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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2024-2031
Treatment of Non-Obese Diabetic (NOD)/Severe-Combined Immunodeficient
Mice (SCID) With flt3 Ligand and Interleukin-7 Impairs the B-Lineage
Commitment of Repopulating Cells After Transplantation of Human
Hematopoietic Cells
By
Ursula Kapp,
Mickie Bhatia,
Dominique Bonnet,
Barbara Murdoch, and
John E. Dick
From the Department of Genetics, Research Institute, Hospital for
Sick Children, Toronto, Ontario, Canada; and Department of
Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario, Canada.
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ABSTRACT |
Until recently, the identification of cellular factors that govern
the developmental program of human stem cells has been difficult due to
the absence of repopulation assays that detect human stem cells. The
transplantation of human bone marrow (BM) or cord blood (CB) into
non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice
has enabled identification of primitive human cells capable of
multilineage repopulation of NOD/SCID mice (termed the
SCID-repopulating cell [SRC]). Here, we examined the effect of
long-term in vivo treatment with various combinations of human
cytokines on the developmental program of SRC. Detailed flow cytometric
analysis of engrafted mice indicated that the vast majority of the
human graft of untreated mice was comprised of B lymphocytes at various
stages of development as well as myeloid and primitive cells; T cells
were not reproducibly detected. Many studies, including murine in vitro
and in vivo data and human in vitro experiments, have suggested that
flt3 ligand (FL) and/or Interleukin-7 (IL-7) promotes T- and
B-cell development. Unexpectedly, we found that treatment of engrafted
mice with the FL/IL-7 combination did not induce human T- or B-cell
development, but instead markedly reduced B-cell development with a
concomitant shift in the lineage distribution towards the myeloid
lineage. Effects on lineage distribution were similar in engrafted mice
transplanted with highly purified cells indicating that the action of
the cytokines was not via cotransplanted mature cells from CB or BM
cells. These data show that the lineage development of the human
graft in NOD/SCID mice can be modulated by administration of
human cytokines providing a valuable tool to evaluate the in vivo
action of human cytokines on human repopulating cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CLINICAL APPLICATION OF hematopoietic
cytokine treatment has increased significantly both in mobilizing
peripheral blood progenitor cells before transplantation and promoting
the generation of mature granulocytic, erythroid, and megakaryocytic
cells after high-dose chemotherapy. However, evaluation of the effects
of cytokines on early human hematopoietic cells has been hampered by
the lack of an in vivo repopulation system that enables detection of
human stem cells. Typically, preclinical studies on the effects of
cytokines involve extrapolation from in vivo studies using other
mammals and in vitro studies with human cells.
We have identified a novel primitive cell, termed the severe-combined
immunodeficient (SCID) mouse-repopulating cell (SRC), which initiates
the human graft after transplantation of human bone marrow (BM) or
umbilical cord blood (CB) by intravenous injection into SCID or
non-obese diabetic SCID (NOD/SCID) mice.1-5 Cell purification and gene-marking studies indicate that the SRC is more
primitive than most clonogenic progenitors and long-term culture-initiating cells (LTC-IC) and the SRC are exclusively found in
the CD34+CD38 cell fraction from human
CB and BM.4,6 Flow cytometric analysis and progenitor
assays from the BM of engrafted mice transplanted with highly purified
cells show that multiple hematopoietic lineages including myeloid,
erythroid, and multilineage progenitors as well as more mature myeloid
cells and B lymphocytes are present, whereas no mature T cells were
detected.6-8 These xenotransplant systems provide the
foundation required for an in vivo system for the preclinical
evaluation of cytokines.
It was necessary in our original studies, using beige/nude/xid (BNX)
or SCID recipients, to treat mice with human cytokines including stem-cell factor (SCF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) to achieve
high-level human cell engraftment.1 However, detailed flow
cytometric studies were not performed on large numbers of these mice.
The increased immunodeficiency and more supportive BM microenvironment of NOD/SCID mice permit a high level of engraftment without the need
for continuous cytokine administration (Gan et al, personal communication). Thus, the NOD/SCID system is ideal to
determine if in vivo cytokine administration can modulate the lineage
development from SRC irrespective of affecting the total level of human
cell engraftment.
We were particularly interested in the effects of cytokines predicted
to act on primitive cells and for combinations that may promote T-cell
development to develop models for HIV and T-cell progenitor assays.
