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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 925-935
Fas-Independent and Nonapoptotic Cytotoxicity Mediated by a Human
CD4+ T-Cell Clone Directed Against an Acute Myelogenous
Leukemia-Associated DEK-CAN Fusion Peptide
By
Hideki Ohminami,
Masaki Yasukawa,
Shin Kaneko,
Yoshihiro Yakushijin,
Yasuhito Abe,
Yoshihito Kasahara,
Yasushi Ishida, and
Shigeru Fujita
From the First Departments of Internal Medicine and Pathology and the
Department of Pediatrics, Ehime University School of Medicine, Ehime,
Japan; and the Department of Pediatrics, Kanazawa University School of
Medicine, Ishikawa, Japan.
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ABSTRACT |
The mechanism underlying the cytotoxicity mediated by a human
CD4+ cytotoxic T-lymphocyte (CTL) clone directed against
a peptide derived from the acute myelogenous leukemia-associated fusion protein, DEK-CAN, was investigated. A DEK-CAN fusion peptide-specific CD4+ Th0 CTL clone, designated HO-1, was established from
the peripheral blood lymphocytes of a healthy individual. HO-1 exerted
direct but not "innocent bystander" cytotoxicity within 2 hours.
The cytotoxicity mediated by HO-1 was completely
Ca2+-dependent. Because HO-1 lysed peptide-loaded
Fas-deficient target cells derived from a patient with a homozygous
Fas gene mutation, its cytotoxicity appeared to be mediated by
a Fas-independent pathway. In addition, its cytotoxicity was only
partially inhibited by treatment with concanamycin A and strontium
ions, which are inhibitors of the perforin-based cytotoxic pathway.
Although membrane-bound type of tumor necrosis factor- (TNF-) was
expressed on HO-1, an anti-TNF- antibody had no effect on
HO-1-mediated cytotoxicity. HO-1 expressed mRNA for apoptosis-inducing
mediators, including perforin, granzyme B, Fas ligand, TNF-, and
lymphotoxin; however, no DNA fragmentation was detected in target cells
incubated with HO-1 by
5-[125I]Iodo-2'-deoxyuridine release assay
and agarose gel electrophoresis of DNA. Although it has been suggested
that the Fas/Fas ligand system is the main pathway by which
CD4+ CTL-mediated cytotoxicity is exerted in murine
systems, HO-1 produced peptide-specific and HLA-restricted cytotoxicity
via a Fas-independent and nonapoptotic pathway. The present study thus
describes a novel mechanism of cytotoxicity mediated by
CD4+ CTL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FUSION PROTEINS RESULTING from
chromosomal translocations can be detected in certain types of
leukemia, for example BCR-ABL in chronic myelogenous leukemia,
AML1-MTG8/ETO in acute myelogenous leukemia (AML) (M2), and PML-RAR
in acute promyelocytic leukemia (M3). The DEK-CAN fusion protein is
produced by the translocation (6;9)(p23;q34) and is associated with a
specific subtype of AML.1 It is well known that patients
with t(6;9) AML treated with conventional chemotherapy have a poor
prognosis2; therefore, new therapies based on novel
concepts are being actively sought. Because fusion proteins are
produced only in leukemia cells and not in normal cells, the area of
fusion between DEK and CAN is a potential target for immunotherapy in
this kind of leukemia. On the basis of this concept, we attempted to
establish human T-cell clones, which specifically react to the DEK-CAN
fusion peptide. In the present study, we succeeded in establishing a
DEK-CAN fusion peptide-specific CD4+ human cytotoxic
T-lymphocyte (CTL) clone and examined the mechanism underlying its
cytotoxic effect.
Various mediators of CTL-mediated cytotoxicity have been identified.
Previous studies have shown that two main pathways account for the
cytotoxicity mediated by CTLs.3-10 The first is granule exocytosis, which is mediated by two types of molecules released into
the extracellular space after contact between CTLs and their target
cells: the pore-forming perforin and the lymphocyte-specific granule
serine esterase granzyme B. The second pathway is a nonsecretory one,
involving interaction of the Fas ligand with the Fas molecule expressed
on the target cells. Both of these cytotoxic pathways result in
apoptosis of the target cells.3,4 In addition, residual
cytotoxic activity observed in mice deficient in both Fas ligand and
perforin suggests the presence of other cytotoxic pathways.11,12 Recent studies have demonstrated that
membrane-bound tumor necrosis factor- (TNF-) and lymphotoxin (LT)
may be mediators of a third cytotoxic pathway.13-15 When
mediated by membrane-bound TNF- and LT, the cytotoxic activity of
CTLs against target cells needs a longer incubation period to become
apparent than when mediated by the granule exocytosis and Fas/Fas
ligand systems.12 Among these cytolytic mediators, the
interaction between Fas and Fas ligand is thought to be the main
pathway by which CD4+ CTLs exert their
cytotoxicity16-18; however, the precise mechanism underlying the cytotoxicity mediated by CD4+ CTLs is still obscure.
We have established a B-lymphoblastoid cell line (B-LCL) from a patient
with a homozygous Fas-gene mutation.19 Because this cell line completely lacks Fas expression, it is useful in studying the
Fas dependency of CTL-mediated cytotoxicity. In the present study, we
clearly show that a DEK-CAN-specific CD4+ CTL clone
exerted antigen-specific and HLA-restricted cytotoxicity via a
Fas-independent pathway, using this Fas-deficient cell line as a
target. In addition, the results of various other experiments using
potent inhibitors of the granule exocytosis pathway, DNA fragmentation
assay, and transmission electron microscopy demonstrated that the
cytotoxicity mediated by CD4+ T cells involves a novel
mechanism. That is, the DEK-CAN peptide-specific and HLA-DR-restricted
cytotoxicity of a human CD4+ CTL clone was mediated by
Fas-independent and nonapoptotic mechanisms. The potential application
of this kind of CTL clone to peptide-based cancer immunotherapy is also discussed.
