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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2738-2747
Graft-Versus-Leukemia Effect and Graft-Versus-Host Disease Can Be
Differentiated by Cytotoxic Mechanisms in a Murine Model of
Allogeneic Bone Marrow Transplantation
By
Nobuhiro Tsukada,
Tetsuji Kobata,
Yoshifusa Aizawa,
Hideo Yagita, and
Ko Okumura
From the Department of Immunology, Juntendo University School of
Medicine, Tokyo, Japan; The 1st Department of Internal Medicine,
Niigata University School of Medicine, Niigata, Japan; and CREST (Core
Research for Evolutional Science and Technology) of Japan Science and
Technology Corp, (JST), Tokyo, Japan.
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ABSTRACT |
Allogeneic bone marrow transplantation (allo-BMT) is associated with
both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL)
effect. In the present study, we examined the contribution of cytotoxic
effector mechanisms, which are mediated by tumor necrosis factor-
(TNF-), Fas ligand (FasL), or perforin, to GVHD and GVL effect in a
murine BMT model. Bone marrow cells plus spleen cells (BMS) from
wild-type, FasL-defective, or perforin-deficient donors were
transferred into lethally irradiated recipients in the parent (C57BL/6)
to F1 (C57BL/6 × DBA/2) BMT model with or without prior inoculation
of DBA/2 leukemia L1210 or P815 mast cytoma cells. The effect of
anti-TNF- antibody administration was also examined. Whereas the
defect or blockade of each cytotoxic pathway could ameliorate lethal
acute GVHD, the GVL effect was differentially affected. The wild-type
BMS recipients died of acute GVHD within 50 days without residual
leukemia cells. The FasL-defective BMS recipients showed 60%<
survival over 80 days without acute GVHD or residual leukemia cells.
Administration of anti-TNF- antibody resulted in early leukemia
relapse and the recipients died within 25 days with massive leukemia
infiltration in the liver. The perforin-deficient BMS recipients died
within 60 days with residual leukemia cells. These results suggest that blockade of the Fas/FasL pathway could be used for ameliorating GVHD
without impairing GVL effect in allo-BMT.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALLOGENEIC bone marrow transplantation
(allo-BMT) has been a potentially curative therapy for patients with a
variety of diseases, including hematologic malignancies.1,2
However, the major obstacle in allo-BMT is acute graft-versus-host
disease (GVHD), which is caused by T cells present in the donor marrow graft.3-6 Although the incidence and the severity of acute
GVHD can be dramatically improved by T-cell depletion or the
combination of immunosuppressive agents such as cyclosporin,
corticosteroid, and methotrexate, the risk of leukemia relapse could be
increased in turn possibly due to the lack of antileukemia effect of
allogeneic T cells infused.7
Molecular mechanisms involved in the pathogenesis of GVHD have been
well studied in the past decades.8 Three cytotoxic pathways
are reported to be involved in the pathogenesis of GVHD; tumor necrosis
factor (TNF)/TNF receptor (TNFR) system, Fas (CD95)/Fas ligand (FasL)
system, and perforin/granzyme system.9-17 Administration of
anti-TNF- antibody ameliorated acute GVHD in a mouse
model,9 and TNFR p55-deficient recipient mice showed a
reduced mortality after allo-BMT.10 Allo-BMT using
FasL-defective gld (generalized lymphoproliferative disease)
mice as a donor did not cause hepatic and cutaneous GVHD.11
Recently, we demonstrated that a metalloproteinase inhibitor, which
inhibits TNF- and FasL release, prevented lethal acute GVHD in the
parent to F1 spleen cell transfer model.12 It has been also
reported that acute GVHD induced by transfer of spleen cells from mice
doubly deficient in perforin and FasL, but not FasL alone, to major
histocompatibility complex (MHC)-mismatched recipients was
significantly ameliorated.13 Similarly, the onset of lethal
GVHD was delayed in allo-BMT from perforin-deficient mice.11 These findings implicated all three of these
cytotoxic pathways in the pathogenesis of GVHD.
The antileukemic effect of allo-BMT, so called graft-versus-leukemia
(GVL) effect, was first reported by Barnes and Loutit in
1956.18 GVL effect is substantiated by clinical evidence that the risk of leukemia relapse is increased in patients who received
T-cell-depleted allo-BMT or BMT from an identical twin and is
considered to be caused by immunocompetent cells in the grafts.19-22 Although T and natural killer (NK) cells in
the donor grafts are thought to play an important role in the GVL
effect,19,23-32 its effector mechanisms are less clear than
GVHD. Thus, it remains to be determined whether the same effector
mechanisms are involved in GVHD and the GVL effect. If GVHD and the GVL
effect are mediated by different mechanisms, it may be possible to
spare the GVL effect while avoiding GVHD. Ideally, the GVL effect
without GVHD is most beneficial for the effective treatment of leukemia
patients after allo-BMT.
