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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4071-4078
RAPID COMMUNICATION
Granulocyte Colony-Stimulating Factor-Mobilized Allogeneic Stem Cell
Transplantation Maintains Graft-Versus-Leukemia Effects Through a
Perforin-Dependent Pathway While Preventing Graft-Versus-Host Disease
By
Luying Pan,
Takanori Teshima,
Geoffrey R. Hill,
David Bungard,
Yani S. Brinson,
Vijay S. Reddy,
Kenneth R. Cooke, and
James L.M. Ferrara
From the Department of Pediatric Oncology, Dana Farber Cancer
Institute, Boston, MA; and the Departments of Internal Medicine and
Pediatrics, University of Michigan Medical School, Ann Arbor, MI.
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ABSTRACT |
Minimization of graft-versus-host disease (GVHD) with preservation
of the graft-versus-leukemia (GVL) effect is a crucial step to improve
the overall survival of allogeneic bone marrow transplantation (BMT)
for patients with hematological malignancies. We and other
investigators have shown that granulocyte colony-stimulating factor
(G-CSF)-mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this
study, we investigated whether G-CSF-mobilized PBSC maintain their GVL
effect in a murine allogeneic transplant model (B6  B6D2F1).
B6 mice (H-2b) were injected subcutaneously with human
G-CSF (100 µg/kg/d) for 6 days and their splenocytes were harvested
on day 7 as a source of PBSC. G-CSF mobilization dramatically improved
transplant survival compared with nonmobilized controls (95% v
0%, P < .001). Systemic levels of lipopolysaccharide and
tumor necrosis factor- were markedly reduced in recipients of
allogeneic G-CSF-mobilized donors, but cytolytic T lymphocyte
(CTL) activity against host tumor target cells p815 was
retained in those recipients. When leukemia was induced in recipients
by coinjection of p815 tumor cells (H-2d) at the time of
transplantation, all surviving recipients of G-CSF-mobilized B6 donors
were leukemia-free at day 70 after transplant, whereas all mice who
received T-cell-depleted (TCD) splenocytes from G-CSF-mobilized B6
donors died of leukemia. When splenocytes from G-CSF-mobilized
perforin-deficient (pfp /) mice were used for
transplantation, 90% of recipients died of leukemia, demonstrating
that perforin is a crucial pathway mediating GVL effects after
G-CSF-mobilized PBSCT. These data illustrate that G-CSF-mobilized
allogeneic PBSCT separate GVL from GVHD by preserving
perforin-dependent donor CTL activity while reducing systemic inflammation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALLOGENEIC BONE MARROW transplantation
(BMT) is a standard therapy for hematological malignancies. An
important benefit of allogeneic BMT is the graft-versus-leukemia (GVL)
effect, a process of tumor eradication by donor cells after
BMT.1-3 However, GVL effects are closely linked with
graft-versus-host disease (GVHD), a major cause of morbidity and
mortality after allogeneic BMT.1,4 Results from a series of
clinical trials demonstrated that donor T cells play a vital role in
both GVL and GVHD, because T-cell depletion (TCD) of the bone marrow
reduced the incidence and severity of GVHD, but increased leukemia
relapse.2,5-7 It is also well recognized that leukemia
relapse is inversely linked to the severity of GVHD after
BMT.1,5 Therefore, separation of GVL and GVHD is a crucial
step to improve the overall survival of allogeneic BMT for hematologic malignancy.