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MATERIALS AND METHODS |
Generation of DEK-CAN fusion peptide-specific T-cell clones.
Peptides were synthesized to a minimum of 90% purity using an
automated peptide synthesizer (Model 432A Synergy; Applied Biosystems Inc, Foster City, CA) with the Fmoc procedure. The sequences of the
16-mer amino acid peptides synthesized were as follows, where "-"
indicates the breakpoint of the DEK-CAN fusion protein1: DEK-CAN, TMKQICKK-EIRRLHQY; DEK, TMKQICKKVYENYPTY; and CAN,
LDPKSQLQEIRRLHQY. Peripheral blood mononuclear cells (PBMCs) from a
healthy individual, whose HLA-DR was HLA-DRB1*0405/1502,
HLA-DRB4*0103/-, were suspended in RPMI 1640 medium supplemented with
10% heat-inactivated human AB serum (referred to hereafter as the
culture medium). These were seeded into round-bottom microtiter plate
wells at a concentration of 1 × 105 cells/0.2 mL
together with DEK-CAN fusion peptide at a concentration of 10 µg/mL.
After 7 days in culture, half of the medium was exchanged for fresh
culture medium and a second stimulation was performed by adding 1 × 105 autologous PBMCs treated with mitomycin C (MMC)
and DEK-CAN peptide at a concentration of 10 µg/mL. After a further 7 days, a third stimulation was performed as for the second stimulation,
4 days after the third stimulation with peptide (day 18 of culture), recombinant human interleukin-2 (IL-2) (Boehringer Mannheim, Mannheim, Germany) was added to each well at a concentration of 10 U/mL. The
growing cells were then transferred to 16-mm diameter wells, and their
proliferative response was examined. The bulk of the cells, which
showed a proliferative response on stimulation with DEK-CAN fusion
peptide, were cloned by a limiting dilution method as described
previously.20 T-cell clones were continuously cultured in
IL-2-containing culture medium, and MMC-treated autologous PBMCs and
DEK-CAN peptide were added to the wells every 2 weeks. This T-cell
clone was designated HO-1.
Alloantigen-specific CD8+ CTLs were also generated as the
control CTLs, which exert granule exocytosis-mediated and
apoptosis-inducing cytotoxicity, as follows: PBMCs from an individual
whose HLA class I and class II were nonidentical to those of a donor
for HO-1 and a patient with Fas deficiency were cocultured with
MMC-treated B-LCL established from HO or that from a patient with Fas
deficiency at a responder to stimulator ratio of 5:1. After 5 days,
CD8+ T cells were isolated using anti-CD8 monoclonal
antibody (MoAb)-coated magnetizable polystyrene beads (DYNAL, Oslo,
Norway). CD8+ T cells were then cultured in
IL-2-containing culture medium and restimulated with MMC-treated B-LCL
cells. Following these procedures, CD8+ CTL lines directed
against allogeneic B-LCL were established.
Establishment of a Fas-deficient B-LCL.
A Fas-deficient B-LCL, designated YK-B1, was established from the
peripheral blood B lymphocytes of a patient with a homozygous Fas gene mutation by in vitro transformation using Epstein-Barr virus.21 A homozygous point mutation was present in the
splice acceptor of intron 3 of the Fas gene of this patient.
This resulted in the skipping of exon 4 and complete loss of Fas
expression.19 The HLA type of the patient was as follows:
HLA-A24/24, B48/48, Cw8/-, DRB1*0401/0401, DRB4*0103/0103,
DQB1*0301/0301, DPB1*0201/0201. A B-LCL, designated YK-B2, was also
established from the peripheral blood B lymphocytes of the patient's
father, who is a heterozygote for the Fas gene mutation. The
B-LCLs were maintained in RPMI 1640 medium supplemented with 10% fetal
calf serum (FCS) and were shown not to be infected with mycoplasma.
Flow cytometric analysis.
The expression of various membrane-bound molecules including Fas,
TNF-, and LT was determined by direct or indirect immunofluorescence using the following antibodies (Abs): fluorescence isothiocyanate (FITC)-conjugated anti-Fas MoAb (MBL, Nagoya, Japan), polyclonal rabbit
anti-TNF- Ab (Genzyme, Cambridge, MA), and polyclonal rabbit
anti-LT- and  Abs, which were prepared by immunizing rabbits
with human recombinant LT- and (Kanegafuchi Chemical Co, Tokyo,
Japan) as described previously.14 Stained cells were analyzed with a flow cytometer (FACSCalibur; Becton Dickinson, San
Jose, CA).
Proliferative response to synthetic peptide.
Proliferative response assays were performed as described previously
with a slight modification.22 Briefly, 2 × 104 clone cells and 2 × 105 MMC-treated
autologous PBMCs or 3 × 104 MMC-treated
HLA-DR gene-transfected murine L cells as a source of antigen
presenting cells (APCs) in 0.2 mL culture medium were seeded into
flat-bottomed microtiter wells, to which synthetic peptide was added.