In the present study, to examine whether the effector mechanisms
leading to the GVL effect and GVHD can be differentiated by their
cytotoxic pathways, we performed a series of experiments using a murine
GVL model. (C57BL/6 × DBA/2) F1 mice, which received a prior
inoculation of L1210 leukemia or P815 mastcytoma cells, were lethally
irradiated and transplanted with bone marrow (BM) cells and spleen
cells from wild-type, gld, or perforin-deficient C57BL/6 mice
or were administered with anti-TNF- monoclonal antibody (MoAb).
This GVL model is similar to human allo-BMT for the patients with
leukemias, because the GVL effect was mediated by donor-derived immunocompetent cells and early leukemia relapse was observed in the
recipients of T-cell-depleted BM or syngeneic BM. Our present results
indicated that, whereas TNF, FasL, and perforin are all involved in the
pathogenesis of GVHD, blockade of the Fas/FasL pathway can markedly
ameliorate lethal acute GVHD without impairing the GVL effect in this
model system. This suggests that selective amelioration of acute GVHD
while preserving the GVL effect can be achieved on the basis of
differential contribution of cytotoxic effector mechanisms to GVHD and GVL.
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MATERIALS AND METHODS |
Mice.
Six- to 8-week-old female C57BL/6 (B6, H-2b) and
(C57BL/6 × DBA/2) F1 (BDF1, H-2b/d) mice
were purchased from Japan SLC (Shizuoka, Japan) and maintained in our
animal facilities. B6 gld/gld (gld) mice were purchased from Jackson Laboratory (Bar Harbor, ME). B6 perforin knock-out (PKO)
mice were purchased from IBL (Gunma, Japan).
MoAbs.
Antimouse TNF- (MP-6 XT22) and antimouse Thy1.2 (30H-12) MoAbs were
obtained from PharMingen (San Diego, CA).
Cells.
L1210 murine T-cell leukemia (DBA/2 origin, H-2d), P815
murine mastcytoma cells (DBA/2 origin, H-2d), L929 murine
fibrosarcoma (C3H/He origin, H-2k), A20 murine B-cell
lymphoma (BALB/c origin, H-2d), and L5178Y murine T-cell
lymphoma cells (DBA/2 origin, H-2d) were obtained from
American Type Culture Collection (Rockville, MD). Cells were cultured
in RPMI1640 medium supplemented with 10% fetal calf serum,
antibiotics, 2-mercaptoethanol, and L-glutamine. Mouse FasL (mFasL)
transfectant (mFasL/L5178Y) was generated by transfection of mFasL cDNA
into L5178Y cells by electroporation as previously
described.33
BMT.
Recipient BDF1 mice were lethally irradiated (12 Gy) using a
60Co irradiator (MBR 1505 R2; Hitachi, Tokyo,
Japan). BM cells were flushed from the shafts of femurs and tibias of
donor mice and single-cell suspensions were prepared. In some
experiments, BM cells were further treated with anti-Thy1.2 MoAb and
rabbit complement and used as T-cell-depleted BM cells (TCD-BM).
Single-cell suspensions of spleen cells from donor mice were used as
the source of GVHD- and GVL-effector cells. Recipients received 2 × 107 BM cells with or without 2.5 × 107 spleen cells from wild-type, gld, or PKO B6
mice in 0.5 mL phosphate-buffered saline (PBS) via the tail vein. The
day of BMT was designed as day 0. For anti-TNF- treatment, 1 mg of
MoAb was administered intraperitoneally (IP) on days
 1 and 0 and then 0.5 mg was administered IP every other day
until day 28. All recipients were maintained on sterilized water
containing antibiotics (2 mg/mL neomycin sulfate) from day 2 to
day 14 post-BMT. Recipient mice were monitored for clinical signs of
GVHD, including survival, weight loss, diarrhea, alopecia, and hantched
posture. A minimum of 5 recipient mice was used in each experimental
group. Representative mice were killed at various time points post-BMT
to harvest tissues for histological examination.
GVL model.
To evaluate GVL effect after BMT, mice were inoculated with 1 × 104 of L1210 or 1 × 105 of P815 cells via
the tail vein 2 days before BMT. Representative mice were killed at
various time points post-BMT to detect residual tumor cells by
histological examination.
