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TRANSPLANTATION
From the Departments of Pediatrics and Medicine,
Memorial Sloan-Kettering Cancer Center, New York, NY; the Department of
Pediatric Oncology, Dana Farber Cancer Institute, Boston, MA; and the
Departments of Internal Medicine and Pediatrics, University of
Michigan, Ann Arbor, MI.
In allogeneic bone marrow transplantation (BMT) donor T cells are
primarily responsible for antihost activity, resulting in graft-versus-host disease (GVHD), and for antileukemia activity, resulting in the graft-versus-leukemia (GVL) effect. The relative contributions of the Fas ligand (FasL) and perforin cytotoxic pathways
in GVHD and GVL activity were studied by using FasL-defective or
perforin-deficient donor T cells in murine parent Allogeneic bone marrow transplantation (BMT) is an
important therapeutic modality for a variety of diseases, including
hematologic malignancies. The therapeutic benefits of allogeneic BMT
are not only derived from the high dose of chemoradiation but also from a graft-versus-leukemia (GVL) effect.1-3 Clinical evidence
for a GVL effect comes from studies demonstrating an increased relapse rate after BMT from an identical twin, autologous BMT, and T-cell depletion of the allogeneic bone marrow (BM) graft (reviewed in Truitt
and Johnson3 and Antin4). Most studies
indicate that the GVL effect is primarily mediated by allogeneic donor
T cells, which recognize either leukemia-specific antigens or
alloantigens expressed on normal and malignant
cells.5-8
Graft-versus-host disease (GVHD) remains the single most important
complication of allogeneic BMT and is defined as a progressive systemic
illness with immunosuppression, cachexia, and specific target organ
disease of the skin, liver, and intestines.9 Although the
complex pathophysiology of acute GVHD involves the conditioning regimen
(radiation or chemotherapy), cytokines, nitric oxide, and non-T
effector cells (reviewed in Krenger et al10), the cytolytic activity of donor T cells is essential for the
development of GVHD activity.
Cytolytic activity of cytotoxic T lymphocytes (CTL) is primarily
mediated through 2 effector mechanisms: the Fas-FasL and perforin-granzyme pathways.11,12 Interaction of FasL,
expressed on the CTL cell surface, with the Fas receptor on the target
cell membrane results in the initiation of the Fas cell death pathway, which involves the activation of a caspase cascade.13 The
perforin-granzyme pathway consists of the exocytosis of lytic granules,
which results in the release of perforin (a pore-forming protein
related to the membrane attack complex of complement) and granzymes
(Ca++-dependent serine esterases that can activate the
caspase cascade).14 Some studies have suggested that the
expression (or secretion) of tumor necrosis factor (TNF) and tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL) can also
contribute to CTL cytotoxicity.15,16
Most attempts to characterize and separate GVHD and GVL activity have
focused on the identification of specific GVHD or leukemia antigens,
donor T-cell dose, selective donor T-cell depletion (such as CD8),
delayed leukocyte infusion (DLI), in vitro polarization of donor T
cells (Th1/Tc1 or Th2/Tc2), and cytokines (such as IL-2). Several
studies have demonstrated the importance of the FasL and perforin
pathways for the development of GVHD.17-24 However, efforts to manipulate GVHD and GVL activity through the effector pathways of T-cell-mediated cytotoxicity (FasL and perforin) have been
limited.24
We hypothesized that donor T cells make differential use of their
cytotoxic effector pathways to mediate GVHD and GVL activity. To test
this hypothesis, we performed experimental BMT studies in 2 murine
parent Cell lines and reagents
Antimurine CD16/CD32 Fc block (2.4G2), antimurine Ly-9.1-fluorescein
isothiocyanate (FITC), antimurine CD4-FITC, -phycoerythrin (PE), and
-PerCP (RM4-5), antimurine CD8 Mice and BMT