Recently, there is increased enthusiasm for the use of granulocyte
colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell
transplantation (PBSCT). Comparison of G-CSF-mobilized PBSCT
(containing a 10- to 20-fold increase in donor CD3+ cells)
and traditional bone marrow grafts demonstrate a surprisingly similar
incidence and severity of acute GVHD.8-11 This relative reduction of acute GVHD may be attributable to immunomodulation of
cells in the donor graft. A decrease in interleukin-2 (IL-2) and
interferon- (IFN-) production to allo-antigen stimulation has
been reported both in human and animal studies.12-15
Monocytes from G-CSF-mobilized human donors have also been reported to
suppress allo-reactivity of T cells in mixed lymphocyte
culture,16-18 perhaps through an IL-10-dependent
mechanism.19 However, recent studies suggest increased
risks of chronic GVHD after G-CSF-mobilized allogeneic
PBSCT.20,21 Because donor T cells are major effectors of
the GVL effect, it is important to investigate whether G-CSF-mobilized PBSC grafts can maintain GVL effects. In this study of a murine PBSCT-leukemia model, we show that T cells from G-CSF-mobilized PBSC
have a markedly diminished capacity to induce acute GVHD, but maintain
their GVL function through a perforin-dependent pathway.
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MATERIALS AND METHODS |
Mice.
Female Ly-5 congenic B6.Ly-5a (H-2b,
CD45.1+) mice were obtained from the Frederick Cancer
Research Facility (Frederick, MD), and female B6D2F1
(H-2bxd, CD45.2+) mice were purchased from the
Jackson Laboratories (Bar Harbor, ME). Female C57BL/6 (B6,
H-2b, CD45.2+) and perforin-deficient mice
(pfp/, B6x129/SvEv, H-2b,
CD45.2+) were purchased from Toconic Laboratory
(Germantown, NY). Ly-5 (CD45) alleles are described according to the
nomenclature of Morse et al.22 Mice were housed in
sterilized microisolator cages and received tap water and normal chow.
Mice used for experiments were between the ages of 10 and 14 weeks and
received autoclaved hyper-chlorinated drinking water during the first 3 weeks posttransplantation.
G-CSF treatment.
Donor mice were injected subcutaneously with recombinant human G-CSF
(Amgen Inc, Thousand Oaks, CA) daily at 100 µg/kg body weight or
saline (control diluent) for 6 days, and splenocytes were harvested on
day 7.
PBSCT.
This protocol has been described previously.13,14 Briefly,
B6D2F1 recipients received 1,100 rad total body irradiation, which was
split into two doses separated by 3 hours to minimize gastrointestinal
(GI) toxicity. Splenocytes (10 × 106) from B6 donors
were injected intravenously into B6D2F1 recipients. Recipients of 5 × 106 TCD B6 splenocytes (treated with 2 cycles of
anti-Thy1.2 and rabbit complement) or 10 × 106 B6D2F1
splenocytes served as non-GVHD controls. For GVL experiments, 5,000 to
25,000 p815 leukemic cells (H-2d, CD45.2+) were
injected together with donor splenocytes. Survival was monitored daily
and recipient body weight was measured weekly. Tumor burden was
determined either by detection of tumor cells in peripheral blood or at
autopsy at the end of experiments. The criteria for tumor-induced death
were defined as either hepatosplenomegaly with macroscopic tumor
nodules in liver and/or spleen or evidence of spinal cord involvement
(hind leg paralysis or pathological demonstration of p815 tumor cells
in the spinal cord). Leukemia-free survival was defined as (1)  0.5%
tumor cells (H-2d+/b + CD45.2+)/CD45.1 in peripheral blood and
(2) no macroscopic tumor nodules in liver, spleen, and spinal cord at
the end of experiments.
Mixed lymphocyte culture.
Splenic T cells were obtained by passage of splenocytes through nylon
wool columns and were cultured with 1 × 105
irradiated B6D2F1 peritoneal cells (2,000 rad) in completed Dulbecco's modified Eagle's medium (DMEM) media in a 96-well
flat-bottomed plate at 37°C in a humidified incubator supplemented
with 7% CO2. All culture media reagents were purchased
from GIBCO BRL (Gaithersburg, MD). Completed DMEM media was
supplemented with 10% fetal calf serum, 50 U/mL penicillin, 50 µg/mL
streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acid, 0.02 mmol/L  -mercaptoethanol, and 10 mmol/L HEPES, pH 7.75. At 48 hours, supernatants were collected for
cytokine levels. For cytokine determination during 2° MLR, splenic
T cells were incubated with irradiated (2,000 rad) B6D2F1 splenocytes
at a 1:2 ratio in a 24-well plate for 6 days and then restimulated with
fresh irradiated (2,000 rad) B6D2F1 peritoneal cells in completed DMEM media in a 96-well flat-bottom plate for 48 hours.