Histopathology.
Liver, spleen, and BM were harvested from recipients at various time
points after BMT. Tissues were fixed in 10% buffered formalin and
paraffin embedded. Sections were stained with hematoxylin and eosin and
examined under microscopy. Peripheral blood was also obtained from
recipients at various time points after BMT. Smeared specimens were
stained with May-Giemsa and examined under microscopy.
Cytotoxic assay.
To analyze the sensitivity to TNF--mediated cytotoxicity, L1210,
P815, and L929 cells (2 × 105/well) were cultured in
96-well flat-bottom microtiter plates in the presence or absence of
serially diluted recombinant mouse TNF- (PharMingen, San Diego, CA)
for 24 hours at 37°C in a 5% CO2 humidified
atmosphere. Cell viability was then measured by alamar Blue method
according to the manufacturer's instructions (Alamar Bioscience, Inc,
Sacramento, CA). To analyze the sensitivity to Fas-mediated
cytotoxicity, 51Cr-labeled L1210, P815, and A20 cells were
cultured with mFasL/L5178Y at the indicated E/T ratios. Cytotoxicity
was measured by the 6-hour 51Cr release assay as described
previously.34
T-cell proliferation assay.
Responder spleen cells (2 × 105/well) from wild-type,
gld, or PKO B6 mice were cultured with 100-Gy irradiated
stimulator L1210 or P815 cells (2 × 104/well) in
96-well round-bottom microtiter plates at 37°C in a 5%
CO2 humidified atmosphere. After 5 days, the cultures were pulsed for 18 hours with 0.5 µCi/well of [3H]thymidine
(3H-TdR) and harvested using a Micro 96 Harvester
(Skartron, Lier, Norway). Incorporated radioactivity was measured in a
 counter (Micro Beta Plus; Wallac, Turku, Finland).
Cytotoxic T lymphocyte (CTL) assay.
To examine the generation of L1210- or P815-specific CTLs, spleen cells
(1 × 106/mL) from wild-type, gld, or PKO B6
mice were cocultured with 100-Gy irradiated L1210 or P815 cells (1 × 105/mL) for 5 days and then restimulated with
irradiated L1210 or P815 cells in the presence of 10 U/mL interleukin-2
(IL-2; Shionogi, Osaka, Japan) for 5 days. CTL activity was tested
against 51Cr-labeled L1210 or P815 cells (5 × 103) at the indicated E/T ratios by the 6-hour
51Cr release assay as described previously.34
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RESULTS |
Contribution of TNF-, FasL, and perforin to lethal acute GVHD.
Our GVL model is based on the acute GVHD model in C57BL/6 (B6) parent
to (C57BL/6 × DBA/2) F1 (BDF1) BMT. Thus, before we examine the
involvement of TNF/TNFR, Fas/FasL, and perforin/granzyme pathways in
the GVL effect, we verified their contribution to acute GVHD in the
present system. The recipients that received B6 BM cells plus 2.5 × 107 spleen cells (B6 BMS) showed typical
manifestations of acute GVHD, such as mortality within 50 days,
progressive body weight loss, hantched posture, patchy alopecia, and
diarrhea, whereas the recipients of BM cells alone (B6 BM),
T-cell-depleted BM cells (B6 TCD-BM), or syngeneic BDF1 BM cells plus
2.5 × 107 spleen cells (BDF1 BMS) regained normal
body weight rapidly after transplantation and showed no manifestation
of acute GVHD. Survival rates of these recipients are shown in
Fig 1A. When anti-TNF- MoAb was
administered to the recipients of wild-type B6 BMS, it successfully
prevented the development of acute GVHD with rapid body weight recovery
and survival over 80 days in all of the recipients (Fig 1B and C).
There was no apparent manifestation of acute GVHD in these recipients,
but liver sections obtained on day 28 showed slight infiltration of
mononuclear cells (data not shown). The recipients of FasL-defective
gld BMS also exhibited long survival (>80 days) without
hantched posture, alopecia, or diarrhea, but recovery of body weight
was slightly impaired as compared with the anti-TNF- group. The
recipients of perforin-deficient PKO BMS exhibited a survival rate of
80% on day 80 with slightly impaired recovery of body weight and
patchy alopecia in the skin, but did not manifest hantched posture or
diarrhea. Liver sections harvested from the recipients of gld
BMS or PKO BMS on day 28 exhibited moderate infiltration of mononuclear
cells around bile ducts (data not shown). Because these recipients
survived over 100 days, this histological manifestation is not
considered to be the cause of mortality. These results indicated that
the TNF-, FasL, and perforin systems all play crucial roles in this
model of lethal acute GVHD.