CD4+ or CD8+ splenic T-cell separation In some BMT experiments, splenic T cells were further selected after RBC lysis and nylon wool passage. Cells were incubated with anti-CD4 (or anti-CD8) antibodies and anti-CD45R antibodies (both coupled to microbeads) for 15 minutes at 4°C. After 2 washes with PBS (containing 1% fetal calf serum), cells were passed through Midimacs columns (Miltenyi). Samples of the negatively selected cells were subsequently analyzed for purity by flow cytometry after staining with PE-labeled anti-CD4 (or anti-CD8) antibodies. Equal numbers of purified CD4+ (or CD8+) splenic T cells were then mixed with B6 BM cells and infused into recipient animals.Assessment of graft-versus-host disease The severity of GVHD was assessed with a clinical GVHD scoring system as first described by Cooke et al.27 Briefly, ear-tagged animals in coded cages were individually scored every week for 5 clinical parameters on a scale from 0 to 2, including weight loss, hunched posture, decreased activity, fur ruffling, and skin lesions. A clinical GVHD index was generated by summation of the 5 criteria scores (0-10). Survival was monitored daily.Leukemia induction and assessment of leukemic death versus death from GVHD Animals received P815 or 32Dp210 cells intravenously in a separate injection on day 0 of BMT. Survival was monitored daily, and the cause of each death after BMT was determined as previously described.28 Briefly, we were able to distinguish lethal GVHD from leukemic death by hepatosplenomegaly (for 32Dp210 leukemia) or macroscopic liver-spleen metastases (P815) at autopsy and by clinical GVHD score (including weight loss). In our previous studies, we established hepatosplenomegaly as a highly specific criterion for death from 32Dp210 leukemia (spleen weight greater than 300 mg, liver weight greater than 1700 mg). In our experiments, we did not find hepatosplenomegaly in animals that had not received 32Dp210 cells. P815-induced death was characterized by the presence of macroscopic liver nodules, spleen nodules, or both. Death from GVHD was defined as the absence of leukemia signs and the presence of GVHD symptoms, as assessed by our clinical GVHD scoring system (described above).At autopsy Cells and serum As described before, splenic T cells were obtained by purification over a nylon wool column; this was followed by RBC removal with ammonium chloride RBC lysis buffer, which resulted in greater than 75% purity. On day 14 after BMT, splenocytes from recipients used in cytotoxicity and proliferation assays were first pretreated with ammonium chloride to remove RBCs, and percentages of CD4+ and CD8+ T cells from donor origin were subsequently determined by 2-color flow cytometry. Splenocytes from naive animals were pretreated with ammonium chloride RBC lysis buffer only before use. Peripheral blood was obtained by retro-orbital or intracardiac puncture after general anesthesia with methoxyflurane (Schering-Plough Animal Health, Union City, NJ).Flow cytometric analysis Splenocytes or murine leukemia cell lines were washed in FACS buffer (PBS/2% BSA/0.1% azide), and 106 cells/mL were incubated for 30 minutes at 4°C with CD16/CD32 Fc block. Subsequently, cells were incubated for 30 minutes at 4°C with primary antibody or antibodies (1 µg/mL) and washed twice with FACS buffer. Stained cells were resuspended in FACS buffer and analyzed on a Facscan flow cytometer (Becton Dickinson, San Jose, CA) with Cellquest (Becton Dickinson) software.Intracellular cytokine staining Briefly, cells were incubated for 4 hours (for PMA-ionomycin stimulation) or for 12 to 15 hours (for MLR) with Brefeldin A (10 µg/mL). Then they were harvested, washed, and stained with primary (surface) fluorochrome (FITC, PerCP, and APC)-conjugated antibodies, fixed and permeabilized with the Cytofix/Cytoperm Kit (Pharmingen, San Diego, CA), and subsequently stained with secondary (intracellular cytokine) PE-conjugated antibody. FACS analysis was conducted by gating for the designated populations. Flow cytometer and software were used as mentioned before.Chromium 51 release assays Target cells were labeled with 100 µCi chromium 51 (51Cr) at 2 × 106 cells/mL for 2 hours at 37°C and 5% CO2. After 3 washes, labeled targets were plated at 104 to 2 × 104 cells/well in U-bottom plates (Costar, Cambridge, MA). Effector cells were prepared (details below) and added at various effector-to-target ratios in a final volume of 200 µL and were incubated for 4 hours (allogeneically stimulated T cells) or 8 hours (activated DO11.10 and mFasL-L5178Y cells) at 37°C and 5% CO2. Subsequently, 100 µL supernatant was removed from each well and counted in a gamma counter to determine experimental release (Cobra, Meriden, CT). Spontaneous release was obtained from wells receiving target cells and medium only, and total release was obtained from wells receiving 1% Triton X-100. Spontaneous release was less than 15% of total release. Percentage cytotoxicity was calculated by the following formula: % cytotoxicity = 100 × (experimental release spontaneous release)/(total release spontaneous release).
To determine FasL-mediated lysis by activated DO11 cells, 96-well plates were coated with antimurine CD3 (145-C11) at 1 µg/mL in PBS for 24 hours at 4°C. Subsequently, DO11.10 cells were incubated with FasL-antibody (MFL3) or isotype control (A19-3) (both obtained from Pharmingen) in these antibody-coated 96-well plates for 3 hours and subsequently coincubated with labeled target cell lines as described above. For allogeneic stimulation, splenocytes (8 × 104) were incubated with irradiated C3FeB6F1 splenocytes (4 × 104) in 96-well plates, and 10 U/mL human IL-2 (Chiron, Emoryville, CA) was added on day 3. Cells were used as effectors on day 7 against lipopolysaccharide (LPS)-stimulated C3FeB6F1 splenocytes. LPS-stimulated cells were obtained by incubating whole splenocytes for 72 hours with 1.6 to 4 µg/mL LPS (Sigma). Proliferation assay Splenic T cells (4 × 105 cells/well; prepared as described above) were incubated for 3 days with irradiated (2000 cGy) C3FeB6F1 splenocytes as stimulators (2 × 105 cells/well) in 96-well plates. Cultures were pulsed during the final 18 hours with 1 µCi/well [3H] thymidine, and DNA was harvested on a Harvester 96 (Tomtec, Hamden, CT).Tumor necrosis factor enzyme-linked immunosorbent assay The TNF enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D Systems. Assays were performed according to the manufacturer's protocol. Briefly, serum samples were diluted at ratios of 1:2 or 1:4 and were incubated in wells coated with specific anti-TNF antibody. After several washes, wells were incubated with a secondary antibody coupled to biotin. The biotin-labeled assays were developed with streptavidin and substrate and were read at 450 nm with a microplate reader (Bio-Rad, Hercules, CA). Recombinant murine TNF was used as a standard. Samples and standards were run in duplicate, and the sensitivity of the assays was 10 to 20 pg/mL.Statistics All values are expressed as mean ± SEM. Statistical analysis of clinical GVHD index scores and weight losses was performed with the nonparametric unpaired Mann-Whitney U test, whereas the Mantel-Cox log rank-test was used for survival data. P < .05 was considered statistically significant.
B6  F1 murine model for
allogeneic BMT with a full mismatch for major histocompatibility
complex (MHC) class I and II (B6 [H-2b] into C3FeB6F1/J
[H-2b/k]) to study the roles of the FasL and perforin
effector pathways in GVHD and GVL activity. The advantages of this BMT
model are that GVL and GVHD activity has been well defined in this
model,30 that it allows the use of CML 32Dp210
(H-2k),25 and that FasL-defective and
perforin-deficient mice on a B6 background can be used as donors. In
all experiments, we used 5 × 106 T-cell-depleted B6
donor BM cells and 1300 cGy split-dose lethal total body irradiation.
In our initial experiments, lethally irradiated C3FeB6F1/J recipients
received T-cell-depleted BM (TCD-BM) with or without
2 × 106 donor B6 splenic T cells. On day 0, 32Dp210
leukemia cells (103 cells) were given with the donor cell
inoculum. T-cell depletion consistently prevented GVHD, but all animals
succumbed to leukemia (Figure 1A). The
addition of T cells to the TCD-BM inoculum induced severe GVHD with
high mortality, but leukemia did not develop in the GVHD survivors,
demonstrating the GVL activity of the donor T cells as previously
described.28
FasL-defective B6.gld donor T cells display diminished GVHD activity but have intact GVL activity We used splenic T cells (2 × 106) from FasL-defective B6.gld and perforin-deficient B6.pfp / mice as donor T-cell inoculum to examine
whether donor T-cell-mediated GVL and GVHD activity can be
differentiated by their cytotoxic pathways. B6.gld
(generalized lymphoproliferative disease) mice have a spontaneous point
mutation in the FasL gene that renders the protein
nonfunctional.31 They have a defect in peripheral tolerance and activation-induced cell death that results in progressive lymphoid hyperplasia (adenopathy and splenomegaly) and auto-antibody production.13 B6.pfp/ mice have a defect
in CTL- and natural killer cell-mediated cytotoxicity32,33 and decreased tumor surveillance.34 Figure 1B-D
demonstrates the development of GVHD and leukemia as determined by
weight loss (Figure 1B), clinical GVHD score (Figure 1C), and overall
survival (Figure 1D). Recipients of B6 TCD-BM only did not have GVHD,
but all animals died of leukemia (Figure 1A). Survival in recipients of
TCD-BM + FasL-defective B6.gld T cells was
significantly better than in the other groups, with only a 6%
mortality rate from GVHD and no leukemic deaths (recipients of
B6.gld T cells vs other recipients;
P < .0001). In contrast, recipients of TCD-BM + perforin-deficient B6.pfp/ T cells had lethal GVHD,
comparable to that in recipients of TCD-BM + normal B6 T cells.
None of these recipients of B6 T cells or B6.pfp/ T
cells had leukemia.
We repeated these experiments without leukemia inoculation at the time
of BMT to eliminate any effects leukemia cells could have on the
development of GVHD. We found again that recipients of
B6.gld T cells had significantly less GVHD than recipients of B6 T cells or B6.pfp Perforin-deficient B6.pfp / T cells. However, 50% (8 of 16) of recipients
of TCD-BM + B6.pfp/ T cells succumbed to leukemia,
whereas none of the recipients of TCD-BM + B6 T cells or
B6.gld T cells had leukemia These results indicate that in
this GVHD/GVL model, the perforin pathway is important for donor
T-cell-mediated GVL activity, whereas FasL-defective donor T cells
displayed intact GVL activity, even at a higher tumor dose.
Interestingly, donor B6 or B6.gld T cells could mediate sufficient GVL activity, even at a dose that resulted in few
GVHD deaths.
B6.gld and B6.pfp / T
cells displayed identical specific proliferative responses to host
antigens that were diminished compared to wild-type B6 cells. Moreover,
we found comparable numbers of donor T cells in the spleen 14 days
after BMT (data not shown). Therefore, B6.gld and
B6.pfp/ T cells seem to be capable of a proliferative
response to C3FeB6F1 host antigens.
We then determined the cytokine profiles of alloreactive T cells from
B6, B6.gld, and B6.pfp We then determined the cytokine response of alloreactive splenic T
cells by analyzing donor splenic T cells from B6, B6.gld, or
B6.pfp In addition, we could not detect any significant expression of IL-4 or
IL-10 in donor splenic T cells from B6, B6.gld, or B6.pfp Antileukemic cytolytic activity from splenic T cells of BMT recipients is dependent on the perforin effector pathway Several studies in murine BMT models have indicated that donor T-cell expansion occurs in the first weeks after allogeneic BMT, with maximal donor T-cell expansion in the spleen by day 10 to 14 after BMT (reviewed in Hakim and Mackall35). To analyze the antileukemia activity of the donor T cells after BMT, we performed in vitro cytotoxicity assays with splenic donor T cells 14 days after BMT. Virtually all splenic T cells recovered from animals 14 days after BMT were of donor origin, as determined by flow cytometric analysis with antibodies against Thy1.2 (pan T-cell marker) and Ly9.1 (present on hematopoietic cells of C3FeB6F1/J host origin but not on B6 donor cells). We found significant lysis of 32Dp210 leukemia by donor T cells from recipients of TCD-BM + B6 T cells or B6.gld T cells, whereas splenic T cells from recipients of TCD-BM only or TCD-BM + B6.pfp/ T cells showed no
cytolytic activity against leukemic target cells (Figure
4A). These results support our in vivo
data that cytolytic activity of donor T cells against 32Dp210 leukemic
cells is mediated through the perforin effector pathway and not through the FasL pathway.
32Dp210 and P815 leukemia cells express the Fas receptor and are sensitive to Fas-induced apoptosis To evaluate whether the preferential use of the perforin pathway for GVL activity by donor T cells was influenced by an intrinsic resistance to Fas-mediated apoptosis of leukemia cells, we analyzed the expression of the Fas receptor and the sensitivity to Fas-mediated apoptosis of 32Dp210 and P815 (a mastocytoma model for the GVL effect that will be further described below) cells. The assessment of Fas sensitivity of Fas-expressing cells has been a topic of much debate. Recent studies have indicated that activation of the Fas receptor by FasL expressed on the surfaces of effector cells will result in a stronger signal more likely to induce apoptosis than Fas activation with soluble FasL or anti-Fas antibodies.26,36-40We first confirmed that both leukemia cell lines express the Fas receptor as described previously by us and others (Figure 4B).24,28 Subsequently, we determined Fas sensitivity of our leukemia cell lines using 2 effector cell lines that express FasL on their cell surfaces. First, we used a T-cell hybridoma cell line (DO11.10) that expresses FasL upon activation through the TCR/CD3 complex and that has been used by us and others as a FasL+ cell line.41 We found that both leukemia cell lines were lysed by FasL-expressing TCR/CD3-activated DO11.10 cells but not by unstimulated DO11.10 cells (Figure 4C). We confirmed that this cytolysis was FasL-dependent by the inhibition of apoptosis with neutralizing anti-FasL antibodies. In addition, we used a lymphoma cell line, L5178Y, that was stably transfected with membrane-bound FasL. When our leukemia cells were exposed to these FasL+ effector cells, we again observed significant cytolysis, whereas leukemia cells incubated with control L5178Y cells were not affected (Figure 4D). Interestingly, the percentage of cytolysis of each leukemia cell line was comparable to that of a highly Fas-sensitive control cell line, LK35.2. These data clearly demonstrate that our leukemia cell lines are Fas-sensitive. Therefore, donor T cells have the opportunity to use the FasL pathway to mediate GVL activity in both our experimental BMT models for GVHD and the GVL effect, but our experiments indicate that they preferentially use the perforin pathway for GVL activity. B6.pfp / T cells can develop
cytotoxic activity against allogeneic C3FeB6F1/J cells, we incubated
splenocytes from B6, B6.gld, and B6.pfp/
mice for 7 days with irradiated B6FeC3F1/J splenocytes as stimulators and subsequently used these cells as effector cells against
51Cr-labeled LPS-stimulated splenocytes from B6FeC3F1 mice.
Effector cells from B6.gld mice had no cytolytic activity
against the allogeneic targets, whereas effector cells from B6 and
B6.pfp/ mice could lyse allogeneic targets (Figure 4E).
These in vitro data correlate with our in vivo observations that B6 and
B6.pfp/ T cells have cytolytic activity against cells
from the recipient and can cause GVHD, whereas B6.gld T
cells are unable to exert cytolytic activity against host cells and
cause less GVHD.
Serum tumor necrosis factor levels do not significantly differ in any of the transplantation groups As discussed in the "Introduction," a number of studies have determined that TNF is a (third) important cytolytic pathway in the pathogenesis of GVHD (reviewed in Hattori et al,23 Herve et al,42 Holler et al,43 Piguet et al,44 Speiser et al,45 and Stuber et al46). To analyze the relative contribution of TNF to the development of GVHD in our murine BMT model, we measured serum TNF levels by ELISA at 14 (or 15) days after BMT in the recipients. As shown in Figure 5, we did not detect any significant differences in the serum TNF levels between the recipients of TCD-BM only, TCD-BM + B6 T cells, TCD-BM + B6.gld T cells, or TCD-BM + B6.pfp/ T
cells. However, these data do not exclude the possibility that membrane-bound TNF on GVHD-GVL effector cells or soluble TNF at the
site of inflammation in GVHD organs could play a role in the development of GVHD and the GVL effect.
FasL pathway is important for GVHD activity by donor CD4+ and CD8+ T cells but is not required for GVL activity by donor CD8+ T cells To analyze the relative contributions of CD4+ and CD8+ donor T cells and their cytolytic pathways in GVHD and the GVL effect, we performed experiments with CD4+ and CD8+ selected T cells. Highly purified donor T cells were obtained by enriching donor splenocytes by RBC lysis and nylon wool passage. Subsequently, we purified CD4+ and CD8+ T cells by negative selection after incubation with anti-B220 (CD45R) and anti-CD4 (or anti-CD8) antibodies coupled to microbeads. We included anti-B220 antibodies in this negative selection step not only to further deplete B cells, but especially to deplete B220+CD3+CD4CD8 T
cells from B6.gld splenic T cells. After this purification, the CD4+ or CD8+ selected T cells were analyzed
by flow cytometry before infusion to ensure that in each group animals
received equal numbers of purified CD4+ or CD8+
T cells.
Lethal GVHD developed rapidly in all recipients of B6 CD4+
T cells (106) and recipients of B6.pfp
GVHD activity is mediated through the FasL effector pathway, and GVL activity is mediated through the perforin pathway in B6D2F1/J recipients To further test our hypotheses regarding the roles of the FasL and perforin pathways in the GVL effect and GVHD, we used another well-established parent F1 murine model for allogeneic BMT with a
full mismatch for MHC class I and II (B6 B6D2F1). In our first experiments, B6D2F1/J recipients received lethal irradiation (1300 cGy)
and were transplanted with B6 TCD-BM (5 × 106 cells)
with or without splenic T cells from normal B6, B6.gld, or
B6.pfp/ mice. In this case, B6D2F1 recipients of
B6.pfp/ T cells had a 31% mortality rate from GVHD,
significantly lower than that of recipients of normal B6 T cells (75%;
P < .04) but significantly higher than that of recipients
of B6.gld T cells (6%; P < .04) (Figure
7A). These results suggest that in this BMT model for GVHD, both cytotoxic pathways are required for maximal GVHD activity by donor T cells, though the FasL pathway seems to be
most important. In additional experiments, we inoculated B6D2F1
recipients with P815 mastocytoma cells (1000 cells/mouse) at the time
of BMT and found a significant delay in deaths from leukemia in
recipients of B6 T cells or B6.gld T cells compared to
recipients of TCD-BM only, but not in recipients of
B6.pfp/ T cells (Figure 7B-C). This indicates again a
role for the perforin pathway in GVL activity, whereas FasL-defective
B6.gld donor T cells displayed GVL activity comparable to
that of normal B6 T cells.
Studies using mice with defects or deficiencies in the
perforin/granzyme pathway or the Fas/FasL pathway have demonstrated that the perforin/granzyme pathway is important in the CTL and natural
killer cell response against virally infected cells and malignant
cells,34,47,48 whereas the FasL pathway is mostly implicated in the regulation of the immune response. For example, perforin-deficient mice display increased vulnerability to a variety of
tumors and infections, whereas both Fas-deficient and FasL-defective mice develop a lymphoproliferative disorder with autoantibody production, lymphoid hyperplasia, and defects in peripheral tolerance and activation-induced cell death.13 Although
pfp Several authors have proposed that 3 cytotoxic pathways are involved in
the pathogenesis of GVHD: FasL/Fas, perforin/granzyme, and
TNF/TNFR.17-24,44,45,52,53 The importance of the Fas/FasL pathway in GVHD was demonstrated in spleen cell transfer models (parent
Several studies with pfp Evidence for the role of the TNF/TNFR pathway in GVHD comes from studies in murine BMT models demonstrating that the administration of anti-TNF antibody to recipients can ameliorate GVHD24,44 and that the TNFR p55-deficient recipients of allogeneic BM with T cells had lower mortality rates, though TNF release and T-cell proliferation and cytotoxicity were similar to levels in control recipients.45 Clinical studies have shown that patients with GVHD have elevated serum TNF levels,43 and phase I/II trials with anti-TNF antibody treatment in patients with refractory GVHD demonstrated some improvement (especially in intestinal GVHD).42 We found in both BMT models, across MHC class I and II disparity, that
the FasL pathway is primarily responsible for GVHD activity. In
C3FeB6F1/J recipients, the use of FasL-defective gld donor T
cells resulted in a virtual elimination of GVHD deaths, whereas
B6D2F1/J recipients had significantly prolonged survival times. In
contrast, the use of pfp Our studies demonstrate that the FasL pathway is not important for GVL activity in either model tested, though we found in both models that leukemia cells displayed Fas on their surfaces and could undergo apoptosis in vitro when exposed to FasL+ effector cells. In both models, we found that the perforin pathway was important for GVL activity. We also demonstrated in vitro a concomitant loss of antitumor cytotoxicity by perforin-deficient lymphocytes. These findings are in agreement with our previous data regarding the importance of the perforin pathway in GVL activity by G-CSF-mobilized donor T cells56 and with the proposed role for the perforin pathway in tumor immunosurveillance, as demonstrated by the decreased tumor surveillance of perforin-deficient mice.34 Our data suggest that alloreactive donor T cells, through an unknown mechanism, select the FasL pathway for their GVHD activity but not for their GVL activity. Thus far, only one other study has evaluated the role of the effector
pathways in GVHD and the GVL effect. Tsukada et al24 used
the B6 Moreover, recipients in the study by Tsukada et al24 received TCD-BM from B6.gld mice that resulted in reconstitution with a FasL-defective hematopoietic system. This could result in the development of the B6.gld-related autoimmune and lymphoproliferative defects in the recipient with potentially lethal outcomes. In our experiments, we elected to use in all transplantation groups TCD-BM from normal B6 mice to avoid the confounding variable of hematopoietic reconstitution with FasL-defective or perforin-deficient stem cells. In conclusion, the in vitro and in vivo data presented here support the hypothesis that donor T cells make differential use of their cytotoxic pathways for GVHD and GVL activity. In the 2 murine BMT models tested, the FasL effector pathway was required for GVHD activity (but not for GVL activity), whereas the perforin pathway was important for GVL activity. This suggests that selective inhibition of a cytolytic pathway may represent a novel strategy for the separation of GVL from GVHD activity.
We thank Dr Geoffrey R. Hill for helpful discussions, Dr Miguel Perales for help with intracellular cytokine staining, Dr Andreas M. Hohlbaum and Dr Ann Marshak-Rothstein for providing the mFasL-transfected L5178Y cell line, and Dr Hai T. Nguyen for histopathologic examination of tissue specimens.
Submitted June 5, 2000; accepted January 10, 2001.
Supported by National Institutes of Health program project grant 5PO1 CA39542 (S.J.B.). M.R.M.v.d.B. is the recipient of a research award from the Amy Strelzer Manasevit Scholars Program for the study of posttransplantation complications, funded by The Marrow Foundation in cooperation with the National Marrow Donor Program.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marcel R. M. van den Brink, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Kettering 1118, Mailbox 111, 1275 York Ave, New York, NY 10021.
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© 2001 by The American Society of Hematology.
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M.-A. Perales, A. Diab, A. D. Cohen, D. W. Huggins, J. A. Guevara-Patino, V. M. Hubbard, M. E. Engelhorn, A. A. Kochman, J. M. Eng, F. Mortazavi, et al. DNA Immunization against Tissue-Restricted Antigens Enhances Tumor Immunity after Allogeneic Hemopoietic Stem Cell Transplantation J. Immunol., September 15, 2006; 177(6): 4159 - 4167. [Abstract] [Full Text] [PDF]
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B. J. Kappel, J. Pinilla-Ibarz, A. A. Kochman, J. M. Eng, V. M. Hubbard, I. Leiner, E. G. Pamer, G. Heller, M. R. M. van den Brink, and D. A. Scheinberg Remodeling specific immunity by use of MHC tetramers: demonstration in a graft-versus-host disease model Blood, March 1, 2006; 107(5): 2045 - 2051. [Abstract] [Full Text] [PDF]
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E. Waldman, S. X. Lu, V. M. Hubbard, A. A. Kochman, J. M. Eng, T. H. Terwey, S. J. Muriglan, T. D. Kim, G. Heller, G. F. Murphy, et al. Absence of beta7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine Blood, February 15, 2006; 107(4): 1703 - 1711. [Abstract] [Full Text] [PDF]
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V. M. Hubbard, J. M. Eng, T. Ramirez-Montagut, K. H. Tjoe, S. J. Muriglan, A. A. Kochman, T. H. Terwey, L. M. Willis, R. Schiro, G. Heller, et al. Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation Blood, November 1, 2005; 106(9): 3285 - 3292. [Abstract] [Full Text] [PDF]
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L. Quemeneur, L. Beloeil, M.-C. Michallet, G. Angelov, M. Tomkowiak, J.-P. Revillard, and J. Marvel Restriction of De Novo Nucleotide Biosynthesis Interferes with Clonal Expansion and Differentiation into Effector and Memory CD8 T Cells J. Immunol., October 15, 2004; 173(8): 4945 - 4952. [Abstract] [Full Text] [PDF]
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P. Reddy, T. Teshima, G. Hildebrandt, D. L. Williams, C. Liu, K. R. Cooke, and J. L.M. Ferrara Pretreatment of donors with interleukin-18 attenuates acute graft-versus-host disease via STAT6 and preserves graft-versus-leukemia effects Blood, April 1, 2003; 101(7): 2877 - 2885. [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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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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O. Alpdogan, C. Schmaltz, S. J. Muriglan, B. J. Kappel, M.-A. Perales, J. A. Rotolo, J. A. Halm, B. E. Rich, and M. R. M. van den Brink Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease Blood, October 1, 2001; 98(7): 2256 - 2265. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-FITC, -PE, and -PerCP (53-6.7),
antimurine CD62L-FITC (MEL-14), antimurine CD122-FITC (TM-B1),
antimurine CD44-APC (IM7), and antimurine Fas-FITC (Jo2), as well as
purified NA/LE antimurine FasL (MFL3) and hamster IgG group 1 
isotype control antibody (A19-3), were all obtained from Pharmingen
(San Diego, CA). Antimurine CD3 (145-2C11) was obtained from the
Monoclonal Antibody Facility at the Sloan-Kettering Institute.
Antimurine CD4 (L3T4), antimurine CD8
-phorbol 12-myristate 13-acetate
(PMA) were obtained from Calbiochem (La Jolla, CA). Tissue culture
medium consisted of RPMI-1640 supplemented with 10% heat-inactivated
fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (as well as 50 µM 2-mercaptoethanol for the culture of
L5178Y and 32Dp210 cells). Murine TNF was obtained from R&D Systems
(Minneapolis, MN).
after fixing in 10% formalin, embedding in paraffin, and
staining with hematoxylin and eosin
) or without (
) irradiated (20 Gy) C3FeB6F1/J splenic stimulator
cells ("Materials and methods"). (B) Splenic T cells from B6 (
),
B6.gld (
) mice were
incubated for 4 hours with PMA (10 ng/mL) and ionomycin (2 µM).
Brefeldin A (10 µg/mL) was added after the first hour of incubation.
Intracellular cytokine expression in CD4 memory cells
(CD4+, CD62L
, or IL-2. Moreover, the level of intracytoplasmic cytokine production (reflected in
the fluorescence intensity) in reactive cells was also comparable in
B6, B6.gld, and B6.pfp
), and 26% in recipients of B6.pfp
. (The effector/target (E/T) ratio was
corrected for the percentage of donor splenic T cells in each group.
(B) Compared to isotype control (nonfilled overlay curve), FACS
staining with anti-Fas antibody (filled curve) demonstrates expression
of Fas on the tumor cell lines 32Dp210 and P815 and on the positive
control LK35.2, a B-cell hybridoma with known high expression of Fas,
but not on the T cell hybridoma cell line DO11.10, which has previously
been shown to be Fas negative in its resting state. (C)
51Cr-labeled tumor cell lines 32Dp210 (