Fluorescence-activated cell sorting (FACS) analysis.
Fluorescein isothiocyanate (FITC)- or R-phycoerythrin (PE)-conjugated
monoclonal antibodies (MoAbs) were purchased from PharMingen (San
Diego, CA). Cells (5 × 105/sample) were first
incubated with MoAb 2.4G2 for 10 minutes at 4°C to block
nonspecific binding to Fc receptors and then with FITC- or
PE-conjugated specific MoAbs for 30 minutes at 4°C. Cells were then
washed twice with phosphate-buffered saline (PBS)/0.2% bovine serum
albumin (BSA) and fixed with PBS/1% paraformaldehyde. Two-color flow
cytometric analysis was performed using a FACScan (Becton Dickson,
Mountain View, CA). Two methods of staining were used to determining
the tumor burden in peripheral blood. Cells were either double-stained
with FITC-conjugated anti-H-2Dd (Cedarlane Lab,
Hornby, Ontario, Canada) and PE-conjugated
H-2Kb or with FITC-conjugated anti-CD45.1 and anti-CD45.2
MoAb. In control experiments, Peripheral blood cells (PBC) from donor
B6 Ly-5a mice were 99.8% CD45.1+ and
H-2b+/d and PBC of B6D2F1 were 99.8%
CD45.2+ and H-2b+/d+, whereas p815 cells were
99.7% CD45.2+ and
H-2Kb/H-2Dd+ (data not shown).
Enzyme-linked immunosorbent assay (ELISA).
The antibodies used in the assays in the IFN-, IL-2, IL-4, IL-10,
and IL-12 p40 assays were purchased from PharMingen (San Diego, CA),
and antibodies used in the tumor necrosis factor- (TNF-) assay
were purchased from Genzyme Corp (Cambridge, MA). All assays were
performed according to the manufacturer's protocol. Briefly, cytokines
were captured by the specific primary MoAb and detected by horseradish
peroxidase-labeled anti-TNF- or by the biotin-labeled
anti-IFN-, anti-IL-2, anti-IL-4, anti-IL-10, or anti-IL-12
followed by strepavidin-horseradish peroxidase. The color reaction was
developed by TMB microwell peroxidase substrate (KPL, Gaithersburg, MD)
and stopped by the addition of an equal volume of 1 mol/L
H2SO4. The absorbance of the assay plate was read at 450 nm using a microplate reader (Model 3550; Bio-Rad Labs,
Hercules, CA). Recombinant murine TNF- (mTNF-),
mIFN-, mIL-2, mIL-4, mIL-10, and mIL-12 p40 were used as standards
for ELISAs. The low limit of sensitivity is 0.1 U/mL for IFN-, IL-2, and IL-4; 15 pg/mL for IL-10 and TNF-; and 1 pg/mL for IL-12 p40.
Limulus amebocyte lysate (LAL) assay.
The serum endotoxin levels were determined by the LAL assay using the
QCL-1000 test kit (BioWhittaker, Walkersville, MD). Assays were
performed according to the manufacturer's protocol. Briefly, serum was
diluted 10-fold with LAL reagent water and heated to 70°C for 5 minutes to remove any nonspecific inhibition to the assay. Samples were
then incubated with equal volumes of LAL for 10 minutes at 37°C and
developed with equal volumes of substrate solution for 6 minutes. The
absorbance of the assay plate was read at 405 nm using a microplate
reader (Model 3550; Bio-Rad Labs). Samples and standards were run in
duplicate and the lower limit of detection was 0.15 U/mL. All units
expressed are relative to the US reference standard EC-6.