In preliminary experiments to determine the optimal concentration of
peptide, a DEK-CAN fusion peptide-specific T-cell clone proliferated
maximally in the presence of more than 10 µg/mL peptide. Therefore,
we used DEK-CAN fusion peptide at a concentration of 10 µg/mL in the
present series of experiments. The culture was incubated at 37°C in
an atmosphere containing 5% CO2 for 72 hours. For the
final 16 hours of incubation, 1 µCi [3H]thymidine
(3H-TdR) (New England Nuclear, Boston, MA) was added to
each well, and the incorporation of 3H-TdR was determined
by liquid scintillation counting. To determine the restriction element
governing the interaction between the T-cell clones and APCs, MoAb L243
(anti-HLA-DR) (ATCC, Rockville, MD), MoAb TÜ169
(anti-HLA-DQ) (Pharmingen, San Diego, CA), or MoAb HI43 (anti-HLA-DP)
(Pharmingen) was added to the wells at an optimal concentration, and
the inhibitory effect of each MoAb on the proliferative response of the
T-cell clones was examined as described previously.20
Cytotoxicity assays.
51Cr-release assays were performed as described
previously.22 Briefly, 1 × 104
51Cr (Na251CrO4) (New
England Nuclear)-labeled target cells suspended in 0.1 mL RPMI 1640 medium supplemented with 10% FCS (referred to hereafter as the assay
medium) were seeded into round-bottomed microtiter wells and incubated
with or without synthetic peptide for 2 hours. Various numbers of
effector cells suspended in 0.1 mL assay medium were then added to each
well. After various incubation periods, 0.1 mL supernatant was
collected from each well. The percentage of specific 51Cr
release was calculated as follows: (cpm experimental release  cpm spontaneous release)/(cpm maximal release cpm spontaneous release) × 100. To examine the Ca2+ dependency of the
cytotoxicity, cytotoxicity assays were performed in the presence of
EGTA (Sigma, St Louis, MO) at various concentrations. To evaluate the
role of perforin in CTL-mediated cytotoxicity, effector T cells were
pretreated with concanamycin A (CMA) (Wako Pure Chemical Industries,
Osaka, Japan), an inhibitor of vacuolar type H+-adenosine
triphosphate (ATPase), at various concentrations for 2 hours, then
incubated with the target cells in the presence of CMA.23
Moreover, to clarify the contribution of the granule exocytosis pathway
to CTL-mediated cytotoxicity, strontium chloride (SrCl2)
(Sigma), which induces degranulation of CTL,24,25 was added
to effector cells at various concentrations. After incubation at
37°C for 18 hours, the effector cells were washed twice and then
51Cr release assays were performed as described above. To
examine the effect of an anti-TNF- Ab on cytotoxicity, effector
cells were incubated with polyclonal rabbit anti-TNF- Ab (Genzyme) at a final concentration of 1:10, which neutralizes 1,000 U TNF- bioactivity for 30 minutes, after which 51Cr release assay
was performed as described above. This procedure inhibited
TNF--dependent cytotoxicity.26,27 Each cytotoxicity assay was performed at least twice, and identical data were obtained.
DNA fragmentation assays.
DNA fragmentation in the target cells was determined by
5-[125I]Iodo-2'-deoxyuridine (125IUdR)
release assays as described previously.28 Briefly, target cells were incubated with 125IUdR (Amersham, Arlington
Heights, IL) for 2 hours at 37°C. Various numbers of effector cells
and 1 × 104 125IUdR-labeled
target cells, which had been incubated with or without peptide for 2 hours, were incubated together in 0.2 mL assay medium in round-bottomed
microtiter wells. After various incubation periods, 0.1 mL supernatant
was removed, and 0.1 mL 0.2% Triton X-100 was added to each well and
mixed by pipetting. The solubilized samples were centrifuged, 0.1 mL
supernatant was collected from each well, and the radioactivity was
counted. The total radioactivity of the target cells was estimated by
directly counting the radioactivity of the cell suspension without
solubilization. The specific 125IUdR release was estimated
as described for the 51Cr release assays.
Cytokine production.
For the assays of cytokine production, 5 × 105 clone
cells and 2 × 106 MMC-treated autologous PBMCs were
suspended in 2 mL assay medium and cultured in 16-mm wells in the
presence or absence of peptide. After 72 hours, the supernatants were
collected from each well and assayed for the production of various
cytokines by enzyme-linked immunosorbent assay (ELISA) (PerSeptive
Diagnostics, Inc, Cambridge, MA).
Detection of cytolytic mediator mRNA expression.
Expression of the mRNAs for various cytolytic mediators in HO-1 was
investigated by reverse transcriptase-polymerase chain reaction
(RT-PCR). Total RNA was extracted from HO-1, which had been stimulated
with peptide 5 days previously (activated phase) and 14 days previously
(resting phase), and cDNA was synthesized by reverse transcription with
Moloney murine leukemia virus reverse transcriptase, as described
previously.29 Amplification of the cDNAs by PCR was
performed using the following primers: perforin, 5'-ACCAGCAATGTGCATGTGTCTGTG-3' and
5'-GAAGGAGGCCGTCATCTTGTGCTT-3'; granzyme B,
5'-TGCAGGAAGATCGAAAGTGCG-3' and
5'-GAGGCATGCCATTGTTTCGTC-3'; Fas ligand,
5'-ATAGGATCCATGTTTCTGCTCTTCCACCTACAGAAGGA-3' and
5'-ATAGAATTCTGACCAAGAGAGAGCTCAGATACGTTGAC-3'; TNF-, 5'-TGAGCACTGAAAGCATGATC-3' and
5'-TTATCTCTCAGCTCCACGCC-3'; and LT-,
5'-ATAAGCTTGATCAGGGAGGACTGGTAACGG-3' and
5'-TAGGTACCTCGCACCACGCACTCATATTC-3'. The expected lengths
of the amplified cDNA sequences for each cytolytic mediator were as
follows: perforin, 459 bp; granzyme B, 180 bp; Fas ligand, 506 bp;
TNF-, 375 bp; and LT-, 635 bp.
Analysis of DNA fragmentation by agarose gel electrophoresis.
