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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3177-3188
Effect of FLT3 Ligand and Granulocyte Colony-Stimulating Factor on
Expansion and Mobilization of Facilitating Cells and Hematopoietic Stem
Cells in Mice: Kinetics and Repopulating Potential
By
Michael Neipp,
Tatiana Zorina,
Michele A. Domenick,
Beate G. Exner, and
Suzanne T. Ildstad
From the Institute for Cellular Therapeutics, Allegheny University of
the Health Sciences, Philadelphia, PA.
 |
ABSTRACT |
We have previously identified a cellular population in murine bone
marrow that facilitates engraftment of highly purified hematopoietic
stem cells (HSC) across major histocompatibility complex (MHC) barriers
without causing graft-versus-host disease. Here we investigated the
effect of flt3 ligand (FL) and granulocyte colony-stimulating factor
(G-CSF) on the mobilization of facilitating cells (FC) and HSC into
peripheral blood (PB). Mice were injected with FL alone (day 1 to 10),
G-CSF alone (day 4 to 10), or both in combination. The number of FC
(CD8+/  TCR /  TCR)
and HSC
(lineage/Sca-1+/c-kit+) was
assessed daily by flow cytometry. Lethally irradiated allogeneic mice
were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF
showed a highly significant synergy on the mobilization of FC and HSC.
The peak efficiency for mobilization of FC (21-fold increase) and HSC
(200-fold increase) was reached on day 10. Our data further suggest
that the proliferation of FC and HSC induced by FL in addition to the
mobilizing effect mediated by G-CSF might be responsible for the
observed synergy of both growth factors. Finally, the engraftment
potential of PBMC mobilized with FL and G-CSF or FL alone was superior
to PBMC obtained from animals treated with G-CSF alone. Experiments
comparing the engraftment potential of day 7 and day 10 mobilized PBMC
indicate that day 10, during which both FC and HSC reached their
maximum, might be the ideal time point for the collection of both
populations. © 1998 by The American Society of Hematology.
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INTRODUCTION |
BONE MARROW transplantation (BMT) is the
treatment of choice for numerous hematologic malignancies1
as well as curative for several nonmalignant hematologic disorders,
including hemoglobinopathies,2,3 aplastic
anemia,4,5 platelet disorders,6 and
deficiencies of soluble enzymes including chronic granulomatous disease
and adenosine deaminase deficiency.7,8 In effect BMT
represents a natural form of gene therapy.9 The replacement
of the defective hematopoietic stem cell (HSC) and its derivatives by
BMT is now also considered as a potential treatment option for the
broad field of autoimmunity.10
In spite of prophylactic immunosuppressive therapy, graft-versus-host
disease (GVHD) remains one of the major limitations in clinical BMT.
Because the effector cells in GVHD are mainly T cells and natural
killer (NK) cells,11,12 GVHD does not occur after
transplantation of purified HSC.13-15 However, while
purified HSC engraft in syngeneic recipients, HSC alone do not engraft in physiologic numbers across major histocompatibility complex (MHC)
barriers.13-15 We have recently identified a cell
population in murine bone marrow that facilitates engraftment of highly
purified HSC in lethally irradiated MHC-disparate allogeneic recipient mice without causing GVHD.14 This facilitating cell (FC)
population is phenotypically characterized by a unique combination of
cell surface molecules, including
CD8+/CD3+/CD45RB+/Thy1+/MHC
class IIdim but
TCR/TCR, and
comprises 0.4% of murine bone marrow.14
There has recently been an increasing interest in the use of mobilized
peripheral blood (PB) progenitor cells (PC) and PB HSC as an
alternative to allogeneic bone marrow grafts. PC and HSC are collected
from the donor in an outpatient setting without general anesthesia, and
reduced engraftment times have been observed. Moreover, larger cell
numbers are available. Another potential advantage reported from animal
models is an enhanced graft-versus-leukemia (GVL) effect after
transplantation of mobilized PB.16 The clinical trials
using granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) mobilized PB
have been encouraging.17-22
A number of hematopoietic growth factors and cytokines including
G-CSF,23,24 GM-CSF,25,26 c-kit
ligand,27,28 interleukin-7 (IL-7),29
IL-8,30 and IL-1231 have shown to expand HSC
and PC in vitro or cause their migration into the periphery in vivo. Another growth factor named flt3 ligand (FL) has now been
identified.32-34 FL binds to its receptor, a member of the
tyrosine kinase receptor family type III. This receptor appears to be
selectively expressed on HSC and progenitor cells.35-37 FL
has been shown to enhance the proliferation of PC and HSC in
vitro32-40 and to mobilize HSC and PC in PB in
vivo.41,42 Interestingly, when FL was used in combination
with G-CSF or GM-CSF, a synergy for PC and HSC mobilization was
observed and mobilized PB showed superior engraftment potential after
transplantation into partially ablated syngeneic murine
recipients.43-45
In the present study we evaluated the ability of FL alone, G-CSF alone,
or the two in combination to mobilize cells of FC phenotype in the
periphery and to study the kinetics of FC and HSC mobilization to
define optimal timing for the collection of both populations. Both
growth factors showed a highly significant synergy on the mobilization
of FC and HSC. The kinetics for mobilization were similar for FC and
HSC, with a peak occurring on day 10. G-CSF alone was not efficient at
mobilizing FC. We further analyzed the distribution of FC and HSC in
hematopoietic sites such as spleen and bone marrow of growth
factor-treated mice at different time points. A dramatic expansion of
both FC and HSC was observed in spleen of FL- and FL + G-CSF-treated animals, whereas no significant changes were detectable
in spleen of mice injected with G-CSF alone. In bone marrow of animals
treated with FL alone, the frequency of FC showed a fivefold increase.