51Cr release assay.
Responder cells from day-6 primary MLR or fresh splenocytes harvested
on day 7 posttransplant were used as effector cells. Two million target
cells were labeled with 100 µCi 51Cr for 2 hours at
37°C and washed 3 times afterwards. Effector cells were incubated
with 10,000 labeled target cells at 37°C for 4 hours at various
effector/target ratios, and 51Cr in supernatant was
determined by a -scintillation counter. p815 cells
(H-2d) were used as allogeneic tumor targets; EL-4 cells
(H-2b) were used as targets syngeneic to the donor. Maximal
and background release was determined by addition to the target cells
of 2% Triton-X (Sigma Chemical Co, St Louis, MO) or media,
respectively. The percentage of specific lysis (%) = 100 × (sample count  background count)/(maximal count background count).
Statistical analysis.
The Mann-Whitney U test was used for the statistical analysis of weight
loss, whereas the Mantel-Cox log rank-test was used to analyzed
survival data. The two-tailed Student's t-test was used to
analyze cytokine and lipopolysaccharide (LPS) data. P = .05 was
considered statistically significant.
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RESULTS |
G-CSF mobilization reduces the severity of acute GVHD.
We first examined the effects of G-CSF mobilization in a murine BMT
model (B6 Ly-5a B6D2F1) that induces acute GVHD
to both major and minor histocompatibility antigens. Injections of
human G-CSF for 6 days at a dose of 100 µg/kg/d increased the yield
of splenocytes by approximately 25%. As shown in
Fig 1, the percentages of T cells
(CD4+, CD8+), B cells (B220+), and
natural killer (NK) cells (NK1.1+) were
similar in control and G-CSF-reated donors, whereas myeloid cells
(Gr-1+) were significantly increased in splenocytes from
G-CSF-treated donors. The percentage of myeloid cells in the bone
marrow doubled in G-CSF-treated donors. B6D2F1 recipient mice were
irradiated with 1100 cGy and transplanted with 10 × 106 splenocytes from either control or G-CSF-treated B6
Ly-5a mice. GVHD induced in this model was severe and
usually lethal. As shown in Fig 2A, all
animals receiving control allogeneic splenocytes died within 2 weeks,
with clinical evidence of GVHD (hunched posture, inactivity, and weight
loss), whereas 95% of mice receiving splenocytes from G-CSF-mobilized
donors survived at day 70 posttransplant. This survival was markedly
superior to that seen in our previous study when splenocytes were
mobilized with a lower dose of human G-CSF.13,14 The
optimal dose of hG-CSF for PBSC mobilization is approximately 10 times
higher in mice than in humans,15,23-25 making the doses
used in this study more clinically relevant. The mortality of acute
GVHD seen in control allogeneic recipients was mediated by donor T
cells, because all mice receiving allogeneic TCD-splenocytes survived
until the end of experiment. Although the severity of acute GVHD in
mice receiving allogeneic G-CSF-mobilized splenocytes was dramatically
reduced, these mice did show signs of moderate GVHD, as measured by
weight loss compared with recipients of allogeneic TCD-splenocytes or
syngeneic splenocytes (Fig 2B).
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| Fig 1.
Effect of G-CSF on granulocyte population and T-cell
phenotype. B6 Ly-5a mice were injected with G-CSF (100 µg/kg/d) or saline for 6 days. BM (n = 15 per group) and
splenocytes (n = 20 per group) were harvested the day after the last
injection. After lysis of red blood cells, cells were stained with
specific Abs and analyzed by FACS. The results represent the mean ± SD from nine experiments. *P < .001 v control mice.
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| Fig 2.