The cloned T cells and B-LCL cells were cocultured at an effector to
target (E:T) ratio of 3:1 for 4 hours in the presence or absence of
DEK-CAN fusion peptide, then harvested by centrifugation. The pellet
was lysed with hypotonic lysing buffer, and the DNA was extracted from
each lysate using a DNA extraction kit (Sepa Gene; Sanko Junyaku Co,
Tokyo, Japan). The samples were electrophoresed in 2% agarose gel. The
gel was stained with ethidium bromide and visualized by
transillumination with ultraviolet light.
Transmission electron microscopy.
Morphologic changes in the target cells after incubation with the
effector T cells were examined by transmission electron microscopy.
After cocultivation of the cloned T cells and peptide-loaded LCL at an
E:T ratio of 3:1 for 4 hours, the cells were fixed with 2.0%
glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4), postfixed with
1% osmium tetroxide, and gradually dehydrated. The samples were
embedded in Epon 812, sectioned, stained with uranyl acetate and lead
citrate, and examined with an H-800 microscope (Hitachi, Ibaragi, Japan).
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RESULTS |
Generation of a T-cell clone directed against the DEK-CAN fusion
peptide and analysis of HLA restriction.
PBMCs from a healthy individual, whose HLA-DR type was DRB1*0405/1502,
DRB4*0103/, were repeatedly stimulated with DEK-CAN fusion peptide
in microtiter wells as detailed in Materials and Methods. From a total
of 192 wells seeded, a T-cell line, which proliferated in response to
stimulation with DEK-CAN peptide in the presence of autologous APCs was
generated. After cloning by a limiting dilution method, a T-cell clone,
designated HO-1, was established. The phenotype of HO-1 was
CD2+ CD3+ CD4+
CD8 CD56. HO-1 proliferated on
stimulation with the 16-mer DEK-CAN fusion peptide, but not in response
to stimulation with the physiological 16-mer counterpart peptides DEK
or CAN (data not shown). The proliferative response of HO-1 to
stimulation with DEK-CAN peptide was inhibited by adding anti-DR MoAb
to the culture medium, but not by adding anti-DQ or anti-DP MoAbs,
suggesting that the proliferative response of HO-1 was restricted by
HLA-DR (Table 1, experiment no. 1). HO-1
appeared to proliferate in response to stimulation with the DEK-CAN
peptide in the presence of allogeneic APCs bearing HLA-DR4, -DR7, and
-DR9, but showed no response in the presence of APCs bearing only
HLA-DR2 or -DR6 (data not shown). Because expression of HLA-DRB4 (DR53)
is associated with HLA-DR4, -DR7 and -DR9, we speculated that the
restriction element for HO-1 might be HLA-DRB4. To verify this
possibility, we used HLA-DRA and HLA-DRB4*0103 gene-transfected murine L cells (L-DR53) as APC. As shown in Table 1,
experiment no. 2, HO-1 proliferated in response to stimulation with the
DEK-CAN peptide in the presence of L-DR53, but not in the presence of
HLA-DRA and DRB1*0401 gene-transfected L cells (L-DR4)
or control L cells transfected with the selection marker Neor gene alone (L-Neo). These data show that the
proliferative response of HO-1 is restricted by HLA-DRB4*0103.
According to a recent study of peptide motifs for the HLA-DR53
molecule,30 the seventh amino acid (K) in the DEK portion
and the tenth amino acid (I) in the CAN portion might be binding motifs
for HLA-DR53 positions 1 and 4, respectively.
Cytotoxic activity against various allogeneic target cells and L-cell
transfectants.
We next examined the ability of HO-1 to lyse DEK-CAN peptide-loaded
target cells. Table 2 shows the
cytotoxicity of HO-1 against autologous and various allogeneic B-LCLs
in the presence or absence of the peptide. HO-1 exerted strong
cytotoxicity (70.0%, 68.4%, and 57.7% specific cytotoxicity at E:T
ratios of 10:1, 5:1, and 2.5:1, respectively) against peptide-loaded
autologous B-LCL. As with the proliferative response, the DEK-CAN
fusion peptide-specific cytotoxicity of HO-1 was restricted by
HLA-DR53, as allogeneic targets bearing HLA-DR53 and L-DR53, but not
HLA-DR53-negative allogeneic or L-Neo cells, were lysed by HO-1.
Direct lysis of target cells.
We addressed the question of whether HO-1 exerts cytotoxicity via the
release of an antigen-nonspecific soluble cytolytic factor into the
culture medium or via direct contact with the target cells. To clarify
this issue, we performed "innocent bystander" experiments, in
which the effector cells were incubated with 51Cr-labeled
HLA-DR53 allogeneic LCL in the presence of unlabeled
peptide-loaded autologous LCL. As shown in
Table 3, HO-1 mediated no apparent
51Cr release from HLA-DR53 allogeneic
cells. These data suggest that HO-1 directly lyses target cells in a
peptide-specific and HLA-restricted manner.
Cytokine production.
HO-1 cells were cultured with autologous MMC-treated PBMCs as APC in
the presence or absence of the peptide, and the supernatants were
analyzed for the production of IL-4, IL-10, TNF-, and interferon- (IFN-). As shown in Table 4, HO-1
secreted all of these cytokines after stimulation with the DEK-CAN
fusion peptide. Therefore, HO-1 can be classified as a Th0 type
CD4+ T-cell clone.
Expression of cytolytic mediators.
Because the mechanism underlying CD4+ CTL-mediated
cytotoxicity remains obscure, we attempted to study the pathway by
which HO-1 cytotoxicity is mediated. First, we examined the expression of cytolytic mediators, which have been reported to be important in
CD8+ and CD4+ CTL- and natural killer (NK)
cell-mediated cytotoxicity, including perforin, granzyme B, Fas ligand,
TNF-, and LT, by RT-PCR. As shown in Fig
1, mRNAs for all of the cytolytic mediators examined were expressed by
HO-1 in both the activated and resting phases.