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| Fig 1.
Mortality and body weight during acute GVHD after
allo-BMT. (A) Lethal GVHD was induced by transfer of B6 BM cells plus
2.5 × 107 spleen cells (B6 BMS) into lethally irradiated
BDF1 mice. Recipients of B6 BM cells (B6 BM), B6 T-cell-depleted BM
cells (B6 TCD-BM), or syngeneic BDF1 BM cells plus spleen cells (BDF1
BMS) manifested no signs of GVHD and almost all recipients survived
more than 80 days, except for 1 recipient of B6 BM. (B and C)
Contribution of TNF--, FasL-, and perforin-mediated cytotoxic
pathways to lethal acute GVHD. Lethally irradiated BDF1 mice were
transplanted with BM cells plus 2.5 × 107 spleen cells
from wild-type B6 (B6 BMS), B6-gld (gld BMS), or
perforin-deficient (PKO BMS) mice. In another group, B6 BMS recipients
were administered with anti-TNF- MoAb (B6 BMS + anti-TNF-). Mortality (B) and body weight (C) were monitored at the
indicated days after BMT. Administration of anti-TNF- MoAb to the
B6 BMS almost completely ameliorated the mortality and the body weight
loss resulted from acute GVHD. Survival of the gld BMS and PKO
BMS recipients was 100% and 80%, respectively, on day 80 after BMT.
The data represent the results of three similar experiments.
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Characterization of L1210 leukemia and P815 mastcytoma cells.
We used L1210 T-cell leukemia and P815 mastcytoma cells in our GVL
model. Both L1210 and P815 are derived from a DBA/2 (H-2d)
mouse. Both tumor cells showed moderate expression of Fas and low
expression of TNF-RI and TNF-RII detected by flow cytometry (data not
shown). Intravenous (IV) inoculation of 1 × 104 L1210 and 1 × 105 P815 cells into
BDF1 (H-2b/d) mice resulted in 100% death of recipients
within 8 and 12 days, respectively, with marked hepatosplenomegaly. B6
(H-2b) mice completely rejected these cells (data not shown).
To determine how L1210 and P815 cells could be eliminated in vivo in
the GVL model, we first examined the susceptibility of L1210 and P815
cells to TNFR- or Fas-mediated cytotoxicity in vitro. L1210 and P815
cells were cultured in the presence of recombinant mouse TNF- or
cultured with mouse FasL transfectant (mFasL/L5178Y) cells. Neither
L1210 nor P815 was susceptible to TNF- in vitro, whereas L929
fibrosarcoma showed high susceptibility to TNF- (Fig 2A). Although both L1210 and P815
cells express Fas, their in vitro susceptibilities to Fas-mediated
cytotoxicity were different. As shown in Fig 2B, P815 cells were
susceptible to Fas-mediated cytotoxicity, whereas L1210 were not. We
next examined whether spleen cells from wild-type, gld, or PKO
B6 mice can equally respond to L1210 or P815 cells. Proliferative
responses of the gld or PKO spleen cells to irradiated L1210 or
P815 cells were comparable with that of wild-type B6 spleen cells (Fig
2C). We also tested CTL activity after the in vitro stimulation of
wild-type, gld, or PKO spleen cells with irradiated target
tumor cells (Fig 2D). PKO-derived CTL exhibited almost no or little
killing activity against both L1210 and P815 cells, whereas killing
activity of CTL from gld mice was almost comparable to that
from wild-type B6 (Fig 2D). These in vitro results suggested that L1210
cells would be killed by predominantly by the perforin pathway but not by Fas or TNF- pathway and that P815 cells would be killed by CTLs
predominantly by the perforin pathway despite their susceptibility to
FasL transfectants.
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| Fig 2.