Survival and weight loss after splenocyte transplant (B6
Ly-5a B6D2F1). B6 Ly-5a donors were
injected with or without G-CSF for 6 days. Total body irradiated B6D2F1
recipients received 1 × 107 splenocytes from control B6
donors (n = 20), G-CSF-mobilized donors (n = 20), or control
B6D2F1 donors (n = 15) or 5 × 106 TCD-splenocytes from
control B6 donors (n = 10). Survival was monitored daily up to day 70 posttransplantation (A). Body weights were measured weekly (B).
*P < .001 v recipients of splenocytes from control B6
donors (A). *P < .001 v recipients of TCD-splenocytes
from control B6 donors or splenocytes from control B6D2F1 donors (B).
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G-CSF mobilization reduces systemic levels of LPS and TNF-.
Both LPS and TNF- are known to be important mediators of acute GVHD
severity.26-28 Consistent with the severe clinical GVHD in
animals receiving control allogeneic splenocytes, the serum LPS levels
in these animals were markedly elevated compared with syngeneic
controls on day 7 posttransplant, a time of maximal elevation
(Fig 3A). By contrast, serum LPS levels in
animals transplanted with G-CSF-mobilized allogeneic splenocytes were
reduced to levels of syngeneic non-GVHD controls (Fig 3A). Serum
TNF- levels were also significantly reduced in the recipients of
G-CSF-mobilized donors, although they remained higher than that seen
in syngeneic non-GVHD controls (Fig 3B).
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| Fig 3.
Serum levels of LPS and TNF- after splenocyte
transplant. Total body irradiated B6D2F1 mice received 1 × 107 splenocytes from control B6 donors, G-CSF-mobilized
donors, or control B6D2F1 donors (n = 5/group). Serum was collected
on day 7 posttransplant, LPS levels were determined by LAL assay (A),
and the TNF- level was determined by ELISA (B). UD, under limit of
detection. *P < .001 v recipients of control B6
donors.
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G-CSF mobilization induces a type 2 cytokine profile with
preservation of CTL activity.
We next examined the effects of G-CSF mobilization on donor T-cell
functions. Consistent with previous reports using a lower G-CSF
dose,13,14 G-CSF treatment led to an increased production of type 2 cytokines (IL-4 and IL-10) with a decreased production of
type 1 cytokines (IL-2 and IFN-) in response to host antigen stimulation. This polarization towards a type 2 cytokine profile was
maintained in 2° MLR despite the absence of exogenous G-CSF at all
times in culture (Table 1). G-CSF also
decreased production of IL-12, reflecting its action on the
antigen-presenting cells (Table 1). T cells from 1° MLR were then
used as effector cells against host type (H-2d) p815
targets or donor type (H-2b) EL4 targets. As shown in
Fig 4, G-CSF mobilization did not reduce the CTL activity of splenocytes, despite the shift in cytokine profile.
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| Fig 4.
CTL activity in vitro. CTL activity was determined by
51Cr release assay. Equal numbers of splenic T cells from a
6-day primary MLR (B6 anti-B6D2F1) were used as effector cells. p815
cells (H-2d) and EL4 cells (H-2b) were labeled
with 51Cr and used as targets. After 4 hours of
coincubation with effector cells, 51Cr in the supernatants
was determined by a -scintillation counter. One of three
representative experiments is presented.
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G-CSF mobilization preserves GVL effects.
To examine the effects of G-CSF mobilization on GVL, animals were
transplanted as described above and 5,000 p815 tumor cells were
injected intravenously together with the donor inoculum. As shown in
Fig 5A, syngeneic recipients all died of
leukemia by 4 weeks posttransplant with macroscopic evidence of tumor
in the liver and spleen. Recipients of allogeneic control donors died
within 2 weeks due to severe GVHD, but necropsy showed no evidence of
tumor. In contrast, 95% of allogeneic recipients of G-CSF-mobilized
donor cells were still alive at day 70 posttransplantation. Eradication
of leukemia was confirmed by absence of CD45.2+ cells in
peripheral blood and lack of tumor in liver and spleen by histology.
The importance of donor T cells in mediating the GVL effect was
confirmed by transplantation of TCD-splenocytes from B6 donors and
5,000 p815 tumor cells. None of recipients showed evidence of GVHD (Fig
2), but they all died by 5 weeks after transplantation with macroscopic
evidence of leukemia (Fig 5B).
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| Fig 5.
Survival after leukemia induction (B6 Ly-5a
B6D2F1). B6 Ly-5a donors were injected with or
without G-CSF for 6 days. (A) Total body irradiated B6D2F1 mice
received 1 × 107 splenocytes plus 5,000 p815 tumor cells
from control B6 donors (n = 15), G-CSF-mobilized donors (n = 20),
or control B6D2F1 donors (n = 13). *P < .001 v
recipients of splenocytes from control B6 donors and control B6D2F1
donors. (B) Total body irradiated B6D2F1 recipients were injected with
5 × 106 TCD-splenocytes plus 5,000 p815 tumor cells from
control or G-CSF B6 donors (n = 8/group). Survival was monitored
daily up to day 70 posttransplantation.
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GVL is mediated through a perforin-dependent pathway.
To further delineate the mechanism of GVL after G-CSF-mobilized PBSCT,
perforin-deficient (pfp/) mice were used as
donors, because perforin has been shown to be an important effector of
Tc2 cytotoxic functions.29,30 CTL activity from
pfp/ was substantially reduced both in vitro
and ex vivo (Fig 6). Splenic T cells after
1° MLR (Fig 6A) or splenocytes from recipients 7 days after
transplantation of 10 × 106 allogeneic splenocytes
(Fig 6B) showed significant decrease in lysis of host-type p815 tumor
targets, and this was unaffected by G-CSF mobilization (Fig 6A and B).
We then examined GVL effects in a murine PSCT-leukemia model. Lethal
irradiated B6D2F1 recipient mice received 10 × 106
splenocytes from G-CSF-mobilized B6 or pfp/
donors with 25,000 p815 tumor cells. As shown in
Fig 7, all syngeneic recipients died with
macroscopic evidence of tumor within 3 weeks. All recipients of
allogeneic G-CSF donors were leukemia-free at day 70 as determining by
FACS staining of peripheral blood cells and macroscopic examination of
tumor-targeted organs. By contrast, 90% of recipients transplanted
with splenocytes from pfp/ donors died with
gross evidence of leukemia. Engraftment of
pfp/ donor cells was complete by day 60 and
expansion of pfp/ donor T cells on day 7 after transplantation was equivalent to wild-type controls
(Table 2). Therefore, the loss of a GVL
effect was not due to diminished donor T-cell expansion or engraftment, but rather lack of perforin activity.
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| Fig 6.
Abolition of CTL activity in pfp/ mice.
CTL activity was determined by 51Cr release assay. (A)
Equal numbers of splenic T cells from a 6-day primary MLR (B6
anti-B6D2F1) were used as effectors. (B) Splenocytes from day 7 posttransplant (n = 5/group) were counted, and equal numbers of T
cells (CD4+ plus CD8+ cells adjusted
according to FACS analysis) were used as effectors.
51Cr-labeled p815 targets (H-2d) were
coincubated with effectors for 4 hours, and 51Cr in the
supernatants was determined by a -scintillation counter.
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| Fig 7.
Survival after leukemia induction (B6 B6D2F1).
Wild-type B6 or pfp/ donors were injected with G-CSF
for 6 days. Total body irradiated B6D2F1 recipients received 1 × 107 splenocytes plus 25,000 p815 tumor cells from
G-CSF-mobilized B6 donors (n = 10) or from G-CSF-mobilized
pfp/ donors (n = 10) or control B6D2F1 donors
(n = 5). Survival was monitored daily until day 70 posttransplantation. *P < .001 v recipients of
splenocytes from G-CSF-mobilized pfp/ donors and
control B6D2F1 donors.
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DISCUSSION |
In this study, we demonstrate that G-CSF-mobilized allogeneic PBSCT
dramatically reduced the severity of acute GVHD while maintaining
perforin-dependent GVL effects in a murine PBSCT-leukemia model. CTL
activity against host antigens in G-CSF-mobilized donor PBSCT is
preserved, although the inflammatory cytokine response is significantly diminished.
The important balance between cytokines derived from type 1 and type 2 T cells in inducing acute GVHD was first demonstrated in the
experimental BMT models.31-33 Elevated levels of type 1 cytokines (IL-12, IL-2, and IFN-) are associated with severe acute
GVHD,34-39 whereas elevated levels of a type 2 cytokine
profile (increased IL-4 and IL-10 production) are
not.31-33,40,41 A correlation of type 1 and type 2 cytokine
profile with the severity of acute GVHD was also reported by Tanaka et
al42 in a clinical study of allogeneic BMT. We and other
investigators have reported that G-CSF mobilization skews T-cell
cytokines toward a type 2 profile upon allo-antigen stimulation and
after experimental allogeneic PBSCT.12-15 G-CSF
mobilization also causes a decreased production of type 1 cytokines
from PBMC upon allo-antigen stimulation compared with before
mobilization,43,44 and increased expression of IL-4 mRNA
has also been reported.45
Type 1 cytokines are known to prime mononuclear cells to secrete
TNF- during GVHD.31,33,46,47 Clinical studies have shown
that elevated serum TNF- levels precede clinical symptoms of acute
GVHD,28,48 and anti-TNF- therapy significantly reduced the severity of acute GVHD.49,50 It has also been reported that G-CSF-mobilized human PBMC produced less TNF- in mixed
lymphocyte cultures than PBMC from same donor pretreated with
G-CSF.17 In this study, serum TNF- levels were
significantly reduced in recipients of G-CSF-mobilized splenocytes
compared with GVHD controls. TNF- has been shown to cause necrosis
in the GI tract during GVHD.51 The endotoxin that
translocates across damaged intestinal mucosa acts as a stimulus to
further TNF- production and may also amplify target organ damage by
enhancing the in vivo clonal expansion and differentiation of
antigen-activated T cells, as shown in other experimental
systems.52
The reduction in severe GVHD using high-dose G-CSF mobilization in this
study was markedly superior to that of previous studies in which a
10-fold lower dose of G-CSF was used.13,14 T-cell responses
toward a type 2 cytokine profile were similar after both high-dose and
low-dose mobilization, suggesting that improved protection from
high-dose mobilization was not due to changes in T-cell cytokine
secretion. However, a reduction in proliferation to host antigens was
observed using enriched splenic T cells from donors mobilized with
high-dose G-CSF (data not shown). In addition, the percentage of
myeloid cells in splenocytes after high-dose G-CSF mobilization was
significantly greater (Fig 1) than that seen after low-dose G-CSF
mobilization.13 These observations are consistent with
studies of G-CSF-mobilized human PBSC that show myeloid components may
play a role in hypo-responsiveness of donor T cells to allo-antigen
stimulation.16-18,53
Donor T cells play a vital role in mediating GVL effects, as
demonstrated by the effectiveness of donor leukocyte infusion to induce
remission after leukemia relapse.54-58 In this study, we
have showed that G-CSF-mobilized donor T cells maintain their CTL
activity against leukemic targets and preserve GVL effects. An improved
GVL effect using G-CSF-mobilized allogeneic PBSCT has been reported in
another murine leukemia model.59 G-CSF-mobilized PBPC have
also been used successfully to treat relapse after allogeneic BMT.60,61 Apoptosis of target cells induced by CTL could be mediated by perforin and/or Fas/FasL pathways, and both pathways may be
involved in the development of GVHD.50,62-69 CTL can be divided into Tc1 and Tc2 subpopulations according to their cytokine secretion pattern. Apoptosis mediated by Tc1 cells depends primarily on
Fas/Fas ligand pathway.29,70,71 IFN- secreted by type 1 T cells has been reported to increase expression of Fas and FasL and
may thereby enhance apoptosis mediated by the Fas/FasL pathway.72,73 However, apoptosis mediated by Tc2 cells is
more dependent on perforin pathway.29,71 Such mechanisms
are consistent with the present study, in which G-CSF mobilization
amplifies a Tc2 response, reduces acute GVHD, and maintains GVL through a perforin-dependent pathway.