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| Fig 1.
Expression of cytolytic mediators in HO-1. Expression of
mRNAs for perforin, granzyme B, Fas ligand, TNF-, and LT- was
investigated by RT-PCR as detailed in Materials and Methods. mRNAs were
extracted from HO-1, which had been stimulated with the DEK-CAN peptide
and autologous APCs for 5 days (lane 1) and for 14 days (lane 2) and
from PBMCs, which had been stimulated with phytohemagglutinin for 3 days (lane 3). Lane M shows 100-bp ladder markers.
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Ca2+-dependent cytotoxicity.
The perforin/granzyme pathway is known to be
Ca2+-dependent, and recent studies have shown that
extracellular Ca2+ is also necessary for the Fas/Fas ligand
system.4,31 With these findings in mind, the cytotoxic
activity of HO-1 was examined in the absence of extracellular
Ca2+. As shown in Fig 2, no
HO-1 cytotoxicity was observed in the presence of the
Ca2+-chelating agent EGTA. Thus, HO-1-mediated
cytotoxicity appears to be completely Ca2+-dependent.
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| Fig 2.
Ca2+-dependent cytotoxicity of HO-1.
HO-1-induced lysis of DEK-CAN peptide-loaded autologous LCL was
examined by 4-hour 51Cr release assay at an E:T ratio of
5:1 in the absence or presence of various concentrations of EGTA.
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Cytotoxicity against a Fas-deficient cell line.
The significance of the Fas/Fas ligand pathway in HO-1-mediated
cytotoxicity was examined by measuring its cytotoxic activity against
Fas-deficient target cells. The results of a flow cytometric analysis
of Fas expression on the B-LCLs used in the present study are shown in
Fig 3A. A complete lack of Fas expression
was detected in the YK-B1 cell line, which was established from a
patient with a homozygous Fas gene mutation. The expression of
Fas on YK-B2, which was established from the patient's father who
carried a heterozygous Fas gene mutation, was decreased as
compared with B-LCL established from a donor for HO-1. Because the
Fas-deficient B-LCL, YK-B1, was positive for HLA-DR53, the degree of
cytotoxicity against peptide-loaded YK-B1 was expected to be reduced if
the Fas/Fas ligand system was involved in the cytolytic pathway.
However, the percentage cytotoxicities against autologous, heterozygous Fas mutant and homozygous Fas mutant target cells were
almost the same (Fig 3B). These data indicate that the HO-1 line has a
Fas-independent cytotoxic pathway.
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| Fig 3.
Cytotoxicity of HO-1 against Fas-positive and -negative
target cells. (A) B-LCLs were established from the donor for HO-1
(Fas+/+), the patient's father
(Fas+/), and the patient with Fas deficiency
(Fas/). Cells were stained with FITC-conjugated mouse
anti-Fas MoAb (shaded histograms) or FITC-conjugated mouse IgG (open
histograms). The fluorescence profiles were analyzed with a flow
cytometer. (B) HO-1-induced lysis of DEK-CAN peptide-loaded (open
columns) and unloaded (shaded columns) target cells was determined by
51Cr release assays over 4 hours at E:T ratios of 10:1,
5:1, and 2.5:1.
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Inhibition of cytotoxicity by CMA and Sr2+.
The significance of the granule exocytosis pathway was examined using
an inhibitor of vacuolar type H+-ATPase, CMA. Recent
studies have shown that CMA is a selective inhibitor blocking only
perforin-based cytotoxicity, but not affecting Fas-dependent
cytotoxicity, due mostly to accelerated degradation of perforin by an
increase in the pH of lytic granules.23 It has been
reported that treatment of CTLs with CMA at a concentration of more
than 10 nmol/L almost completely inhibits perforin-based cytotoxic
activity.23,32 As treatment of HO-1 cells with CMA at
concentrations of more than 100 nmol/L produced toxic effects, CMA was
used at concentrations of 1 to 100 nmol/L. The results shown in
Fig 4A demonstrate that while pretreatment
of HO-1 with CMA produced some inhibition of peptide-specific
cytotoxicity against both autologous B-LCL (Fas+/+) and
Fas-deficient B-LCL (Fas/), this inhibition
was only partial. On the other hand, cytotoxicity of
alloantigen-specific CD8+ CTLs directed against
Fas-deficient target cells, which was considered to be mediated via
granule exocytosis pathway, was completely inhibited by treatment with
CMA at a concentration of more than 10 nmol/L, as reported
previously.23,32
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| Fig 4.
Effect of CMA and Sr2+ on cytotoxicity
mediated by HO-1. (A) HO-1 cells were preincubated with various
concentrations of CMA for 2 hours. The CMA-treated or untreated HO-1
cells were then cocultured with 51Cr-labeled autologous LCL
(Fas+/+ LCL) and Fas-deficient LCL
(Fas/ LCL), which had been loaded with the DEK-CAN
peptide in the presence or absence of CMA at E:T ratios of 10:1 ( ),
5:1 ( ), and 2.5:1 ( ) for 4 hours. Effect of CMA on cytotoxicity
mediated by alloantigen-specific CD8+ CTLs directed
against Fas+/+ LCL and Fas/ LCL was
also examined. (B) HO-1 cells were preincubated with various
concentrations of SrCl2 for 18 hours. The
Sr2+-treated or untreated HO-1 cells were then cocultured
with 51Cr-labeled autologous LCL (Fas+/+
LCL) and Fas-deficient LCL (Fas/ LCL), which had been
loaded with the DEK-CAN peptide at E:T ratios of 10:1 (), 5:1 (),
and 2.5:1 () for 4 hours. The effect of Sr2+ on
cytotoxicity mediated by alloantigen-specific CD8+ CTLs
directed against Fas+/+ LCL and Fas/
LCL was also examined.
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It has been reported that Sr2+ causes degranulation and
release of granule contents from mast cells, basophils, and large
granular lymphocytes and induces inhibition of NK cell
activity.24 Recent studies have also demonstrated that
pretreatment of human CTL clones with Sr2+ selectively
inhibits the cytotoxicity mediated by the granule exocytosis
pathway.25 In view of these findings, we examined the
effect of Sr2+ on cytotoxicity mediated by HO-1. As shown
in Fig 4B, the treatment of HO-1 resulted in only partial inhibition of
cytotoxicity against peptide-loaded target cells, while cytotoxicity
mediated by alloantigen-specific CD8+ CTLs was completely
abrogated by treatment with Sr2+ at concentrations of more
than 25 mmol/L. Taken together with the results of the RT-PCR, which
showed the expression of perforin and granzyme B mRNAs in HO-1, these
results suggest that the granule exocytosis pathway is involved to some
extent, but that other mechanisms may also exist and may be more
important in HO-1-mediated cytotoxicity.
Expression of membrane-bound forms of TNF-.
TNF- and LT have been reported to exist in both membrane-bound and
secretory forms. Membrane-bound TNF- and LT are known to be
expressed on some CD8+ CTLs, CD4+ CTLs and
lymphokine-activated killer cells, and exert cytotoxicity against
appropriate target cells.13-15,33 Flow cytometric analysis showed that TNF- was expressed; however, membrane-bound LT- and
were not detected on the surface of HO-1
(Fig 5A). Negative reactivity of HO-1
against anti-LT- and Abs did not result from uselessness of
these Abs, as lymphokine-activated killer cells14 and other
CTL clones33 were stained with these Abs. To evaluate the
roles of membrane-bound TNF- in HO-1-mediated cytotoxicity against
peptide-loaded autologous (Fas+/+) and Fas-deficient
(Fas/) B-LCLs, inhibition assay using
anti-TNF- Ab was performed. As shown in Fig 5B, no inhibition of
cytotoxicity was induced by the addition of anti-TNF- Ab,
suggesting that membrane-bound TNF- is not involved in
HO-1-mediated antigen-specific cytotoxicity.
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| Fig 5.
Expression of membrane-bound forms of TNF- and LT on
HO-1 cells and the effects of anti-TNF- Ab on cytotoxicity. (A)
HO-1 cells were stained by indirect immunofluorescence with rabbit
anti-TNF- IgG, rabbit anti-LT- IgG, or rabbit anti-LT- IgG,
then with FITC-conjugated goat anti-rabbit IgG (shaded histograms).
Control stainings were performed using normal rabbit IgG and
FITC-conjugated goat anti-rabbit IgG (open histograms). (B)
HO-1-induced lysis of DEK-CAN peptide-loaded autologous LCL
(Fas+/+ LCL) and Fas-deficient (Fas/
LCL) was examined in 51Cr release assays conducted over 4 hours at E:T ratios of 10:1, 5:1, and 2.5:1 in the presence of normal
rabbit IgG or anti-TNF- rabbit IgG. The effect of Ab against
TNF- was also examined using TNF--dependent killer cells,
MY-3.11, and L929 as TNF--sensitive target cells.
|
|
Kinetics of 51Cr and 125IUdR release assays.
The results of kinetic studies of 51Cr release, which
reflects membrane damage, and 125IUdR release, which
reflects DNA fragmentation in target cells, are shown in
Fig 6. 51Cr release from target
cells incubated with HO-1 was first detected after 1 hour of
incubation, then increased rapidly. The 51Cr release level
was almost maximal after 4 hours of incubation. In contrast, a low
level of 125IUdR release was detected after 1 hour of
incubation and did not increase further during continuous culture. As
shown in Fig 6B, rapid DNA fragmentation from target cells is induced
by alloantigen-specific CD8+ CTLs via perforin/granzyme
system. Taken together with the data shown in Fig 4, we strongly
suggest that the granule exocytosis system is not the main pathway
governing HO-1-mediated cytotoxicity.
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| Fig 6.
Kinetics of 51Cr and 125IUdR
release from target cells mediated by HO-1. (A) HO-1 cells were
cocultured with 51Cr- or 125IUdR-labeled
autologous LCL (HO-LCL), which had been loaded with the DEK-CAN peptide
at E:T ratios of 10:1 (), 5:1 (), and 2.5:1 () for various
incubation periods. (B) 51Cr and 125IUdR
release from HO-LCL mediated by alloantigen-specific CD8+
CTLs was also examined. The percentage 51Cr and
125IUdR release levels were determined as described in
Materials and Methods.
|
|
Agarose gel electrophoresis of DNA.
We investigated whether apoptosis is induced in target cells after
coculture with HO-1 by agarose gel electrophoresis of their DNA. As
shown in Fig 7, a ladder pattern, which
reflects DNA fragmentation into oligonucleosome units, was rarely
detected in DNA from a mixture of target cells and HO-1 cells, which
had been incubated at an E:T ratio of 3:1 for 4 hours. Taken together
with the results of the 51Cr and 125IUdR
release assays, it was concluded that HO-1 mediates rapid cytotoxicity
without inducing apparent DNA fragmentation in target cells.
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| Fig 7.
Agarose gel electrophoresis of cellular DNA. HO-1 cells
were cocultured with peptide-free autologous LCL (lane 1), DEK-CAN
peptide-loaded autologous LCL (lane 2), or DEK-CAN peptide-loaded
HLA-DR53-negative allogeneic LCL (lane 3) at an E:T ratio of 3:1 for 4 hours. The DNA was extracted from each sample and electrophoresed in
2% agarose gel. DNA extracted from B-LCL and alloantigen-specific
CD8+ CTLs, which had been cocultured for 4 hours was also
electrophoresed as the control for fragmented DNA (lane 4). Lane M
shows 100-bp ladder markers.
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Transmission electron microscopy of target cells.
CTLs are well known to mediate apoptosis in target cells. This is
characterized morphologically by condensation of the nuclear chromatin,
shrinkage and blebbing of the cytoplasm, and the formation of apoptotic
bodies. Therefore, we investigated morphologic changes in target cells
cocultured with HO-1 by transmission electron microscopy. As shown in
Fig 8, severe degeneration of the
mitochondria occurred in the target cells after incubation with HO-1
cells; however, none of the aforementioned characteristics of apoptosis were detected, suggesting that the cytotoxicity of HO-1 against peptide-loaded target cells was mediated by nonapoptotic mechanisms.
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| Fig 8.
Transmission electron micrographs of DEK-CAN
peptide-loaded autologous LCL cocultured with HO-1. (A) A target cell,
which is in contact with a CTL (arrow). Note the degeneration of the
mitochondria in the target cell. (B) A target cell, which was lysed by
HO-1 cells. Note the degeneration of the mitochondria and rough
endoplasmic reticula in the target cell. Original magnification: (A),
×6,300; (B), ×7,200.
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| |
DISCUSSION |
In the present study, we established a human CD4+ CTL clone
directed against an AML-associated DEK-CAN fusion peptide, designated HO-1. The characteristics of the peptide-specific and
HLA-DR-restricted cytotoxicity mediated by HO-1 were as follows:
first, HO-1 produced direct, but not "innocent bystander,"
cytotoxicity over a short culture period, suggesting that its
cytotoxicity was not mediated by soluble cytolytic factor(s), but by
direct contact with the target cells. Second, the cytotoxicity of HO-1
was Ca2+-dependent. Third, HO-1 lysed peptide-loaded
Fas-deficient cells, which were established from a patient with a
homozygous Fas gene mutation, indicating that the HO-1 line has
a Fas-independent cytotoxic pathway. Fourth, treatment of HO-1 with CMA
and Sr2+, which are inhibitors of the granule exocytosis
pathway, resulted in only partial inhibition of its cytotoxicity.
Fifth, although HO-1 expressed the membrane-bound form of TNF-, a
neutralizing Ab against TNF- had no effect on HO-1-mediated
cytotoxicity. Sixth, no apparent DNA fragmentation was detected in
target cells by agarose gel electrophoresis, and transmission electron
microscopy of the target cells showed a necrotic appearance rather than
apoptosis, suggesting that the main pathway involved in HO-1-mediated
cytotoxicity was not apoptosis. These characteristics of CTL-mediated
cytotoxicity have not been shown previously.
The mechanisms underlying cell-mediated cytotoxicity have mainly been
studied in CD8+ CTLs and NK cells, and two main pathways,
granule exocytosis mediated by perforin and granzymes and the Fas/Fas
ligand system, have been identified.3-10 Antigen-specific
and major histocompatibility complex (MHC)-restricted cytotoxicity was
formerly thought to be mediated only by CD8+ CTLs, but it
is now well known that some CD4+ T cells can also exert
cytotoxic activity34-39; thus, the mechanisms underlying
CD4+ CTL-mediated cytotoxicity are of considerable
interest. These mechanisms have mainly been studied in murine systems
using various mutant and knockout mice. Recent studies have shown that
CD4+ CTLs derived from gld/gld mice, which lack the
expression of a functional Fas ligand, do not exert Fas-mediated
cytotoxicity.17 In addition, it has been reported that
CD4+ Th1 cells, which lyse target cells derived from
wild-type mice, do not show cytotoxicity against targets derived from
Fas-deficient lpr/lpr mice.18 These reports
strongly suggest that the Fas/Fas ligand system acts as the main
pathway for CD4+ CTL-mediated cytotoxicity. In contrast to
the extensive studies in murine systems, the mechanisms involved in
human CD4+ CTL-mediated cytotoxicity have not been studied
precisely due to the lack of a suitable experimental system. In the
present study, we first examined the Fas-dependency of human
CTL-mediated cytotoxicity using Fas-deficient target cells derived from
a patient with a homozygous Fas gene mutation, reflecting the
situation in lpr/lpr mice.19,40,41 The results
obtained using this experimental system unexpectedly showed that the
HLA-DR-restricted cytotoxicity mediated by a DEK-CAN peptide-specific
human CD4+ CTL clone was Fas-independent.
The granule exocytosis model mediated by perforin and granzymes, the
other important mechanism involved in CTL-mediated cytotoxicity, is
thought to be the main pathway in CD8+ CTLs and NK cells.
It has recently been reported that perforin expression is detectable in
some CD4+ T cells, and that the granule exocytosis pathway
also plays an important role in CD4+ CTL-mediated
cytotoxicity.33,42-45 As addition of the
Ca2+-chelating agent EGTA to the assay medium resulted in
complete inhibition of cytotoxicity, HO-1-mediated cytotoxicity
appeared to be completely Ca2+-dependent. Because
perforin-mediated cytotoxicity depends on extracellular
Ca2+, we addressed the question of whether the
perforin/granzyme system is involved in HO-1-mediated cytotoxicity. To
clarify this issue, we performed an experiment using CMA and
Sr2+, which inhibit the granule exocytosis pathway, and
found that treatment of HO-1 with optimal concentrations of CMA and
Sr2+ resulted in only partial inhibition of cytotoxicity.