Many studies on murine stem cells, both in vitro9-11 and in
vivo,12,13 and on in vitro analyses of human cells14-17 have suggested that SCF18 and flt3
ligand (FL) act on primitive hematopoietic cells. The tyrosine-kinase receptors for both of these ligands are expressed on
early cells including CD34+ human cells.18-20
Although these two factors alone have little effect on the
proliferation of primitive cells, both are potent synergistic factors
able to induce marked proliferation of both myeloid, erythroid, and
lymphoid lineages.18,21 With respect to lymphocyte
development, murine studies have shown that the combination of FL and
IL-7 induced marked in vitro proliferation of
CD43+B220low B cells and fetal
thymocytes.9,22 Moreover, FL and IL-7 promoted stromal-independent expansion and differentiation of human fetal pro-B
cells in vitro.23 IL-7 alone is an important factor in the
development of both T and B cells,24,25 although some
reports have shown that IL-7 together with other myeloid factors can
support myelopoiesis.26-28
Here, we report that long-term intraperitoneal administration of some
combinations of SCF, FL, IL-3, IL-7, GM-CSF to NOD/SCID mice
transplanted with human hematopoietic cells can modulate the lineage
distribution of the human graft in vivo. Treatment of mice with FL and
IL-7, alone or in combination, did not result in increased numbers of T
cells or B cells, rather there was a marked reduction in the number of
CD19+B cells with a concomitant increase in the proportion
of myeloid cells. Because the results we observed were not predicted
from some earlier in vitro and in vivo murine studies or in vitro human studies, this study shows that the in vivo NOD/SCID repopulation model
can be used to evaluate the biological effect of cytokine treatment on
the developmental capacity of the engrafting human cells within the
complexity of an in vivo setting.
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MATERIALS AND METHODS |
Human cells.
One human BM sample was obtained from a harvest of a normal donor for
allogeneic transplantation in accordance with procedures approved by
the Human Experimentation Committee at the Ontario Cancer Institute
(OCI), Toronto, Ontario, Canada. Mice transplanted with BM cells are
indicated in Fig 1 by circles. Samples of
CB were obtained from discarded placental and umbilical tissues. Both
BM and CB samples were diluted (1 to 3) in Iscove's modified Dulbecco's medium (IMDM; GIBCO, BRL, Gaithersburg, MD) and enriched for mononuclear cells by centrifugation on Ficoll-paque (Pharmacia, Baie d'Urfe, Quebec, Canada).
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| Fig 1.
Effect of cytokine treatment on human cell engraftment in
the BM of NOD/SCID mice. The proportion of CD45+ human
cells present in the BM was determined by flow cytometry 6 to 8 weeks
after the transplant of unseparated mononuclear CB or BM cells. Each
dot represents the level of human cells detected in the BM of a single
mouse. BM was used in one single experiment and indicated by open
circles. Both the frequency of successful engraftment and the
log10 mean level of human cells present in the group of
engrafted mice are shown.
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Purification of cell populations.
As previously described,6 mononuclear cells were stained
with a mixture of lineage-specific antibodies provided by the manufacturer (Stem Cell Technologies, Vancouver, Canada), followed by
addition of secondary antibody conjugated to metal colloid. Cells were
then eluted through a magnetized column to enrich for cells not
expressing lineage markers (Lin). These cell
fractions were then stained with anti-hu CD34-fluorescein isothiocyanate (FITC) and anti-hu CD38-phycoerythrin (PE), and analyzed
and sorted on a FACStar Plus (Becton Dickinson, San Jose, CA) equipped
with 5 W argon and 30 mW helium neon lasers. Fluorescence of FITC and
PE excited at 482 nm (0.38 W) and 633 nm (30 mW), respectively, as well
as known forward- and side-scatter properties of normal live human
hematopoietic cells were used to establish sorting gates. Data
acquisition and analysis were performed using LYSIS II software (Becton
Dickinson). The purity of CD34+Lin cells
after FACS was 98% to 99% and the purity of
CD34+/CD38 cells ranged from 96%
to 98%.
Transplantation of human cells into NOD/SCID mice.