In this study, we demonstrated that G-CSF-mobilized grafts reduce
severity of acute GVHD by disruption of cytokine cascade involved in
development of acute GVHD. More importantly, G-CSF-mobilized grafts
maintain their GVL effects through a perforin-dependent pathway.
Therefore, G-CSF mobilization may offers a novel approach to the
separation of GVL effects from GVHD. Studies are currently in progress
to determine the effects of G-CSF mobilization of donor cells in
chronic GVHD and immune reconstitution.
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ACKNOWLEDGMENT |
The authors thank Dr Anastasia Skandalis for her valuable discussions
and Scott Bressler and Vicki Mosher for their technical support.
| |
FOOTNOTES |
Submitted January 18, 1999; accepted March 22, 1999.
Supported in part by National Institutes of Health Grants No. CA 39542 and HL 55709.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to James L.M. Ferrara, MD, Bone Marrow
Transplant Program, University of Michigan Cancer Center, 1500 E
Medical Center Dr, Ann Arbor, MI 48109; e-mail: ferrara{at}umich.edu.
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A. H. Elmaagacli, S. Basoglu, R. Peceny, R. Trenschel, H. Ottinger, A. Lollert, V. Runde, H. Grosse-Wilde, D. W. Beelen, and U. W. Schaefer
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J. E. Levine, T. Braun, S. L. Penza, P. Beatty, K. Cornetta, R. Martino, W. R. Drobyski, A. J. Barrett, D. L. Porter, S. Giralt, et al.
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M. Korbling and P. Anderlini
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Blood,
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[Abstract]
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M. J. Smyth, J. M. Kelly, V. R. Sutton, J. E. Davis, K. A. Browne, T. J. Sayers, and J. A. Trapani
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D. Przepiorka, T. L. Smith, J. Folloder, P. Anderlini, K.-W. Chan, M. Korbling, B. Lichtiger, F. Norfleet, and R. Champlin
Controlled trial of filgrastim for acceleration of neutrophil recovery after allogeneic blood stem cell transplantation from human leukocyte antigen-matched related donors
Blood,
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[Abstract]
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C. Schmaltz, O. Alpdogan, K. J. Horndasch, S. J. Muriglan, B. J. Kappel, T. Teshima, J. L. M. Ferrara, S. J. Burakoff, and M. R. M. van den Brink
Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect
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[Abstract]
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T. Teshima, N. Mach, G. R. Hill, L. Pan, S. Gillessen, G. Dranoff, and J. L. M. Ferrara
Tumor Cell Vaccine Elicits Potent Antitumor Immunity after Allogeneic T-Cell-depleted Bone Marrow Transplantation
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M. H. Hsieh and R. Korngold
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M. R. Bishop, S. R. Tarantolo, Z. S. Pavletic, J. C. Lynch, M. E. Morris, D. Zacharias, J. O. Armitage, and A. Kessinger
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G. R. Hill and J. L. M. Ferrara
The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation
Blood,
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[Abstract]
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V. Reddy, J. Moreb, P. Mehta;, F. Dazzi, R. M. Szydlo, and J. M. Goldman
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TOP
ABSTRACT
INTRODUCTION