Characterization of L1210 murine leukemia and P815 murine
mastcytoma cells. (A) L1210 and P815 cells were cultured in the
presence of recombinant TNF- for 24 hours and then cell viability
was analyzed using the alamar Blue method. Both L1210 and P815 were
resistant to TNF-, whereas L929 was susceptible to TNF-. (B)
51Cr-labeled L1210 or P815 cells were cocultured with
murine FasL transfectant (mFasL/L5178Y) cells at the indicated E/T
ratios for 6 hours and then cytotoxicity was measured by
51Cr-release assay. (C) Proliferative response of spleen
cells obtained from wild-type, gld, or PKO B6 mice against
allogeneic L1210 or P815 cells. Spleen cells (2 × 105)
were cultured with irradiated L1210 or P815 cells (2 × 104) for 5 days and pulsed with 3H-TdR during
the last 16 hours. Spleen cells were prepared from one mouse of each
strain and were used immediately after preparation. Data are
represented as the mean ± SD of triplicated samples. Spleen cells
obtained from each mouse responded equally to L1210 or P815 cells. (D)
Killing of L1210 cells by CTL derived from wild-type, gld, and
PKO B6 mice. Spleen cells were collected after 5 days of coculture with
irradiated L1210 cells as described in (B) and restimulated with
irradiated L1210 cells in the presence of 10 U/mL IL-2 for 5 days. CTL
activity was tested against 51Cr-labeled L1210 or P815
target cells at the indicated E/T ratios. Although killing activity of
CTL derived from gld mice was only slightly diminished as
compared with that from wild-type mice, PKO-derived CTL exhibited no
significant cytotoxicity against L1210 cells. Data are represented as
the mean ± SD of triplicated samples. A representative of three
experiments is shown.
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Establishment of the GVL model.
IV inoculation of 1 × 104 L1210 leukemia cells into
BDF1 mice resulted in 100% mortality within 8 days with marked
hepatosplenomegaly when the recipients received only irradiation 2 days
after inoculation without BMT (Fig 3A).
When BMT was performed 2 days after leukemia inoculation, all
recipients of B6 TCD-BM and BDF1 BMS died within day 12 and 14, respectively, with marked hepatosplenomegaly. Liver sections obtained
from these recipients showed massive leukemia infiltration
(Fig
4B). Thus, these two groups of recipients exhibited poor GVL effect.
The recipients of B6-BM survived longer than these two groups and did
not manifest apparent hepatosplenomegaly. However, these recipient mice
finally died within 60 days, probably due to the leukemia, because
their liver sections obtained on day 28 showed residual leukemia cells
(Fig 4C) with only mild infiltration of mononuclear cells around the
bile ducts and almost all recipients of B6 BM without leukemia
inoculation survived more than 80 days with no apparent manifestation
of GVHD (Fig 1A). In contrast, the recipients
of B6 BMS exhibited a similar survival to the recipients without
inoculation of leukemia cells and sections of the liver obtained on day
28 showed manifestations of severe acute GVHD, but no residual leukemia
cell (Fig 4D). Similar results were observed by using another tumor
P815 cells (Fig 3B). These results indicated that the transfer of B6
BMS exerted both GVL effect and lethal acute GVHD in this system.
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| Fig 3.
GVL effect after allo-BMT. GVL effect was evaluated in
BDF1 mice that were inoculated IV with 1 × 104 L1210
cells (A) or 1 × 105 P815 cells (B) 2 days before the BMT
as described in Fig 1A. (A) Mice inoculated with L1210 and P815 cells
died within 8 and 10 days, respectively, when they received irradiation
alone without BMT (no BMT). Transfer of B6 TCD-BM or syngeneic BDF1 BMS
resulted in early leukemia relapse. Recipients of B6 BM or BMS survived
longer than the above-noted three groups, but all mice died within 60 days. Data represent the results of three similar experiments.
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| Fig 4.
Histopathological examination of the liver
obtained from normal BDF1 and the recipients of B6 TCD-BM, B6 BM, or B6
BMS in the GVL experiment shown in Fig 3A. (A) Liver section from an
age-matched normal BDF1 mouse. (B) Massive leukemia cell infiltration
was observed in the liver section from the recipients of B6 TCD-BM at
the time of death. (C) Liver section obtained from the recipients of B6
BM on day 28 after BMT showed residual leukemia cells among normal
liver tissue. (D) Liver section from the recipients of B6 BMS on day 28 after BMT showed severe mononuclear cell infiltration, but there was no
residual leukemia cell in any fields of the section. Each figure shows
a representative of three similar experiments. Similar results were
also observed by using another tumor P815 cells. Original magnification × 400.
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Contribution of TNF-, FasL, and perforin to the GVL effect.
We showed that lethal acute GVHD could be ameliorated by modulation of
either TNF-, FasL, or perforin system (Fig 1B). Considering clinical
application, it is important to examine how these modulations affect
the GVL effect. We then used gld or PKO B6 mice as the BMS
donors and anti-TNF- MoAb in our GVL model (Fig 5A
and B). Interestingly, after inoculation of L1210 cells, all B6 BMS
recipients that received anti-TNF- MoAb died within 12 days with
marked hepatosplenomegaly (Figs 5A and 6A), and the liver sections
obtained from these mice showed massive infiltration of leukemia cells (Fig 6B). On the other hand, the recipients of
gld BMS exhibited survival rates of 100% on day 28 and 60% on
day 80 (Fig 5A). No residual leukemia cell was observed in liver,
spleen, BM, and peripheral blood in representative mice killed on day
28 or mice that died on days 24, 35, 48, and 55 (not all data shown).