Eight-week-old NOD/LtSz-scid/scid (NOD/SCID) mice were bred from
breeding pairs originally obtained from L. Shultz (Jackson Laboratory,
Bar Harbor, ME), and maintained in the defined-flora animal facility
located at the OCI. All animals were handled under sterile conditions
and maintained under microisolators. Fifteen  20 × 106 unseparated CB or BM cells, 1800 to 6000 CD34+/CD38 cells, or 16 × 10480×104
CD34+Lin cells were transplanted by
tail-vein injection into sublethally irradiated mice (375 cGy using a
137Cs irradiator) according to our standard protocol as
previously described.1,3,6 Human cytokines were injected
every second day intraperitoneally: huSCF, 10 µg, huIL-3 and
huGM-CSF, 6 µg (Amgen, Thousand Oaks, CA); FL, 10 µg and huIL7, 10 µg (Immunex, Seattle, WA). Mice were killed 6 to 8 weeks after
transplantation, and the BM from the femora, tibiae, humeri, and iliac
crests of each mouse was flushed into IMDM containing 10% fetal calf
serum (FCS).
Analysis of human cell engraftment in transplanted mice.
Genomic DNA was isolated from the BM and spleens of transplanted mice
by standard extraction protocols.1 EcoRI-digested DNA was separated by agarose-gel electrophoresis, transferred onto a
positively charged nylon membrane, and probed with a labeled human
chromosome 17-specific  -satellite probe (p17H8). The level of human
cell engraftment was determined by comparing the characteristic 2.7 kb
band with those of human:mouse DNA mixtures as controls (limit of
detection 0.05% human DNA). The presence of human progenitors in the
BM of transplanted mice was determined by plating BM cells in
methylcellulose cultures under conditions that are selective for the
growth of human cells.
Flow cytometric analysis of murine BM.
To prepare cells for flow cytometry, contaminating red cells were lysed
with a 6% ammonium chloride solution and the remaining cells were
washed in phosphate-buffered saline (PBS) containing 5% FCS.
Approximately 106 cells were resuspended in 1 mL of PBS+5%
FCS containing 5% human serum (to block Fc receptors) for 30 minutes
at 4°C, washed, then incubated with monoclonal antibodies at a
concentration of 5 µg/mL for 30 minutes at 4°C. CD45 was
conjugated to PerCP; CD34, CD14, CD20, sIgM, CD3, CD4, and CD15 were
conjugated to FITC; and CD38, CD33, CD19, CD8, CD7, and CD13 were
conjugated to PE. Anti-CD45, -CD34, and -CD38 antibodies were purchased
from Becton Dickinson, the antibody against surface IgM (sIgM) was from
Cedar Lane (Hornby, Ontario, Canada), whereas all other antibodies were
obtained from Coulter (Burlington, Ontario, Canada). Cells were then
washed three times in PBS + 5% FCS and analyzed on a FACScan (Becton Dickinson). For each mouse analyzed, an aliquot of cells was also stained with mouse IgG conjugated to FITC, PE, and PerCP as an isotype
control. BM cells from an untransplanted NOD/SCID were stained in
parallel as an additional negative control. Fluorescence levels
excluding greater than 98% of the cells in these negative controls
were considered to be positive and specific for human staining.
Clonogenic progenitor assays.
Human clonogenic progenitors present in the BM of engrafted mice were
detected by plating mononuclear cells in methylcellulose cultures as
described under conditions selective for the growth of human
progenitors.4
Statistics.
Comparison of treatment groups was performed using the Student's
t test or the Mann-Whitney Rank Sum test, when appropriate. P values below .05 were considered significant. The analyses
were done using the SigmaStat statistical software (SigmaStat for
Windows, Jandel Corp, San Rafael, CA).
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RESULTS |
Effect of cytokine treatment on human cell engraftment.
Groups of sublethally irradiated mice were transplanted with
mononuclear cells from CB or BM and treated with the following cytokine
combinations: FL, IL-7, FL/IL-7, SCF/FL/IL-7, SCF/FL/IL-3, or
SCF/GM-CSF/IL-3 every second day for 6 to 8 weeks. The murine bone
marrow was examined for the presence of human cells by staining with a
human-specific CD45 antibody. The results obtained by flow cytometry
were confirmed by Southern blotting using a human-specific -satellite probe (data not shown). As shown in Fig 1, the frequency of successful engraftment, obtained from analysis of 79 mice from 10 different donors, was similar among the different treatment groups. If
only engrafted mice are analyzed, the mean level of engraftment amongst
the different treatment groups ranged from 4% to 17%, with the lowest
levels of human cells found in the groups treated with FL/IL-7 and
FL/IL-7/SCF. Analysis of 27 mice (from six donors) that were engrafted
with purified CD34+Lin or
CD34+CD38 cells showed similar frequency
and levels of engraftment (data not shown).