In addition, a 125IUdR release assay showed that HO-1
induced only partial DNA fragmentation in the target cells within 1 hour, and no further 125IUdR release was detected during
continuous culture. Taking into consideration the evidence that
51Cr release, which reflects membrane damage, increased
continuously over 4 hours of incubation, we suggest that the granule
exocytosis pathway certainly plays a role in HO-1-mediated
cytotoxicity, but that the main pathway is mediated by cytolytic
mediator(s) other than the perforin/granzyme system. It has been
reported that mice deficient in both granzyme B and Fas ligand can
produce residual cytotoxic activity, which seems to be mediated by
membrane damage induced by perforin alone.46-48 Although
this finding suggests that HO-1-mediated cytotoxicity may be due to a
perforin-dependent, but granzyme-independent mechanism, this
possibility is unlikely, as RT-PCR and Western blotting showed that
granzyme B mRNA and protein were expressed in HO-1.
The residual cytotoxic activity seen in CTLs deficient in both a
functional Fas ligand and perforin suggests the presence of a third
pathway of CTL-mediated cytotoxicity.11,12 Recent studies
have shown that TNF- and LT are major candidates as mediators of
such Fas ligand- and perforin-independent cytotoxicity. Because it has
recently been reported that both TNF- and LT are expressed on some
CTLs in their membrane-bound forms and that they induce cytotoxicity
through contact with target cells,13-15,27 the expression of these cytokines in HO-1 was examined. mRNAs for both TNF- and LT
were found to be expressed in HO-1 and membrane-bound form of TNF-
was detectable by flow cytometry. However, it is well known that
cytotoxicity mediated by TNF- and LT needs a longer culture period
to become apparent than that mediated by the perforin/granzyme and
Fas/Fas ligand systems.12 In the present study, HO-1
exerted strong cytotoxicity within 2 hours, whereas it usually takes
more than 10 hours to detect TNF-- and LT-mediated cytotoxicity. In addition, the addition of a neutralizing Ab against TNF- did not
have any effect on the cytotoxic activity of HO-1, and the B-LCL
appeared to be relatively resistant to cytotoxicity mediated by soluble
form of TNF- (data not shown). These findings strongly suggest that
TNF- and LT are not involved in HO-1-mediated antigen-specific cytotoxicity. Similar results were reported for a study of TNF- by
Liu et al,49 who found that human immunodeficiency
virus-specific CD4+ CTL clones produced membrane-bound and
secreted forms of TNF-, although these molecules were not required
for cytolysis of the target cells.
On the basis of the findings described above, we conclude that the
mechanism underlying HO-1-mediated cytotoxicity is independent of the
previously reported main pathways of CTL-mediated cytotoxicity, ie,
granule exocytosis mediated by perforin and granzymes, the Fas/Fas
ligand system, and membrane-bound TNF- and LT. It is noteworthy that
although HO-1 expressed mRNAs for all major cytolytic mediators,
including perforin, granzyme B, Fas ligand, TNF-, and LT, its
cytotoxic activity was not mediated by these mediators, except for weak
perforin activity. The main cytolytic mechanism(s) of HO-1 may thus act
predominantly against these conventional cytotoxic pathways.
Whether leukemia-associated fusion peptide-specific CD4+
CTL clones are able to inhibit the growth of leukemia cells is a point of particular interest. However, because the frequency of AML with
t(6;9) is low (<1% of all cases of AML), we were unable to obtain
t(6;9) AML cells bearing HLA-DRB4*0103, and thus could not investigate
the cytotoxic and growth inhibitory effects of HO-1 on such leukemia
cells. It has been shown that cancer-associated intracellular proteins,
such as ras p21, can be recognized by CD4+ T cells in the
context of MHC class II antigens.50,51 In addition, recent
studies have shown that chronic myelogenous leukemia-associated BCR-ABL
fusion peptide-specific CD4+ T cells can specifically
recognize leukemia cells in an HLA class II-restricted
manner.52 These findings strongly suggest that DEK-CAN
fusion peptide-specific CD4+ T cells should be able to
react against leukemia cells, as the leukemic cells in most cases of
t(6;9) AML are positive for HLA-DR.53 Although the Fas
antigen is frequently expressed on AML cells, these cells have been
reported to be relatively resistant to anti-Fas antibody-induced
apoptosis.54 This finding suggests that CTL-mediated cytotoxicity acting via the Fas/Fas ligand system may be relatively ineffective against leukemia cells, thus a Fas-independent pathway of
cytotoxicity mediated by CTLs could be anticipated to inhibit leukemia
growth effectively. Considering these possibilities, we strongly
suggest that adoptive transfer of CTL clones possessing the
characteristics of cytotoxic activity described in this study may be a
hopeful prospect in immunotherapy for leukemia.
| |
ACKNOWLEDGMENT |
We thank Drs Yasuharu Nishimura (Kumamoto University, Kumamoto, Japan)
and Takahiko Horiuchi (Kyushu University, Fukuoka, Japan) for providing
the cell lines and for their helpful suggestions. We also thank Dr
Seiji Matsuda (Ehime University, Ehime, Japan) for his
helpful comments on the electron micrographs.
| |
FOOTNOTES |
Submitted May 18, 1998; accepted September 17, 1998.
Supported by grants from the Ministry of Education, Science, Sports and
Culture of Japan, the Ministry of Health and Welfare of Japan, the
Mochida Foundation for Medical and Pharmaceutical Research, the Inamori
Foundation, and the Suzuken Memorial Foundation.
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 Masaki Yasukawa, MD, PhD, The First
Department of Internal Medicine, Ehime University School of Medicine,
Shigenobu, Ehime 791-0295, Japan; e-mail: yasukawa{at}m.ehime-u.ac.jp.
| |
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Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis |
Top
Abstract
Introduction