Liver sections obtained from representative mice on day 28 showed focal infiltration of mononuclear cells in portal areas, but there was no
leukemia infiltration (Fig 6C). Although the cause of death of these 4 mice that died within 80 days was not clear, cachexia and/or infection
were suspected because these mice showed a slight body weight loss and
some mice showed manifestation of infection when they died. In fact,
serum TNF- levels on day 7 were relatively higher in recipients
inoculated with tumor cells before BMT than recipients without tumor
cells (data not shown). All recipients of PKO BMS died within 60 days
and their liver sections on day 28 showed residual leukemia cells (Fig
6D). These residual leukemia cells might contribute to the mortality,
because the recipients of PKO BMS could survive more than 80 days
without leukemia inoculation (Fig 1B). All recipients of gld or
PKO BMS received anti-TNF- MoAb died within day 12 (data not
shown). Similar results were obtained by using another tumor P815
cells, which are sensitive to in vitro Fas-mediated cytotoxicity, in
contrast to L1210 (Fig 5B). These results indicated that TNF- and to
a lesser extent perforin play critical roles in the GVL effect in the
present system and that the blockade of FasL could spare the GVL effect while preventing lethal acute GVHD.
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| Fig 5.
Contribution of TNF--, FasL-, or perforin-mediated
cytotoxicity to the GVL effect. GVL effect was evaluated in BDF1 mice
that were inoculated IV with 1 × 104 L1210 or 1 × 105 P815 cells 2 days before the BMT as described in Fig
1B. (A) Administration of anti-TNF- MoAb to the recipients of B6
BMS resulted in early death within 16 days. Six of 10 gld BMS
recipients survived more than 80 days, whereas all B6 BMS or PKO BMS
recipients died within 51 days. (B) Similar results were also observed
by using another tumor P815 cells. Administration of anti-TNF- MoAb
resulted in early death within 24 days. Six of 8 gld BMS
recipients survived more than 80 days, whereas all B6 BMS or PKO BMS
recipients died within 47 days. Data represent the results of three
similar experiments.
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| Fig 6.
Histopathological examination of the GVL
experiments shown in Fig 5A. (A) Administration of anti-TNF- MoAb
resulted in early death with marked hepatosplenomegaly (left) compared
with an age-matched normal mouse (right). The liver section obtained at
the time of death showed massive leukemia infiltration (B). (C) Liver
section obtained from gld BMS recipients on day 28 showed focal
mononuclear infiltration around portal area, but there was no residual
leukemia cells in any fields of the section. Residual leukemia cell was
not detected in spleen, BM, or peripheral blood. (D) Residual leukemia
cells were observed in small areas of the liver section from the PKO
BMS recipients. Each figure shows a representative of three similar
experiments. Similar results were also observed by using another tumor
P815 cells. Original magnification × 400.
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| |
DISCUSSION |
It is now established that mature T cells in the marrow graft are
responsible for both initiating acute GVHD and exerting GVL
effect.3-6,19,22 A dramatic decrease in the incidence of GVHD after clinical allo-BMT could be achieved by employing various marrow manipulation protocols designed to eliminate T cells from the
donor graft.35-39 However, one potential disadvantage of
this approach is that the transfer of T-cell-depleted marrow has been associated with a higher incidence of leukemic
relapse.7,22,40-42 Although it has been reported that both
T and NK cells were responsible for the GVL effect,23-32 it
remains still unclear whether GVHD and the GVL effect are mediated by
distinct lymphocyte subpopulations and can be separately mediated. In a
human in vitro study, ricin-conjugated anti-CD25 MoAb selectively
depleted response of donor lymphocytes to recipient lymphoblasts while
conserving response against leukemia cells.23 It has been
shown that IL-2 could be administered to recipient mice to enhance the
antitumor effect of NK cells without exacerbation of GVHD and that NK
cells rather suppressed GVHD at least in part through the production of
transforming growth factor- (TGF-).24 In
contrast to these previous reports describing the cellular mechanisms
of the GVL effect, our present study is the first demonstration that
GVHD and the GVL effect can be differentiated by their molecular
mechanisms of the cytotoxic pathway at the effector phase. In our GVL
model, blockade of the Fas/FasL interaction, using FasL-defective
gld mice as a donor, efficiently prevented lethal acute GVHD
without impairing the GVL effect. Blockade of the perforin pathway,
using PKO mice as a donor, could ameliorate acute GVHD but failed to
exert sufficient GVL effect. Thus, these results suggest that blockade
of the Fas/FasL pathway could prevent lethal acute GVHD while
preserving sufficient GVL effect in patients with certain leukemias.