This phenomenon was not observed in animals that received G-CSF alone
or in combination with FL. The engraftment potential of HSC and FC
mobilized by FL and FL + G-CSF into fully ablated MHC-disparate
recipients was superior to that for G-CSF treated donors alone.
| |
MATERIALS AND METHODS |
Animals.
Four- to 6-week-old male C57BL/10SnJ (B10, H-2b) and
B10.BR.SgSnJ (B10.BR, H-2k) mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). Animals were housed in a barrier
animal facility at the Institute for Cellular Therapeutics, Allegheny
University of the Health Sciences, Philadelphia, PA, and cared for
according to specific Allegheny University and National Institutes of
Health animal care guidelines.
Growth factors.
Recombinant human FL was kindly provided by Immunex (Immunex Corp,
Seattle, WA) and diluted in 0.1% mouse serum albumin (MSA; Sigma, St
Louis, MO) at a concentration of 100 µg/mL. Recombinant human G-CSF
was purchased from Amgen (Amgen Inc, Thousand Oaks, CA). Growth factors
were diluted in saline before injections to a total volume of 500 µL,
and B10.BR mice were injected once daily subcutaneously (SC). Mice
received either 10 µg FL/d alone from day 1 to 10, 7.5 µg G-CSF/d
alone from day 4 to 10, or a combination of FL and
G-CSF.42-44 Control animals were injected with saline only.
Tissues.
PB was obtained daily from the tail vein of growth factor-treated
animals. After individual count of peripheral blood mononuclear cells
(PBMC) with a hemocytometer, cells were stained for flow cytometric
analysis to study the kinetics of FC and HSC mobilization. In separate
experiments PB was collected on days 0, 7, and 10 from growth
factor-treated anesthetized animals via cardiac puncture into
heparinized tubes and pooled for each group for reconstitution of
allogeneic recipients. At the same time points, spleens and long bones
were obtained and single-cell suspensions were prepared for flow
cytometric analysis. Splenocytes were isolated by gently flushing the
organ with media 199 (MEM; Life Technologies, Rockville, MD). Red blood
cells (RBC) were lysed using ammonium chloride lysing buffer (ACK;
prepared in our laboratory). Bone marrow was obtained from tibiae and
femurs as described previously.46 Briefly, bones were
flushed with MEM. Bone marrow cells were resuspended and filtered
through a sterile nylon mesh. After centrifugation, cells were
resuspended in MEM and counted.
Monoclonal antibodies.
Anti-H-2Kb-phycoerythrin (PE) (AF6-88.5);
anti-H-2Kk-fluorescein isothiocyanate (FITC) and -Biotin
(36-7-5); anti-GR1-FITC (RB6-8C5); anti-Mac-1 (CD11b)-FITC (M1/70);
anti-CD8-FITC and -APC (53-6.7); anti-CD11b-FITC (M1/70);
anti-B220/CD45R-FITC (RA3-6B2); anti-TCR-FITC and -PE
(H57-597); anti-TCR-FITC and -PE (GL3); anti-NK1.1-PE (PK 136);
anti-Sca-1 (Ly-6A/E)-PE (D7); and anti-c-kit (CD117)-Biotin (2B8)
were purchased from Pharmingen (San Diego, CA). Streptavidin-APC was
purchased from Becton Dickinson (Mountain View, CA).
Detection of FC and HSC by flow cytometry.
The mobilization kinetics of FC and HSC in PB were analyzed daily for
individual animals. Aliquots of 100 µL PB were incubated with
monoclonal antibodies (MoAbs) for 30 minutes on ice. Cells were washed
twice in fluorescence-activated cell sorter (FACS) medium (prepared in
laboratory). Cells labeled with biotinylated MoAb were counterstained
with streptavidin-APC for 15 minutes. RBC were lysed using ammonium
chloride lysing buffer. PBMC were washed twice and fixed in 2%
paraformaldehyde (Tousimis Research Corporation, Rockville, MD). Flow
cytometric analysis was performed using a FACSCalibur (Becton
Dickinson) as described previously.14 For analysis of FC
and HSC, a minimum of 1 × 105 events were collected.
FC were defined as cells residing in a wide lymphoid gate with a dim to
intermediately positive expression of CD8 but negative for expression
of TCR and TCR. For enumeration of HSC, cells positive for
Sca-1 (Ly-6A/E) and negative for lineage markers
(lin) were gated. Gated cells were then analyzed for
their expression of c-kit (CD117).
Lin/Sca-1+/c-kit+ cells were
defined as HSC. Statistical analysis of flow data was performed using
CELL Quest Software, Version 3.0.1 (Becton Dickinson). The percentage
of FC and HSC of total PBMC was determined and the absolute number of
FC and HSC per microliter blood was calculated based on individual PBMC
counts. In addition, the percentage of FC and HSC in spleen and bone
marrow was determined at different time points under treatment with FL
and/or G-CSF.
Reconstitution of allogeneic recipients with mobilized PB.
To investigate the repopulating potential of FC and HSC in mobilized
PB, allogeneic B10 mice were lethally irradiated with a single dose of
950 cGy total body irradiation (TBI; 117.18 cGy/min) from a cesium
source (Nordion, Ontario, Canada). On day 7 or 10 of mobilization, PB
was obtained from B10.BR mice, pooled for each treatment group, and
counted. Three to 5 hours following irradiation, animals were
reconstituted with mobilized whole blood containing 1 × 106, 2.5 × 106, or 5 × 106 PBMC diluted in MEM to a total volume of 1 mL via the
lateral tail vein. Radiation controls as well as control animals
reconstituted with equal numbers of PBMC from unmobilized PB were
prepared. Reconstituted animals were monitored daily to detect failure
of engraftment as indicated by excessive body weight loss, and survival was calculated based on the life-table method. In addition, PBMC counts
were performed 10, 20, and 30 days following reconstitution.
Characterization of chimeras by flow cytometry.
Thirty days and 6 months after reconstitution, recipients were analyzed
for evidence of donor cell engraftment by flow cytometry to detect the
percentage of PBMC bearing H-2Kb (recipient) and
H-2Kk (donor) markers. PB was collected from the tail vein
into heparinized vials. After thoroughly mixing, 100 µL PB was
incubated with H-2Kb-PE and H-2Kk-FITC MoAb
for 30 minutes on ice. RBC were lysed using ammonium chloride lysing
buffer. PBMC were washed twice and fixed in 2% paraformaldehyde
(Tousimis Research Corporation). Lymphocytes, granulocytes, and
monocytes were gated based on forward and side scatter and analyzed for
anti-H-2Kb or anti-H-2Kk expression. PB from
unmanipulated B10 and B10.BR mice served as controls.
To confirm HSC engraftment, the presence of multilineage chimerism was
assessed using three-color flow cytometry 6 months after
reconstitution. PB was obtained and stained with FITC- and PE-labeled
lineage MoAbs and biotinylated H-2Kk MoAb, counterstained
with streptavidin-APC, as described above.
Statistical analysis.
Statistical analyses were performed using unpaired two-tailed
Student's t-test, and P values < .05 were considered
as significant. The 6-month survival of transplanted animals was
assessed using Kaplan-Meier estimates. The 30-day and 6-month survival
of different groups was compared using the Wilcoxon test, and P
values < .05 were considered as significant.
| |
RESULTS |
Kinetics of mobilization of FC and HSC in PB.
We evaluated the effect on the total number of PBMC in PB after
administration of FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or
a combination of both growth factors. Animals treated with G-CSF and FL
alone showed a threefold and fourfold increase of PBMC, respectively
(Fig 1A). Combined administration of both growth factors showed a synergistic effect, because PBMC increased significantly and a maximum (22-fold increase) was observed on day 10. PBMC of animals injected with carrier only remained at baseline levels.
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| Fig 1.
Kinetics of mobilization of PBMC, HSC, and FC under
treatment with FL alone ( ), G-CSF alone ( ), FL + G-CSF ( ),
or carrier ( -). FL (10 µg/mouse) was injected SC for 10 days and
G-CSF (7.5 µg/mouse) from day 4 to 10. (A) PB was obtained daily and
PBMC were counted. The percentage of HSC
(lineage/Sca-1+/c-kit+) and
FC
(CD8+/TCR/TCR)
was analyzed by flow cytometry, and absolute numbers of (B) HSC and (C)
FC were calculated based on individual PBMC counts. Results represent
the mean (SEM) of two different experiments (n = 5 per group). PBMC
or absolute numbers of FC and HSC that differed significantly from
controls are marked (* P < .005 or ** P < .0005).
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To assess the potential of growth factor administration on mobilization
of FC and HSC, the absolute number of FC and HSC under treatment with
FL alone, G-CSF alone, and a combination of FL and G-CSF was determined
(Fig 1B and C). Although G-CSF alone resulted in a 17-fold increase of
primitive HSC, only a modest effect on the mobilization of FC was
noted. In contrast, FL as a single agent caused a 7-fold increase of FC
and 36-fold increase of HSC, respectively, and peak levels for both
populations occurred on day 9. A maximal elevation of both FC and
primitive HSC was detectable when both growth factors were combined. An
increase of HSC was detectable on day 6, and a plateau reflecting a
more than 200-fold increase or an absolute number of approximately 400 HSC/µL PB was reached from day 9 to 11. The number of cells of FC
phenotype increased on day 5 and a peak level representing a 21-fold
increase was observed on day 10. In contrast to HSC, the number of FC
declined rapidly after day 10.
Because the majority of cells in PB after FL + G-CSF treatment were
neutrophilic granulocytes, the relative percentage of CD8+
T cells decreased significantly from 8.7% on day 0 to 1.3% on day 10 (Fig 2). In striking contrast, the
percentage of FC remained at a constant level, whereas an increase in
the percentage of HSC from 0.01% on day 0 to 0.36% on day 10 was
observed. A similar observation was made when mice were treated with FL
alone. CD8+ T cells in G-CSF-treated animals showed only a
slight decrease (8.7% to 5.4%), and no significant changes in the
percentage of FC and HSC were observed.
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| Fig 2.
Percentage of HSC, FC, and CD8+ T cells in
PB during treatment with (A) FL alone (n = 5), (B) G-CSF alone (n = 5), or (C) FL + G-CSF (n = 5). FL (10 µg/mouse) was injected SC
from day 1 to 10 and G-CSF (7.5 µg/mouse) from day 4 to 10. PB was
stained for HSC ( )
(lineage/Sca-1+/c-kit+), FC
()
(CD8+/TCR/TCR),
and CD8+ T cells ( )
(CD8+/TCR+). Results show the mean
(SEM) percentage before and on day 7 and day 10 of growth factor
administration. Percentages of HSC, FC, or CD8+ T cells
that differed significantly from day 0 values are marked (* P < .05; ** P < .005; or *** P < .0005).
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Distribution of FC and HSC in spleen and bone marrow of mice treated
with growth factors.
To address whether the observed increase in absolute numbers of FC and
HSC in PB was due to mobilization of preexisting cells or due to de
novo hematopoiesis, splenocytes and bone marrow cells from FL-,
G-CSF-, and FL + G-CSF-treated mice were analyzed by flow cytometry,
and percentages of FC and HSC were determined. Mobilization of mature
cells from the bone marrow into the periphery occurred in all growth
factor-treated animals as indicated by a significant reduction of the
percentage of CD8+ T cells (Fig
3A, B, and C). In the bone marrow of animals that received G-CSF alone,
only a marginal increase in the percentage of HSC was present, whereas
the frequency of FC decreased significantly during mobilization. In
animals treated with FL and G-CSF, a significant increase in the
percentage of HSC was observed on day 7. However, on day 10 of
mobilization, the frequency of HSC in bone marrow decreased to baseline
levels. Interestingly, in mice treated with FL alone an 18-fold and
5-fold increase in the percentage of HSC and FC was detected,
respectively, indicating proliferation and/or lack of
mobilization of FC and HSC in the absence of G-CSF.
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| Fig 3.
Distribution of HSC, FC, and CD8+ T cells
in (A through C) bone marrow and (D through F) spleen of B10.BR mice
treated with FL alone (10 µg/mouse; day 1 to 10); G-CSF alone (7.5 µg/mouse; day 4 to 10); or FL + G-CSF. Animals (n = 6 per group)
were euthanized before, on day 7, or day 10 of growth factor
administration. Long bones and spleens were obtained and processed for
each individual animal. Bone marrow cells and splenocytes were analyzed
for the percentage of HSC ()
(lineage/Sca-1+/c-kit+), FC
()
(CD8+/TCR/TCR),
and CD8+ T cells ()
(CD8+/TCR+) by flow cytometry.
Results represent the mean (SEM) percentage on total bone marrow and
total splenocytes. Percentages of HSC and FC that differed
significantly from day 0 values are marked (* P < .05 or
** P < .005).
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In spleen, the frequency of both FC and HSC increased over time under
treatment with FL alone or FL in combination with G-CSF (Fig 3D and F).
Cells of FC phenotype increased significantly from 1% on day 0 to 11%
on day 10 in animals treated with FL alone and from 1% on day 0 to 7%
on day 10 in animals that received a combination of both growth
factors. The percentage of primitive HSC in spleen of FL- and FL + G-CSF-treated mice showed a 20-fold (0.09% on day 0 to 1.79% on day
10) and an 18-fold increase (0.09% on day 0 to 1.62% on day 7),
respectively. In striking contrast, under treatment with G-CSF alone,
the percentage of FC remained unchanged over time, whereas the
frequency of HSC was slightly elevated (Fig 3E).
Short-term engraftment potential of mobilized PBMC in allogeneic
recipients.
To determine the short-term engraftment potential of HSC and FC
mobilized by treatment with FL, G-CSF, or FL + G-CSF, allogeneic B10
mice were lethally irradiated and reconstituted with whole PB
containing varying numbers of PBMC. Recipients were transplanted with
either 1 × 106, 2.5 × 106, or 5 × 106 PBMC from donors treated
with growth factors for 7 or 10 days. The cell number of mobilized HSC
and FC per kilogram body weight of recipients is shown in
Table 1. Control animals received equal amounts of PBMC from untreated B10.BR mice. The 30-day survival of
transplanted animals as a function of PBMC dose and time-point of
collection of PB is shown in Fig 4. Animals
reconstituted with 1 × 106 PBMC collected
on day 7 from donors treated with FL, G-CSF, or FL + G-CSF showed a
33% survival at day 30. Control animals injected with 1 × 106 PBMC from untreated donors died within 12 days from
irradiation-induced aplasia (Fig 4A). At a cell dose of 2.5 × 106 PBMC, the 30-day survival rate increased to 67% after
treatment with FL alone and 100% after treatment with FL + G-CSF. No
significant difference between both cell doses was observed for the
G-CSF treatment group and control group (Fig 4B). The 30-day survival of irradiated recipients was superior when PB from FL- or FL + G-CSF-treated animals was collected after 10 days of growth factor administration. As few as 1 × 106 PBMC mobilized with
FL alone rescued more than 80% of recipients, whereas with FL + G-CSF, 100% of transplanted animals survived (Fig 4C). At a PBMC
dose of 2.5 × 106, 100% of animals transplanted with
FL- and FL + G-CSF-mobilized PB were alive after 30 days (Fig 4D).
When 1 × 106 and 2.5 × 106 PBMC
mobilized with G-CSF as a single agent were given, the 30-day survival
rate was only 20% and 33%, respectively. All control animals injected
with PB from untreated donors died within 14 days from TBI-induced
aplasia. Irradiation controls that received 950 cGy TBI without PBMC
injection died within 10 days (data not shown). When the dose of PBMC
collected on day 7 or day 10 was further increased to 5 × 106, no further improvement of the 30-day survival rate was
observed for all treatment groups (data not shown).
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| Fig 4.
Survival (30 days) of lethally irradiated recipients
(B10) transplanted with mobilized PB from donor mice (B10.BR). Donors
were treated once daily with FL alone (), G-CSF alone (), FL + G-CSF (), or carrier only ( ). PBMC were obtained from donors
after 7 days (A and B) or 10 days (C and D) of growth factor
administration and pooled for each group. Recipients were injected with
1 × 106 or 2.5 × 106 PBMC 3 to 5 hours
after irradiation (4 to 7 mice per group). There was a significantly
greater survival of mice reconstituted with PBMC from FL- and FL + G-CSF-treated donors when compared with unmobilized or
G-CSF-mobilized PBMC. Differences between groups that reached
statistical significance are marked (a: FL + G-CSF v control,
P <0.05; b: FL + G-CSF v G-CSF, P < .05;
c: FL v control, P < .05; d: FL + G-CSF v
control P < .005; e: FL + G-CSF v G-CSF, P
<0.05; f: FL v control, P < .005; g: FL v
G-CSF, P < .05).
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To study the time course of HSC and PC engraftment, PBMC from
individual animals transplanted with mobilized PB collected on day 10 were counted 10, 20, and 30 days following reconstitution. Engraftment,
defined as a PBMC count of  500 PBMC/µL, was observed in 67% of
recipients 20 days after reconstitution with 1 × 106
and 2.5 × 106 PMBC from FL- and FL + G-CSF-treated
donors (Table 2). After 30 days, 100% of
these animals had a PBMC count of 500 PBMC/µL. When 5 × 106 PBMC from FL- and FL + G-CSF-treated
animals were injected, engraftment occurred as early as on day 10 in 1 out of 3 and 3 out of 3 recipients, respectively. In striking contrast,
animals reconstituted with equal amounts of PBMC from G-CSF-treated or
unmanipulated donors did not present PBMC counts of 500 PBMC/µL at
any of the time points tested (data not shown).
Flow cytometric analysis of PB obtained from transplanted animals 30 days following reconstitution was performed, and the lineage derivation
of PBMC was determined based on cell size and granularity. To exclude
contamination with radio-resistant or repopulating cells of host
origin, PB was stained with MoAbs specific for host (H-2Kb)
and donor (H-2Kk) MHC class I antigen. In engrafted
recipients 91.2% ± 4.0% of PBMC were located in the granulocyte
gate, whereas 5.6% ± 3.1% and 0.5% ± 0.1% of PBMC resided
in the lymphocyte or monocyte gate, respectively
(Fig 5). More than 95% of PBMC were of
donor origin.
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| Fig 5.
Flow cytometric analysis of PB obtained from a
representative chimera 30 days after reconstitution with mobilized PB.
B10 mice (H-2Kb) were lethally irradiated and transplanted
with varying numbers of PBMC from growth factor-treated B10.BR donors
(H-2Kk; 4 to 7 mice per group). PB from unmanipulated B10
and B10.BR mice served as controls. Lineage derivation of PBMC was
analyzed based on forward and side scatter, and the percentage of cells
residing in a lymphocyte (R1), monocyte (R2), or granulocyte gate (R3)
was calculated. (A) The majority of PBMC in engrafted recipients were
located in the granulocyte gate, (B and C) while most of PBMC from
untreated controls resided in the lymphocyte gate. (D through F) In
addition PB was stained with MoAb specific for recipient
(H-2Kb) and donor (H-2Kk) MHC class I, and
gated populations were analyzed by two-color flow cytometry. (D) Gated
lymphocytes from engrafted recipients expressed exclusively donor MHC
class I. Positive staining for donor but negative staining for
recipient MHC class I was also observed when gated granulocytes and
monocytes were analyzed (data not shown).
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Long-term engraftment of mobilized HSC in allogeneic recipients.
Long-term survival (>6 months) was 79% and 67% in animals
transplanted with PBMC from FL- and FL + G-CSF-treated donors,
respectively (Fig 6). This survival rate
was comparable to that of recipients (n = 25) reconstituted with 1 × 106 untreated bone marrow cells from naïve
B10.BR donors. The majority of recipients reconstituted with mobilized
PB developed clinical signs of acute GVHD within 30 to 60 days after
transplantation as indicated by diarrhea and loss of body weight.
However, GVHD was self-limiting in most of these animals. In striking
contrast, long-term survival of animals transplanted with PBMC
mobilized with G-CSF alone was significantly lower, and the estimated
survival after 6 months was only 13%. None of the recipients
transplanted with PBMC from carrier-treated B10.BR donors survived for
more than 14 days.
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| Fig 6.
Long-term survival (>6 months) of lethally irradiated
and transplanted recipients was calculated using Kaplan-Meier
estimates. B10 mice received 1 × 106 to 5 × 106 PBMC from B10.BR donors treated with FL alone, G-CSF
alone, or FL + G-CSF (n 6 per group). Controls were
transplanted with similar numbers of PBMC from untreated donors or
1 × 106 bone marrow cells. Survival between
different groups was compared using the Wilcoxon test, and significant
differences are marked (* P < .0001). The follow-up ranged
from 3 to 6 months.
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Representative animals transplanted with PB from donors treated with
growth factors for 10 days were analyzed after 6 months, and the level
of donor chimerism as well as the presence of multiple hematopoietic
lineages was assessed by flow cytometry using lineage-specific MoAbs.
All long-term surviving animals tested showed >99% donor chimerism
and multiple lineages including B-cells, TCR+ T
cells, TCR+ T cells, NK cells, macrophages, and
granulocytes of donor origin were present in these animals
(Table 3 and
Fig 7). Percentages of analyzed
donor-derived cell lines were comparable to those of naive B10.BR mice.
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Table 3.
Characterization of Long-Term Surviving Animals (B10)
Reconstituted With Mobilized PB From B10.BR Donors Treated With FL
Alone, G-CSF Alone, or FL + G-CSF
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| Fig 7.
Assessment of long-term engraftment of mobilized HSC and
FC by three-color flow cytometry. PB was obtained from lethally
irradiated B10 mice (H-2Kb) 6 months after reconstitution
with PBMC from growth factor-treated B10.BR mice (H-2Kk)
and stained with lineage- and donor-specific MoAbs. Unmanipulated B10
and B10.BR mice served as controls (data not shown). Figure shows
results of a representative long-term surviving chimera. (A)
Lymphocytes (R1) and granulocytes/macrophages (R2) were gated based on
forward and side scatter. Engraftment of multiple donor derived cell
lines including (B) B-cells, (C) T cells, (D) NK cells, (E)
granulocytes, and (F) macrophages were detectable 6 months after
transplantation indicating HSC engraftment.
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| |
DISCUSSION |
It has been previously shown that FL mobilizes large numbers of PBMC
into the circulation of mice.41,42 A maximum effect was
observed when FL was injected at a daily dose of 10 µg subcutaneously for 10 days, resulting in an increase of PBMC to 4 × 104/µL. In subsequent experiments a synergistic effect
was observed when FL was used in combination with
G-CSF.44,45 In these studies both FL and G-CSF were
initiated on day 1, and a peak of PBMC as high as 1 × 105/µL was observed on day 8. FL in combination with
GM-CSF was much less effective.44 Because it was previously
reported that PBMC mobilized by G-CSF alone peaked on day 5 to
6,24 we initiated G-CSF treatment on day 4 to allow for
maximal synergy of both growth factors. In fact, a peak of PBMC under
treatment with FL + G-CSF was detected on day 10 and an average of 1.75 × 105 PBMC/µL was counted. This peak represents an
almost twofold increase in PBMC numbers when compared with the
experiments performed by Brasel et al.44 Thus, we show in
this study that optimized timing of growth factor administration can
further enhance the synergy between G-CSF and FL.
However, the peak of PBMC does not necessarily reflect the ideal time
point for the collection of the desired cellular population from
mobilized PB. Therefore, we were mainly interested in the kinetics of
mobilization of FC and HSC. FC have been previously shown to be
critical in engraftment of murine allogeneic HSC across MHC
barriers.14,47,48 Although in our previous studies 1,000 HSC (lineage/Sca-1+/c-kit+)
purified from bone marrow engrafted routinely in lethally irradiated syngeneic mice, even a 10-fold increase in HSC failed to rescue allogeneic recipients from irradiation-induced aplasia. When as few as
30,000 FC (CD8+/TCR/CD3+)
positively selected by cell sorting were added to 10,000 purified HSC,
100% of allogeneic recipients engrafted and none of these animals
developed GVHD.14
When G-CSF alone was injected, the number of cells with facilitating
phenotype
(CD8+/TCR/TCR)
in PB of those animals was not increased significantly compared with
carrier-treated controls. In contrast, FL as a single agent elevated
(sevenfold) the absolute numbers of FC over time, and a peak occurred
on day 9. Mobilization of FC by FL + G-CSF resulted in a highly
significant synergy. Beginning on day 5, a continuous increase of FC
was observed and a maximum (21-fold over controls) was reached on day
10. Interestingly, a similar pattern was observed for mobilization of
HSC. When both factors were used in combination, a more than 200-fold
increase of HSC occurred from day 9 to 11. G-CSF alone and FL alone
were less effective (17-and 36-fold). Previous work has also shown that
certain inbred mouse strains, including C57BL mice, show a limited
elevation of progenitor cells in PB after mobilization with G-CSF
alone.49 Our results in terms of HSC mobilization are in
accordance with data presented by others identifying HSC/progenitor
cells based on in vitro colony assays.43-45 In these
studies a synergy of FL and G-CSF on the mobilization of burst-forming
unit-erythroid (BFU-E), colony-forming unit-granulocyte-macrophage
(CFU-GM), colony-forming unit-granulocyte, erythroid, monocyte,
megakaryocyte (CFU-GEMM), and colony-forming unit-spleen (CFU-S) into
PB was observed.43-45 However, different doses and timing
of growth factor administration as well as different methods to assess
the frequency of HSC make a direct comparison difficult.
Preliminary data from our laboratory suggests that FC may provide a
tropic effect to maintain the HSC in a primitive state. Although HSC
alone undergo apoptosis in vitro, the addition of FC maintains the HSC
in G0 (manuscript in preparation). The fact that the kinetics for mobilization of FC and HSC are similar may suggest that the two are in close proximity in the hematopoietic microenvironment.
To understand the mechanism for the observed synergy of both growth
factors on the mobilization of HSC and FC, we assessed the frequency of
HSC (lineage/Sca-1+/c-kit+)
and FC (CD8+/TCR) in spleen and bone
marrow under treatment with both growth factors alone or in
combination. As shown previously, G-CSF mobilized both mature and
progenitor cells into the periphery as indicated by declining
cellularity in bone marrow.24 The latter might be
responsible for the slightly increased frequency of HSC in bone marrow
observed in our G-CSF-treated animals rather than proliferation of
those cells. Interestingly, when FL was used as a single agent, a
highly significant increase in HSC (18-fold) and FC (5-fold) in bone
marrow was observed on day 10. This is in accordance with results from
Brasel et al,42 who reported 8.2-fold higher numbers of
low-density
lineage/Sca-1+/c-kit+ HSC in
bone marrow after treatment with 10 µg FL for 10 days when compared
with untreated controls.42 In striking contrast, when we
injected FL and G-CSF simultaneously, the percentage of HSC showed a
fourfold expansion on day 7, but dropped to pretreatment levels on day
10, whereas the frequency of FC in bone marrow remained unchanged. This
suggests that the proliferation of HSC and FC caused by FL in
combination with the mobilizing effect of G-CSF might be responsible
for the potent synergy of both growth factors to elevate HSC and FC
numbers in PB.
In addition to the growth factor-mediated effects in bone marrow,
there was a significant increase in cellularity and frequency of HSC/PC
reported in spleens of FL-treated mice.42,45,50 We observed
in our study a highly significant expansion of cells with FC phenotype
and HSC in spleens of FL- and FL + G-CSF-treated mice. The percentage
of FC in the spleen of mice injected with FL alone increased from less
than 1% on day 0 to 11% on day 10. This increase seems to be specific
for certain cell types, such as FC and HSC, because the frequency of
CD8+ T cells declined after FL administration. A similar
observation was made by Brasel et al,42 who showed that an
increase of CD8+/Thy-1 cells in spleens
of FL treated mice occurred.42 The same group reported
later that a dramatic increase of dendritic cells was present in spleen
under treatment with FL.50 When splenocytes from FL-treated
mice were depleted of T cells, B cells, NK cells, and cells of
erythroid lineage using MoAbs and complement, these cells could be
divided into five groups based on their expression of the dendritic
cell markers CD11c and CD11b. Interestingly, more than 50% of cells of
population D (CD11cbright/CD11bdim) and E
(CD11cbright/CD11b) coexpressed
CD8.50,51 Whether these two populations mediate a
graft-facilitating effect is currently under investigation in our
laboratory. However, El-Badri et al52 showed recently that dendritic cells isolated from murine bone marrow did not facilitate engraftment of purified HSC across MHC barriers.52
Nevertheless, our preliminary data indicate that
CD8+/TCR cells from PB and spleens of FL + G-CSF-treated animals highly purified by cell sorting facilitate
engraftment of allogeneic bone marrow across MHC barriers (unpublished
observation).
This observation is further confirmed by the superior engraftment
potential of PB from B10.BR donors mobilized with FL and FL + G-CSF in
fully ablated allogeneic B10 mice. One hundred percent and 83% of
animals transplanted with 1 × 106 FL + G-CSF- and
FL-mobilized PBMC obtained on day 10 were rescued from
irradiation-induced aplasia, respectively. The transplanted PBMC
contained 0.63 or 0.64 × 105 HSC and 2.10 or 1.21 × 105 FC after treatment with FL + G-CSF and FL
alone, respectively. In contrast, similar amounts of HSC and FC present
in G-CSF-mobilized PB rescued only 33% of lethally irradiated animals,
indicating a qualitative disadvantage of these cells after treatment
with G-CSF alone. When PBMC were obtained on day 7 of growth factor administration, the engraftment potential was less efficient. Therefore, the collection of PB on day 10 at which the highest numbers
of HSC and FC in PB were detectable seems to be favorable. Moreover,
the long-term repopulating potential of HSC and FC from FL- and FL + G-CSF-treated allogeneic donors was superior when compared with donors
treated with G-CSF alone. However, the development of GVHD as a result
of high numbers of T and NK cells in whole PB has limited the long-term
survival in those animals. Additional functional studies need to be
performed in the future to determine whether mobilized facilitating
cells defined by phenotypic criteria will function as effectively as
those purified from naïve bone marrow in previous studies.
In summary, treatment of mice with FL results in proliferation of cells
with FC phenotype in bone marrow and spleen. In animals treated with a
combination of FL and G-CSF, both FC and HSC were most efficiently
mobilized into PB and peak levels for both populations were detected on
day 10. This strategy might be useful in the clinical setting
especially when HLA disparities between donor and recipient exist and
FC are needed to achieve HSC engraftment. However, improved collection
and processing of mobilized PB to contain mainly FC and HSC yet reduce
the amount of contaminating T cells and NK cells might be necessary to
avoid GVHD and enhance the engraftment potential.
| |
FOOTNOTES |
Submitted March 4, 1998;
accepted June 10, 1998.
Supported by Deutsche Forschungsgemeinschaft (M.N.) (Ne 620/1-2; Ex
11/1-1), and National Institutes of Health Grant Nos. DK 52294 and 2R01
DK52294-06AZ (S.T.I.).
Address reprint requests to Suzanne T. Ildstad, MD, Director, Institute
for Cellular Therapeutics, Professor of Surgery, Allegheny University
of the Health Sciences, Broad & Vine Streets, Mail Stop 490, Philadelphia, PA 19102.
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.
| |
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Abstract
Introduction
Abstract