Mobilization and/or induction of hematopoiesis in
extramedullary sites such as the spleen has been observed in mice
treated with cytokines, especially by IL-7.29-31 To
determine if the distribution of human cells present in engrafted
NOD/SCID mice could be changed due to cytokine treatment, we analyzed
both the BM and spleen from 20 mice transplanted with three different
donor CB samples that were untreated or treated with FL and FL/IL-7.
The overall level of human cell engraftment of the BM of this untreated
group was somewhat higher than the average shown in Fig 1. There was a
lower level of human cells present in the spleens from these mice.
Similar to the overall data shown in Fig 1, cytokine treatment of this
group of mice resulted in a lower level of human cells in the BM
(Fig 2). Concomitantly, the human cell
engraftment of the spleen was also reduced with cytokine treatment. The
correlation of human cell engraftment in the spleen and BM indicates
that the reduction of human cells in the BM, from the FL- and
FL/IL-7-treated mice, was not accompanied by a redistribution of human
cells to the spleen.
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| Fig 2.
The distribution of human cells in the BM and spleen
after cytokine treatment. Three different CB samples were injected into
20 NOD/SCID mice and treated with the indicated cytokines. The amount
of human cells present in the BM and the spleen was evaluated by DNA
analysis 6- to 8-weeks post-transplant. Log10 mean levels
of human cells are compared. The number of mice in each treatment group
is shown in brackets.
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Effect of cytokine treatment on human myeloid clonogenic progenitor
cells.
To determine if cytokine treatment could affect the number of
progenitors present in treated and untreated mice, progenitor assays
were performed on the BM of engrafted NOD/SCID mice. Total number of
colony-forming cells (CFC) ± standard error
of mean (SEM)/2 × 105 cells in each group were: no
treatment (n = 19), 149 ± 16; IL-7 (n = 10), 124 ± 25; FL (n = 11), 66 ± 30; FL/IL-7 (n = 7), 60 ± 8; SCF/FL/IL-7 (n = 4),
57 ± 25. Thus, the CFC content of the different treatment
groups correlated with the level of human cell engraftment of the BM.
Effect of cytokine treatment on the lineage composition of the graft.
Flow cytometric analysis was performed on human cells present in
engrafted mice from the various treatment groups to determine if
cytokine treatment affected the development of specific lineages (Fig 3). The proportion of lymphocytes,
myeloid cells, and CD34+ cells present in mice transplanted
with mononuclear CB cells and treated with the indicated cytokines was
determined (Fig 3A). Lymphocytes made up the majority of the human
cells in engrafted NOD/SCID mice that were untreated or treated with
IL-7 (Fig 3A). By contrast, mice treated with the combinations FL/IL-7
and FL/IL-7/SCF had significantly decreased proportions of lymphocytes
(average of 15%, compared with 60% in the untreated group) with
concomitant increases in the proportion of myeloid cells (average of
40%, compared with 9% in the untreated group). The mice treated with FL alone or SCF/GM-CSF/IL-3 had equal proportions of myeloid and lymphoid cells, the increase in the proportion of myeloid cells was
significant for both treatment groups. The CD34+ cells
remained similar amongst the treatment groups (~10%). Calculation of
the total number of lymphocytes, myeloid, and CD34+ cells
present in these mice confirmed the reduction in the number of
lymphocytes and shows that the absolute myeloid cell content was not
increased in the FL/IL-7 treatment groups (Fig 3B). Similar trends were
observed in the NOD/SCID mice transplanted with
CD34+Lin or
CD34+CD38 cells indicating that the
effect of the cytokine treatment on lymphocytes was not due to action
on cotransplanted mature cells (Fig 3C).
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| Fig 3.
Determination of the proportion of lymphocytes,
myelocytes and primitive cells in engrafted mice treated with
cytokines. The percentage of lymphoid, myeloid, and CD34+
cells in the region of human CD45+ cells was measured by
flow cytometry for each engrafted mouse in each treatment group. The
lymphoid gate that contained >90% of CD19+ B
lymphocytes and the myeloid gate that contained >90%
CD33+ cells were established based on FSC and
SSC characteristics.6 The histogram shows the
mean ± SEM for each cell fraction. (A) Flow cytometric analysis of
engrafted (>~1% human cells) mice shown in Fig 1. In all cases,
mice were transplanted with mononuclear CB cells except for the
FL/SCF/IL-7 group that also contained two of four mice analyzed, which
were transplanted with 1 BM sample. (B) Total number of cells present
in the BM of the mice shown in A. This calculation was made by
multiplying the percentage of cells with the total cell count obtained
from four long bones. (C) Mice transplanted with purified
CD34+Lin or
CD34+CD38 cells. Statistically significant
differences with the untreated control mice are shown with the *.
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To examine the effect of cytokine treatment on the lymphocyte
development, detailed flow cytometric analysis of engrafted mice was
performed. A representative experiment of four mice transplanted with
mononuclear cells from one CB sample is shown in
Fig 4A and a detailed analysis on all mice
is shown in Table 1A.
Regardless of treatment, mice were engrafted at high levels (11% to
67% CD45+ cells). Without cytokine treatment, B cells made
up 84% of the human cells present in the BM of the engrafted mouse as
indicated by the presence of CD19+ cells. With the
exception of the few mice with mature T cells (data not shown),
normally more than 90% of cells in the lymphocyte gate were
CD19-expressing B cells at different stages in B-cell development.
Approximately, 5% to 10% of the CD19+ cells coexpressed
CD34 indicating the presence of immature pro-B cells (data not shown),
whereas, the detection of CD19+CD20+ cells
shows that human pre-B cells were present (Fig 4A). A low proportion of
these lymphocytes (8%) were mature and expressed sIgM (Table 1).
Treatment with IL-7 had little effect on B-cell development, whereas
the FL and FL/IL-7 groups had significantly lower levels (36% and 7%,
respectively) of CD19+ and
CD19+CD20+ cells in the human graft compared
with untreated mice (84%) (Fig 4A). Similar effects of FL/IL-7
treatment on lymphocyte development were observed in mice transplanted
with purified cells (Fig 4B). The similar reduction in B cells in mice
transplanted with unseparated or purified cells indicates that the
cytokine action on lineage development was not via cotransplanted
nonrepopulating mature cells when whole CB or BM was used. The mice
from all treatment groups in Fig 4 were well engrafted and there was no
correlation between engraftment level and B-cell content showing that
the effect of human cytokine treatment was specific to B-cell
differentiation, rather than a general effect on human cell
engraftment.
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| Fig 4.
Flow cytometric analysis of the human B-cell population
present in engrafted NOD/SCID mice treated with various cytokines. (A)
Four NOD/SCID mice were transplanted with one CB donor and the BM was
analyzed 6-weeks later for the presence of CD19+
and/or CD20+ human B lymphocytes. These analyses
were performed on gated CD45+ human cells. The level of
human cell engraftment is shown in brackets for each mouse. In all
cases the mice were treated with the indicated cocktail of cytokines
every other day for the entire experiment. Lymphocytes (red) were
distinguished from blasts (green) and myeloid cells (blue) according to
their size and morphology using forward and side-scatter
characteristics. (B) Mice were transplanted with
CD34+CD38 cells for the no
GF, SCF/IL-3/GM-CSF, FL/SCF/IL-3 treatment groups, or
CD34+Lin cells for the IL-7/FL treatment
group.
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Mature T cells that express CD3 and express CD4 and/or CD8 were
found sporadically in the BM of 7 out of 63 mice (11%) that were
engrafted with human cells from unseparated CB or BM (data not shown).
However, no T cells were detected in mice transplanted with purified
CD34+/CD38 or
CD34+Lin cells suggesting that some of
the detected T cells could be derived from committed T-cell precursors,
rather than from SRC. The detection of T cells did not correlate to any
particular treatment group. From these data we conclude that human
T-cell differentiation in NOD/SCID mice could not be promoted by
treatment with FL and IL-7.
Detailed analysis of myeloid cells from untreated mice showed that from
15% to 44% of the human cells expressed CD33 when transplanted with
either mononuclear or purified cells, respectively (Table 1A and B).
Significant proportions of CD14+ monocytes and
CD15+ granulocytes were also detected showing various
stages of myeloid cell development in engrafted mice. As the proportion
of CD19+ cells was reduced in the treatment groups, the
proportion of each of these myeloid lineages increased concomitantly by
several-fold indicating that there was no differential increase in one
specific myeloid lineage (Table 1).
To determine if primitive cells were affected by cytokine treatment, we
measured the levels of CD34+ cells present in engrafted
mice. As reported earlier,3,6 the proportion of
CD34+ cells present in the BM of engrafted NOD/SCID mice is
higher (10%) than in the original CB sample (0.5%). No significant
differences were found in the numbers of human CD34+
progenitors comparing the different treatment groups indicating that
long-term cytokine treatment did not alter the already elevated levels
(Table 1, Fig 3).
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DISCUSSION |
The data reported here indicate that long-term human cytokine treatment
can modulate the lineage distribution of human hematopoietic cells
present in NOD/SCID mice transplanted with human unseparated CB or
purified CD34+Lin fractions. B cells
comprised the major population in engrafted NOD/SCID mice that were not
treated with cytokines, confirming recent reports.7,8
Treatment with our standard cocktail of SCF/GM-CSF/IL-3 resulted in
mice in which the human graft was composed of equal proportions of
myeloid cells and B cells. The most dramatic effect of cytokine
treatment was obtained in mice treated with FL/IL-7 or FL/SCF/IL-7 in
which the proportion of human B cells was reduced by fivefold in the
BM, compared with untreated mice. Treatment with FL alone was
intermediate, with equal proportions of myeloid and B cells present,
indicating that FL and IL-7 were acting synergistically to effect
B-cell development in engrafted NOD/SCID mice. The reduced quantity of
B cells in the BM was not due to redistribution to the spleen because
there was no proportional increase in human cells in the spleens of treated mice. Although one of our original goals was to enhance T-cell
development, no cytokine combinations affected T-cell development in
the NOD/SCID mice. Mature T cells were only detected in a small proportion of engrafted mice that were transplanted with unfractionated CB and cytokine treatment did not increase the frequency of mice that
contained T cells. Because T cells were never detected in mice
transplanted with purified CB cell fractions that were depleted of T
cells, it is likely that the T cells occasionally detected in mice
transplanted with unseparated CB are derived from the expansion of
committed T cells that were cotransplanted with the primitive
repopulating cells.
The marked reduction in human B-cell development and the absence of an
effect on T-cell development after in vivo FL/IL-7 treatment of
engrafted NOD/SCID mice was not generally predicted from prior in vitro
murine experiments with these cytokines. For example, IL-7 treatment in
liquid culture of cell fractions, enriched for primitive B- and T-cell
progenitors, stimulated growth and differentiation resulting in the
generation of mature T cells, T-cell progenitors, and B-cell
progenitors.9,17,23-25 However, it is noteworthy that when
primitive Sca+Lin cells were used as the
target, IL-7 promoted myeloid progenitor growth.26
IL-7-gene-deleted mice have reduced numbers of B and T cells
indicating the important role this cytokine plays in lymphoid development.32 Similarly, FL plays an important role in
lymphoid development although it is more pleiotropic and can synergize with many factors to affect virtually all hematopoietic lineages. FL
receptor gene deleted mice show some impairment in the commitment and/or differentiation of pluripotent stem cells and a major
depletion of pro-B cells.12 When FL is combined with IL-7,
in vitro culture studies showed that stimulation of the growth of
murine CD43+B220loCD24
lymphoid cells occurs.22 However, the optimal effect was
seen when stroma was added indicating that some other factors were also
important in promoting B lymphopoiesis. The combination of FL/IL-7 also
stimulated the clonal growth of single
Sca1+Lin cells resulting in B cells
after 12 days.33 However, other studies using
unfractionated murine BM show that FL/IL-7 can stimulate the in vitro
proliferation of clonogenic murine myeloid cells.26,28,33 Taken together, these murine data indicate that although the
predominate action of FL and IL-7 is on stimulation of lymphoid
development, this cytokine combination can also promote the
proliferation of myeloid progenitors under different conditions. It
appears that the specificity of a particular cytokine treatment may be
influenced by the defined conditions of in vitro culture and the nature
of the purified target cells (eg, pluripotential or lineage committed).