In the present parent to F1 BMT system, all the TNF-, FasL, and
perforin pathways were shown to contribute to the mortality of acute
GVHD. These results are almost consistent with previous reports but
different in some points.9-11,13-17 In contrast to the report by Baker et al,11 almost all recipients of
gld or PKO BMS showed long survival (>80 days) and did not
manifest severe cachexia (Fig 1B and C). Liver sections from the
gld BMS recipients on day 28 after BMT showed moderate
mononuclear cell infiltration around bile ducts (data not shown),
whereas minimal inflammatory change was observed in the recipients of
gld T cells in their report.11 These differences
may result from different BMT systems (B6 to BDF1 BMT v B6 to
LP/J BMT).
Recently, two predominant molecular mechanisms of lymphocyte-mediated
cytotoxicity have been shown.43-47 The perforin/granzyme pathway is the dominant one for CD8+ CTL and NK cells that
play a classical defense role against infected or transformed host
cells.43,45 On the other hand, the Fas pathway needs Fas
receptor expression on target cells and may be principally involved in
regulating immune responses such as deletion of activated or
self-reactive T cells.43,44 It has been reported that
perforin is a key molecule for tumor surveillance by CD8+ T
cells and NK cells in vivo, whereas the Fas system may play only a
minor role even if tumor cells were transfected with Fas receptor.48,49 In our present study, L1210 cells are
resistant to Fas-mediated cytotoxicity and the GVL effect was thought
to be predominantly mediated by the perforin pathway. In this regard, most human tumor cells have been shown to be resistant to Fas-mediated apoptosis even if Fas receptor is expressed on these
cells.50-55 Dirks et al54 showed that most
myeloid cell lines were resistant to anti-Fas antibody-induced
apoptosis. Karawajew et al55 reported that Fas receptor was
expressed by most of leukemic blasts obtained from patients with T- and
B-lineage acute lymphoblastic leukemia, but that cross-linking of these
receptors by anti-Fas antibody was either not able to initiate
apoptosis or induced low rates of apoptosis. In addition, our
hypothesis that blockade of the Fas/FasL interaction prevent lethal
acute GVHD without impairing the GVL effect was also confirmed by using
Fas-sensitive P815 cells in our GVL model. These results suggest that
the Fas/FasL pathway seems not to be a major pathway when CTL kills
leukemia cells. Therefore, it is considered that the perforin pathway
may play an essential role in elimination of residual leukemia cells after allo-BMT and thus the perforin pathway should be preserved for
exerting the GVL effect. It is also advantageous to spare the perforin
pathway for preventing infectious disease, which is one of major
complications after allo-BMT, because infected host cells are
eliminated predominantly by this pathway. Because the Fas/FasL system
makes only a minor contribution to the GVL effect, it should be
impaired to reduce acute GVHD.
Our present results also showed a marked impairment of the GVL effect
in the recipients receiving anti-TNF- MoAb. The L1210 cells used
were not susceptible to recombinant TNF- in vitro (Fig 2A). It has
been reported that the soluble form (sTNF) and membrane-bound form
(mTNF) of TNF- exhibit different cytotoxicity.56-58 mTNF, but not sTNF, can induce target cell apoptosis via TNFR p75,
whereas both can induce apoptosis via TNFR p55. Because flow cytometric
analysis showed the expression of both TNFR p55 and p75 on the surface
of L1210 cells (data not shown), L1210 might be killed by mTNF
expressed on CD8+ CTL and/or activated macrophages.
Alternatively, it is possible that TNF- serves as an
immunoregulatory factor. Because TNF- is thought to be involved in
both induction and effector phases of GVHD,59,60
administration of anti-TNF- MoAb might diminish not only direct
cytotoxic activity of TNF-, but also T-cell activation responsible
for the GVL effect, resulting in the abrogation of both GVHD and the
GVL effect.
