus of flt3L/ mice revealed a marked reduction
in both myeloid-related and lymphoid-related DC. However, there were
detectable numbers of DC in the flt3L/ mice, and on
purification the splenic DC were as efficient at stimulating naive
allogeneic T-cell proliferation in an MLR as DC isolated from the
flt3L+/+ mice (Figure 7). As has previously been
described,33 the lymphoid-related DC were less effective
than the myeloid-related DC at stimulating CD4+ T-cell
proliferation. It remains to be determined whether the reduced numbers
of DC will have an effect on infectious agent or tumor cell challenge
in vivo. In vitro studies show that the addition of flt3L to a
combination of cytokines can augment the generation and expansion of
myeloid-related34,35 and lymphoid-related DC.36
A number of other cytokines have been implicated in DC development
including GM-CSF, tumor necrosis factor- (TNF), and transforming
growth factor-1 (TGF1). DC can be generated in vitro with GM-CSF
alone37 or TNF plus GM-CSF,38-40 but mice lacking GM-CSF do not have reduced numbers of lymphoid tissue DC.41 Mice deficient in TGF1 lack Langerhans DC, which
reside in the epithelia, implying a critical role for TGF1 in the
development of this subset of DC.42 One or more of these
cytokines may substitute for the lack of flt3L to generate the residual
DC present in flt3L/ mice.
The flt3L/ mice have a marked reduction in NK cells in
the spleen (Figure 8A) demonstrating that flt3L is critical for NK cell
generation. NK numbers were reduced 5.3-fold and this resulted in an
almost complete loss in the ability of poly-I:C-stimulated flt3L/ splenocytes to lyse Yac-1 lymphoma targets (Figure
8B). Administration of flt3L to C57BL/6 mice results in increased
numbers of NK cells in numerous tissues,43 and
administration of flt3L to severe combined immunodeficient (SCID) mice
(which lack T and B cells) also expands splenic NK cells (D.H.L.,
R.E.M., and K.B. unpublished observation). These flt3L-expanded NK
cells appear to be functional because SCID mice inoculated with
fibrosarcoma cells and treated with flt3L exhibit slower tumor
growth26 and this effect can be abrogated if the mice are
treated in vivo with an anti-NK cell antibody (PK136) (D.H.L. and
R.E.M., unpublished observation).
A number of reports have demonstrated that flt3L has a role in the
expansion of immature B cells that develop in the BM.13-15 Flt3 receptor expression has been detected in the earliest B-cell progenitors, the pre-progenitor B cells, and as development progresses, flt3 receptor expression is down-regulated.44 The
significant reduction in BM-derived B-cell progenitors we noted in the
flt3L/ mice is similar to that described for the
flk-2/ mice.29 However, preliminary
immunization experiments with TNP-KLH plus alum (T-cell-dependent antibody response) indicated that flt3L/ mice generated
TNP-specific antibodies at levels similar to flt3L+/+ mice. Normal
B-cell function was also indicated by the presence of normal levels of
circulating polyclonal immunoglobulins in the serum of unchallenged
flt3L/ mice, with the exception of IgM, which was
elevated (2.8-fold increase). The significance of this is unclear.
Peripheral B cells in the spleen and LN expressed normal levels of
cell-surface IgM (data not shown), and after immunization no
differences in serum IgM concentrations were noted.
Thymic cellularity in the flt3L/ mice was unaffected.
Flt3L is expressed in the thymus45 and primitive thymocytes
express the flt3 receptor4 implying a role for flt3L in
thymocyte development. However, like the flt3L/ mice
described here, the flk-2/ mice have normal thymic
cellularity, though reduced numbers of primitive thymocytes are noted
in neonatal mice.29 Although thymic cellularity was normal,
the absolute number of T cells present in the lymphoid tissues was
reduced. Factors produced by other circulating cells, which are
reduced in the flt3L/mice, may be required to maintain normal numbers of circulating T cells.
There is no evidence that there are other ligands for the flt3
receptor, nor is there any evidence that flt3L binds to any other
protein besides the flt3 receptor. Thus it would be expected that mice
carrying targeted disruptions in either the ligand or the receptor gene
would have an identical phenotype. It is therefore not clear why such
differences are seen between the flt3L/ mice described
here and the flk-2/ mice previously
described.29 We have observed that 10 days of flt3L
administration to flt3L/ mice partially restores DC and
NK numbers (data not shown), confirming that these defects are due
specifically to the flt3L mutation. The DC and NK lineages were not
discussed in the manuscript describing the flk-2/ mice,
so it remains possible that there are defects in these lineages in the
flk-2/ mice. However, the decreased leukocyte
cellularities in the PB and hematopoietic tissues, as well as decreased
numbers of myeloid precursors, were not observed in the
flk-2/ mice, though the more primitive stem cells, as assayed by competitive repopulation, are deficient. The possibility exists that the differences are strain dependent, because the flk-2/ mice were created on a 129 background, and the
flt3L/ mice were created on a C57BL/6 background.
Strain-dependent phenotypes have been described for gene knock-out
mice. Epidermal growth factor receptor knock-out mice have varying
phenotypes and life expectancies depending on the strain in which the
mutation is introduced.46-48
We conclude that flt3L is a multipotent cytokine with effects on the
expansion of hematopoietic progenitors of both the myeloid and lymphoid
lineage. Flt3L appears to be particularly important for the generation
of B-cell precursors, splenic NK cells, and lymphoid tissue DC because
these cell types were the most severely reduced in flt3L/ mice.
| |
Acknowledgments |
The authors acknowledge the expert editorial assistance of Anne Aumell,
the contributions of the animal facility technical staff and Joann
Schuh for reading the histology slides. In addition we thank Doug
Williams for helpful discussions.
| |
Footnotes |
Submitted August 27, 1999; accepted January 31, 2000.
Reprints: Hilary McKenna, Immunobiology Department, Immunex
Corporation, 51 University St, Seattle, WA 98101; e-mail: mckenna{at}immunex.com.
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]
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C. P. Kalberer, U. Siegler, and A. Wodnar-Filipowicz
Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells
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T. E. Toliver-Kinsky, C. Y. Lin, D. N. Herndon, and E. R. Sherwood
Stimulation of Hematopoiesis by the Fms-Like Tyrosine Kinase 3 Ligand Restores Bacterial Induction of Th1 Cytokines in Thermally Injured Mice
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[Abstract]
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K. M. Murphy, M. Levis, M. J. Hafez, T. Geiger, L. C. Cooper, B.D. Smith, D. Small, and K. D. Berg
Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay
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[Abstract]
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M. Prlic, B. R. Blazar, M. A. Farrar, and S. C. Jameson
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G. Miller, V. G. Pillarisetty, A. B. Shah, S. Lahrs, and R. P. DeMatteo
Murine Flt3 Ligand Expands Distinct Dendritic Cells with Both Tolerogenic and Immunogenic Properties
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G. Sailaja, S. Husain, B. P. Nayak, and A. M. Jabbar
Long-Term Maintenance of gp120-Specific Immune Responses by Genetic Vaccination with the HIV-1 Envelope Genes Linked to the Gene Encoding Flt-3 Ligand
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J. Aliberti, O. Schulz, D. J. Pennington, H. Tsujimura, C. R. e Sousa, K. Ozato, and A. Sher
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Blood,
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[Abstract]
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P. Brawand, D. R. Fitzpatrick, B. W. Greenfield, K. Brasel, C. R. Maliszewski, and T. De Smedt
Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs
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S. Frohling, R. F. Schlenk, J. Breitruck, A. Benner, S. Kreitmeier, K. Tobis, H. Dohner, and K. Dohner
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D. G. Gilliland and J. D. Griffin
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R. J. Steptoe, J. M. Ritchie, and L. C. Harrison
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M. Levis, J. Allebach, K.-F. Tse, R. Zheng, B. R. Baldwin, B. D. Smith, S. Jones-Bolin, B. Ruggeri, C. Dionne, and D. Small
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L. M. Kelly, Q. Liu, J. L. Kutok, I. R. Williams, C. L. Boulton, and D. G. Gilliland
FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model
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Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential
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[Abstract]
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J. M. Lean, K. Fuller, and T. J. Chambers
FLT3 ligand can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function
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Absence of the Wild-Type Allele Predicts Poor Prognosis in Adult de Novo Acute Myeloid Leukemia with Normal Cytogenetics and the Internal Tandem Duplication of FLT3: A Cancer and Leukemia Group B Study
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[Abstract]
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F. Colucci, E. Rosmaraki, S. Bregenholt, S. I. Samson, V. Di Bartolo, M. Turner, L. Vanes, V. Tybulewicz, and J. P. Di Santo
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Identification of a leukemic counterpart of the plasmacytoid dendritic cells
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[Abstract]
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S. Gillessen, N. Mach, C. Small, M. Mihm, and G. Dranoff
Overlapping roles for granulocyte-macrophage colony-stimulating factor and interleukin-3 in eosinophil homeostasis and contact hypersensitivity
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[Abstract]
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E. Chklovskaia, C. Nissen, L. Landmann, C. Rahner, O. Pfister, and A. Wodnar-Filipowicz
Cell-surface trafficking and release of flt3 ligand from T lymphocytes is induced by common cytokine receptor {gamma}-chain signaling and inhibited by cyclosporin A
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Top
Abstract
Introduction