The effects of FL/IL-7 on the in vitro culture of human cells have not
been studied as extensively as in murine systems, however, many
similarities exist. FL treatment of CD34+ cells alone
resulted in the long-term maintenance of clonogenic progenitors,
whereas a potent synergistic effect on virtually all lineages was
observed when other cytokines (eg, IL-3, IL-6, EPO, etc) were combined
with FL.16 FL alone is also able to induce the
proliferation of primitive CD34+CD38
cells and improve long-term maintenance of progenitors during liquid
culture.34 Treatment of human fetal
CD34+CD19+ pro-B cells in liquid culture with
FL/IL-7 resulted in promotion of pro-B cell growth and differentiation
into pre-B cells and mature sIgM+ cells. The addition of
IL-3 plus coculture with stromal cells resulted in even greater B-cell
production and higher levels of mature B cells,23
suggesting that additional costimulatory molecules are important for
maximal B-cell stimulation. Interestingly, the combination of SCF and
IL-7 resulted in marked increases in myeloid progenitors, although in
contrast to the murine system, no effects were observed when IL-7 was
used alone. Thus, the combination of cytokines that are added together
with IL-7 in the liquid culture can greatly affect the lineage
specificity of growth stimulation. Overall, this in vitro data would
not have predicted the reduction in B-cell development we observed in
vivo.
Although no in vivo murine studies have been reported on the
coadministration of FL and IL-7, the most directly comparable study to
ours was the report from Brasel et al13 on short-term (10d)
treatment of mice with human FL. The peripheral blood lymphocytes increased by threefold whereas the monocytes increased by 78-fold and
the granulocytes by 10-fold. In the BM, the increases were more modest,
B cells increased about twofold whereas monocytes and granulocytic
cells increased by fivefold. Thus, the increased proportion of myeloid
cells is consistent with our data, whereas the increased B-cell
development was not. Because in vivo systems are inherently complex, it
is difficult to identify a specific explanation for these contrasting
results. There are several possibilities. (A) These data may reflect
inherent differences in the cytokine requirements for B-cell commitment
between murine and human hematopoietic cells. (B) The murine data are
derived from steady-state mice, whereas our NOD/SCID system is a
repopulation system and our mice received a longer duration of
treatment. (C) There may be subtle differences in the response of
primitive human repopulating cells to these cytokines compared with
murine stem cells. Because engraftment in the NOD/SCID recipients is
derived from the primitive human SRC, it is possible that the cytokines
normally present in engrafted mice when combined with FL, direct
myeloid development at the expense of B-cell development. (D) Finally,
because these cytokines are not species-specific it is possible that
they are impacting on the human cells indirectly via the murine
hematopoietic or stromal cells. However, because the murine FL study
showed increases in B cells under similar treatment conditions, one
would need to postulate that the human B-cell commitment is
differentially affected compared with murine B cells by the factors
acting "indirectly."
In conclusion, this study shows the importance of examining the effects
of cytokine treatment on primitive human cells in an in vivo
repopulation setting. Although in vivo systems, such as the NOD/SCID
system, are inherently complex compared with defined in vitro cultures,
they provide a means to ascertain the net biological effect of cytokine
treatment, in the milieu of all the other cytokines and stromal factors
that are present in vivo, on primitive human hematopoietic populations.
This system may be a useful preclinical tool to evaluate the biological
consequence of cytokine treatment on human hematopoiesis being proposed
for clinical trials.
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FOOTNOTES |
Submitted October 2, 1997;
accepted May 11, 1998.
Supported by grants to J.E.D. from the Medical Research Council of
Canada (MRC), the National Cancer Institute of Canada (NCIC) with funds
from the Canadian Cancer Society, the Canadian Genetic Diseases Network
of the National Centers of Excellence, an MRC Scientist award (J.E.D.),
postdoctoral fellowships from the Deutsche Krebshilfe (U.K.), the NCIC
(M.B.), and the Human Frontier Science Organization Program and the
French Cancer Research Association (D.B.).
Address correspondence to John E. Dick, MD, Department of
Genetics, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8; e-mail:
dick{at}sickkids.on.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank S. Lyman at Immunex for gifts of FL and IL7, I. McNiece at
Amgen for SCF, L. McWhirter for providing cord blood specimens, N. Jamal and H. Messner for providing bone marrow samples, and members of
the laboratory for critically reviewing the manuscript.
| |
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Abstract
Introduction
Abstract