In summary, we have established a murine GVL model that enabled us to
demonstrate the differential contribution of cytotoxic effector
mechanisms to the GVL effect and GVHD. Although only two murine tumor
cell lines were tested in the present study, this model suggested that
GVHD and the GVL effect can be separately modulated. In our present
system, blockade of the Fas pathway is thought to be most suitable for
preventing lethal acute GVHD without impairing the GVL effect. Further
studies with various tumors are needed to generalize this notion. It is
also necessary to investigate the contribution of Fas/FasL interactions
to the GVL effect other than the direct cytotoxicity such as by
promoting the expansion and maturation of donor T cells as previously
described by Via et al.14 A better understanding of the
molecular mechanisms involved in GVHD and the GVL effect would allow
the development of new strategies to prevent GVHD while enhancing the
beneficial GVL effect, which will decrease the incidence of leukemic
relapse after allo-BMT. We hope our present results provide a new
prospect in the field of clinical BMT.
| |
ACKNOWLEDGMENT |
The authors thank K. Saito, K. Seino, K. Takeda, T. Kodama, and K. Kayagaki for technical assistance and helpful suggestions.
| |
FOOTNOTES |
Submitted May 18, 1998; accepted December 10, 1998.
Supported by grants from the Ministry of Education, Science and Culture
and the Ministry of Health, Japan.
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 Ko Okumura, MD, The Department of
Immunology, Juntendo University School of Medicine, 2-1-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan; e-mail:
kokumura{at}med.juntendo.ac.jp.
| |
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J. Leukoc. Biol.,
September 1, 2003;
74(3):
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[Abstract]
[Full Text]
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E. Orsini, R. Bellucci, E. P. Alyea, R. Schlossman, C. Canning, S. McLaughlin, P. Ghia, K. C. Anderson, and J. Ritz
Expansion of Tumor-specific CD8+ T Cell Clones in Patients with Relapsed Myeloma after Donor Lymphocyte Infusion
Cancer Res.,
May 15, 2003;
63(10):
2561 - 2568.
[Abstract]
[Full Text]
[PDF]
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C. Schmaltz, O. Alpdogan, S. J. Muriglan, B. J. Kappel, J. A. Rotolo, E. T. Ricchetti, A. S. Greenberg, L. M. Willis, G. F. Murphy, J. M. Crawford, et al.
Donor T cell-derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation
Blood,
March 15, 2003;
101(6):
2440 - 2445.
[Abstract]
[Full Text]
[PDF]
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P. Reddy, T. Teshima, G. Hildebrandt, U. Duffner, Y. Maeda, K. R. Cooke, and J. L. M. Ferrara
Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation
Blood,
October 16, 2002;
100(9):
3429 - 3431.
[Abstract]
[Full Text]
[PDF]
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A. Janin, C. Deschaumes, M. Daneshpouy, J. Estaquier, J. Micic-Polianski, P. Rajagopalan-Levasseur, K. Akarid, N. Mounier, E. Gluckman, G. Socie, et al.
CD95 engagement induces disseminated endothelial cell apoptosis in vivo: immunopathologic implications
Blood,
April 15, 2002;
99(8):
2940 - 2947.
[Abstract]
[Full Text]
[PDF]
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U. F. Hartwig, M. Robbers, C. Wickenhauser, and C. Huber
Murine acute graft-versus-host disease can be prevented by depletion of alloreactive T lymphocytes using activation-induced cell death
Blood,
April 15, 2002;
99(8):
3041 - 3049.
[Abstract]
[Full Text]
[PDF]
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J. Liu, B. E. Anderson, M. E. Robert, J. M. McNiff, S. G. Emerson, W. D. Shlomchik, and M. J. Shlomchik
Selective T-cell subset ablation demonstrates a role for T1 and T2 cells in ongoing acute graft-versus-host disease: a model system for the reversal of disease
Blood,
December 1, 2001;
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3367 - 3375.
[Abstract]
[Full Text]
[PDF]
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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
Unlocking the secrets of cytotoxic granule proteins
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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
Blood,
May 1, 2001;
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[Abstract]
[Full Text]
[PDF]
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M. H. Hsieh and R. Korngold
Differential use of FasL- and perforin-mediated cytolytic mechanisms by T-cell subsets involved in graft-versus-myeloid leukemia responses
Blood,
August 1, 2000;
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[Abstract]
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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,
May 1, 2000;
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[Abstract]
[Full Text]
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G. R. Hill, T. Teshima, V. I. Rebel, O. I. Krijanovski, K. R. Cooke, Y. S. Brinson, and J. L. M. Ferrara
The p55 TNF-{alpha} Receptor Plays a Critical Role in T Cell Alloreactivity
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January 15, 2000;
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION