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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3355-3361
CD4+ Cytotoxic T-Cell Clones Specific for bcr-abl b3a2
Fusion Peptide Augment Colony Formation by Chronic Myelogenous Leukemia
Cells in a b3a2-Specific and HLA-DR-Restricted Manner
By
Masaki Yasukawa,
Hideki Ohminami,
Shin Kaneko,
Yoshihiro Yakushijin,
Yasuharu Nishimura,
Koiti Inokuchi,
Tsuyoshi Miyakuni,
Shinji Nakao,
Kenji Kishi,
Ichiro Kubonishi,
Kazuo Dan, and
Shigeru Fujita
From the First Department of Internal Medicine, Ehime University
School of Medicine, Ehime; the Division of Immunogenetics, Department
of Neuroscience and Immunology, Kumamoto University Graduate School of
Medical Sciences, Kumamoto; the Division of Hematology, Third
Department of Internal Medicine, Nippon Medical School, Tokyo; the
Department of Internal Medicine, Okinawa Prefectural Hospital, Okinawa;
the Third Department of Internal Medicine, Kanazawa University School
of Medicine, Ishikawa; the First Department of Internal Medicine,
Niigata University School of Medicine, Niigata; and the Third
Department of Internal Medicine, Kochi Medical School, Kochi, Japan.
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ABSTRACT |
Although it is well known that CD8+
cytotoxic T lymphocytes (CTLs) play an important role in the
suppression of cancer cell growth, the significance of
CD4+ CTLs in resistance to cancer is obscure. In an
attempt to elucidate the role of CD4+ CTLs in
immunosurveillance of chronic myelogenous leukemia (CML), we examined
the immunologic functions of bcr-abl b3a2 fusion peptide-specific CD4+ CTL clones. Seven CD4+ T-cell clones
that responded to stimulation with b3a2 peptide, but not with b2a2
peptide or physiological counterparts bcr b3b4 and abl 1A-a2 peptides,
were established from two healthy individuals. Restriction elements of
these clones were HLA-DRB1*0901. These CD4+ T-cell clones
exhibited b3a2 peptide-specific and HLA-DRB1*0901-restricted cytotoxicity and produced interleukin-3 (IL-3), IL-4, IL-10,
interferon- , tumor necrosis factor- , and granulocyte-macrophage
colony-stimulating factor in response to bcr-abl peptide stimulation,
indicating they were Th0 clones. The numbers of HLA-DRB1*0901-positive
b3a2, but not those of b2a2-positive or HLA-DRB1*0901-negative CML cell colonies increased when CML cells were cultured with b3a2-specific CD4+ CTL clones. These data suggest that
bcr-abl-specific CD4+ CTLs recognize CML cells in an
antigen-specific and HLA-DR-restricted manner, and that they do not
inhibit, but in fact augment, CML cell growth.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
TRANSLOCATION BETWEEN two distinct genes
resulting in formation of a new chimeric protein occurs specifically in
various types of leukemia. The translocation t(9;22) (q34;q11) that
gives rise to a hybrid bcr-abl gene encoding a 210-kD fusion
protein is detected in over 95% of patients with chronic myelogenous
leukemia (CML).1,2 Two major types of bcr-abl chimeric
protein, ie, the products of b2a2 fusion between exon 2 of the
bcr gene and exon 2 of the abl gene, and b3a2 fusion
between exon 3 of the bcr gene and exon 2 of the abl
gene have been found to be expressed in CML cells.3 Because
chimeric protein is only expressed in leukemic cells, not in normal
cells, the fusion sequence may act as a potential target for a
T-cell-mediated immune response to leukemia.
Recently, synthetic peptides spanning the fusion point between bcr and
abl have been shown to bind to certain types of HLA class I molecule
and induce bcr-abl fusion peptide-specific CD8+ cytotoxic T
lymphocytes (CTLs) in vitro.4-8 Induction of
CD4+ T lymphocytes that proliferate specifically in
response to stimulation with bcr-abl fusion peptide in an HLA class
II-restricted manner has been also achieved by stimulating peripheral
blood lymphocytes of healthy individuals with synthetic bcr-abl fusion
peptide.9-12 Although these recent findings support the
concept that the bcr-abl fusion peptide is immunogenic in the
T-cell-mediated immune response, the majority of previous studies
involved experiments using synthetic peptide-loaded antigen presenting
cells (APCs) and whether T lymphocytes can recognize naturally
processed bcr-abl fusion peptide in the context of major
histocompatibility complex (MHC) molecules on CML cells is obscure.
There is no doubt that CTLs play an important role in protection
against tumor development. It was thought that antigen-specific cytotoxicity was mediated solely by HLA class I-restricted
CD8+ CTLs, but it is now well known that some
CD4+ T lymphocytes also exert antigen-specific cytotoxicity
in an HLA class II-restricted manner.13-16 Although it is
well established that CD8+ CTLs play an important role in
resistance to tumor cell growth, the significance of CD4+
CTLs in immunosurveillance of tumors is obscure. The recent
demonstration that infusing donor lymphocytes depleted of
CD8+ T lymphocytes could induce remissions with a low rate
of severe graft-versus-host disease in patients with CML who had
relapsed after allogeneic bone marrow transplantation17
suggests that CD4+, but not CD8+, T lymphocytes
are essential for a graft-versus-leukemia effect in patients with CML.
In this study, we established bcr-abl b3a2-specific and
HLA-DRB1*0901-restricted CD4+ CTL clones from two healthy
individuals and investigated their immunologic functions by focusing on
their effects on CML cell growth. Unexpectedly, our results showed that
bcr-abl fusion peptide-specific CD4+ CTL clones did not
inhibit, but actually augmented, CML cell colony formation in vitro in
an HLA-DR-restricted fashion. These data strongly suggest that HLA
class II-restricted cytotoxicity mediated by CD4+ CTL does
not itself inhibit CML cell growth effectively and that CD4+ T lymphocytes can recognize the bcr-abl fusion peptide
in the context of HLA class II molecules expressed on CML cells,
resulting in the production of growth factors for CML cells.
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MATERIALS AND METHODS |
Generation of bcr-abl 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
17-mer amino acid peptides synthesized (underlined amino acids indicate
the breakpoints with a new amino acid) were as follows: bcr-abl b2a2,
IPLTINKEEALQRPVAS; bcr-abl b3a2, ATGFKQSSKALQRPVAS; bcr b3b4, ATGFKQSSNLYCTLEVD; and abl 1A-a2, SSSSCYLEEALQRPVAS. Peripheral blood mononuclear cells (PBMCs) from healthy individuals suspended in RPMI 1640 medium supplemented with 10% heat-inactivated human AB type serum (referred to hereafter as the culture
medium) and synthetic peptide at a concentration of 10 µg/mL were
seeded in round-bottom microtiter plate wells at a concentration of 1 × 105 cells/0.2 mL. After 7 days in culture, half of
the medium was exchanged for fresh culture medium and a second
stimulation was performed by addition of 1 × 105
autologous PBMCs treated with mitomycin C (MMC) as APCs and peptide at
a concentration of 10 µg/mL. After a further 7 days, a third stimulation was performed as for the second stimulation. Four days
after the third stimulation with peptide, human recombinant interleukin-2 (IL-2; Boehringer-Mannheim, Mannheim, Germany) was added
to each well at a concentration of 10 U/mL. Then, the growing cells
were transferred from the microtiter wells to 16-mm diameter wells and
their proliferative responses were examined. Bulk cells showing a
proliferative response to stimulation with bcr-abl fusion peptide were
cloned by the limiting dilution method described previously.18 T-cell clones were cultured continuously in
IL-2-containing culture medium and MMC-treated autologous PBMCs and
bcr-abl fusion peptide were added to the wells every 2 weeks.
Proliferative response to synthetic peptide.
Proliferative response of T cells to stimulation with peptide was
examined as described previously19 with a slight
modification. Briefly, 2 × 104 T cells and 2 × 105 MMC-treated PBMCs or 3 × 104
MMC-treated HLA-DR gene-transfected murine L cells as a source of APCs in 0.2 mL culture medium were seeded into flat-bottom microtiter wells, to which synthetic peptide was added. In preliminary experiments to determine the optimal concentration of peptide, bcr-abl
b3a2 fusion peptide-specific T-cell clones proliferated maximally in
the presence of over 10 µg/mL peptide. Therefore, we used synthetic
peptide at a concentration of 10 µg/mL in this series of experiments.
The culture was incubated at 37°C in a 5% CO2
incubator for 72 hours. For the final 16 hours of incubation, 1 µCi
of [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(s) governing the interaction between
T-cell clones and APCs, monoclonal antibody (MoAb) L243 (anti-HLA-DR; American Type Culture Collection, Rockville, MD), TÜ169
(anti-HLA-DQ; Pharmingen, San Diego, CA), or HI43 (anti-HLA-DP;
Pharmingen) was added to each well at its optimal concentration, and
the inhibitory effect of each MoAb on the proliferative response of the
T-cell clones was examined as described previously.19
Cytotoxicity assays.
51Cr-release assays were performed as described
previously.20 Briefly, 1 × 104
51Cr (Na251CrO4; New
England Nuclear) -labeled Epstein-Barr virus-transformed B
lymphoblastoid cell lines (B-LCLs) suspended in 0.1 mL RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS) (referred to hereafter as
the assay medium) were seeded into round-bottom microtiter wells and
incubated with or without synthetic peptide at a concentration of 50 µg/mL for 2 hours. Then, various numbers of effector cells suspended
in 0.1 mL assay medium were added to each well. After incubation for 4 hours, 0.1 mL supernatant was collected from each well and its
radioactivity was determined using a gamma counter. The percentage of
specific 51Cr release was calculated as follows: (cpm
experimental release  cpm spontaneous release)/(cpm maximal
release cpm spontaneous release) × 100. Each cytotoxicity
assay was performed at least twice and yielded identical data.
Cytokine production.
Cloned T cells were collected from the wells and washed twice with RPMI
1640 medium to remove IL-2 and any other cytokines present in the
culture medium. Then 5 × 105 T 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 supernatant was collected from each well
and assayed for the production of various cytokines by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, MN).
Detection of cytolytic mediator mRNA expression.
Expression of various cytolytic mediator mRNAs in the T-cell clones was
investigated by reverse transcriptase-polymerase chain reaction
(RT-PCR). The total RNA was extracted from each T-cell clone that had
been stimulated with peptide 5 days previously and cDNA was synthesized
by reverse transcription with Moloney murine leukemia virus RT, as
described previously.21 Amplification of cDNAs by the 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-ATAGGATCCATGTTTCTGCT- CTTCCACCTACAGAAGGA-3 and 5-ATAGAATTCTGACCAAGA- GAGAGCTCAGATACGTTGAC-3; tumor
necrosis factor- (TNF-), 5-TGAGCACTGAAAGCATGATC-3
and 5-TTATCTCTCAGCTCCACGCC-3; and lymphotoxin- ,
5-ATAAGCTTGATCAGGGAGGACTGGTAACGG-3 and
5-TAGGTACCTCGCACCACGCACTCATATTC-3. The expected lengths
of the amplified cytolytic mediator cDNAs were as follows: perforin,
459 bp; granzyme B, 180 bp; Fas ligand, 506 bp; TNF-, 375 bp; and lymphotoxin-, 635 bp.
Colony formation assay.
The effects of the T-cell clones on CML cell growth were examined by
performing the colony formation assays described
previously22 with a slight modification. Bone marrow cells
were isolated from patients with chronic-phase CML when the Ph
chromosome was detected in all bone marrow cells analyzed and had been
cryopreserved until use. Cloned T cells and CML bone marrow cells were
suspended in assay medium at an E:T ratio of 5:1, centrifuged at 1,000 rpm for 3 minutes to ensure close cell contact, and then coincubated in
assay medium at 37°C for 4 hours. To examine the role of HLA-DR in
the interaction between cloned T cells and CML cells, CML bone marrow
cells were preincubated with anti-HLA-DR MoAb before centrifugation with cloned T cells. Control CML bone marrow cells were centrifuged and
incubated without T-cell clone in the same manner. After incubation, 5 mL Iscove's Modified Dulbecco's medium containing 1%
methylcellulose, 5% GCT conditioned medium, 1% bovine
serum albumin, 30% FCS, 10 4 mol/L
2-mercaptoethanol, and 3 U/mL erythropoietin (Stem cell CFU Kit;
Baxter, Deerfield, IL) was added to the cell pellet at the final CML
cell concentration of 1 × 105 cells/mL. Then, each
cell suspension was placed in triplicate 24-mm wells and cultured at
37°C for 12 to 14 days, after which the numbers of colony-forming
unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid
(BFU-E) were counted using an inverted microscope. For the control
assay, bone marrow cells were isolated from a HLA-DRB1*0901-positive
patient with a nonhematopoietic disorder and cocultured with cloned T
cells as described above. To examine the effect of soluble factors
produced by T-cell clones on CML cell growth, the culture supernatants
of T-cell clones that had been stimulated with peptide for 2 days were
obtained, and then added to the CML bone marrow cells. The significance of differences between values for two groups was determined using the
paired two-tailed Student's t-test and those at P < .05 were considered significant.
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RESULTS |
Generation of T-cell clones directed against bcr-abl fusion peptide and
analysis of HLA restriction.
PBMCs from two healthy individuals, MY and TO, whose HLA-DR types were
DRB1*0901/*1406 and *0405/*0901, respectively, were stimulated
repeatedly with b2a2 or b3a2 fusion peptide in microtiter wells, as
described in Materials and Methods. Of a total of 192 wells per donor
seeded with PBMCs, three and four CD4+ T-cell clones that
proliferated in response to stimulation with b3a2 peptide in the
presence of autologous APCs were generated from MY and TO,
respectively. A CD4+ T-cell clone that showed a
proliferative response to stimulation with b2a2 peptide in an
HLA-DRB1*1406-restricted manner was also generated from PBMCs of MY,
but this clone stopped growing during the study. Thus, seven
CD4+ T-cell clones, designated MY-1, MY-2, MY-3, TO-1,
TO-2, TO-3, and TO-4, that proliferated in response to stimulation with
b3a2 fusion peptide were used for further experiments. Southern blot analysis of the T-cell receptor- genes of these T-cell clones showed
distinct rearrangement patterns (data not shown), indicating that these
seven T-cell clones originated from different T cells. Because
identical results were obtained with each of these seven T-cell clones,
only the data for MY-1 and TO-1 are presented. MY-1 and TO-1
proliferated when stimulated with the 17-mer b3a2 peptide, but did not
do so in response to stimulation with b2a2 or physiological 17-mer
counterpart peptides bcr b3b4 or abl 1A-a2. The proliferative responses
of MY-1 and TO-1 to b3a2 peptide were inhibited by adding the anti-DR
MoAb to the culture medium, but not by adding the anti-DQ or anti-DP
MoAb, suggesting that the proliferative responses of MY-1 and TO-1 were
restricted by HLA-DR (Table 1, Exp. 1). The
restriction elements of both MY-1 and TO-1 seemed to be HLA-DR9, as
both clones proliferated in response to peptide stimulation in the
presence of allogeneic APCs bearing HLA-DR9, but not in the presence of
HLA-DR9-negative allogeneic APCs (data not shown). To examine this
further, we used HLA-DRA and HLA-DRB1*0901
gene-transfected murine L cells (L-DR9) as APCs. As shown in Table 1,
Exp. 2, MY-1 and TO-1 proliferated in response to stimulation with the
b3a2 peptide in the presence of L-DR9, 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 responses of MY-1 and TO-1 were restricted by
HLA-DRB1*0901. In view of the results of a recent study on peptide
motifs for the HLA-DR9 molecule,23 the fourth amino acid
(F) and the seventh amino acid (S) may be binding motifs for HLA-DR9
positions 1 and 4, respectively.
Cytotoxic reactivities of T-cell clones against peptide-loaded and
-unloaded target cells.
Next, we examined the ability of T-cell clones to lyse b3a2
peptide-loaded target cells. Table 2 shows
the cytotoxicities of MY-1 and TO-1 to autologous and various
allogeneic B-LCLs in the presence and absence of the peptide. MY-1 and
TO-1 were strongly cytotoxic to b3a2 peptide-loaded autologous B-LCL.
As with their proliferative responses, the b3a2 fusion peptide-specific
cytotoxicities of MY-1 and TO-1 were restricted by HLA-DRB1*0901, as
allogeneic targets bearing HLA-DRB1*0901, but HLA-DRB1*0901-negative
allogeneic cells were not lysed by MY-1 and TO-1.
Cytokine production by bcr-abl-specific CD4+ T-cell
clones.
MY-1 and TO-1 were cultured with autologous MMC-treated PBMCs as APCs
in the presence or absence of peptide and the supernatants were
analyzed for the production of IL-3, IL-4, IL-10, interferon- (IFN-), TNF-, and granulocyte-macrophage colony-stimulating factor (GM-CSF). As shown in Table 3, MY-1
and TO-1 secreted all six cytokines after stimulation with b3a2 fusion
peptide and, therefore, they were both classified as Th0 type
CD4+ T-cell clones.
Expression of cytolytic mediators by T-cell clones.
Because the mechanism underlying CD4+ CTL-mediated
cytotoxicity is obscure, we examined the expression of cytolytic
mediators reported to be important in cytotoxicity mediated by
CD8+ and CD4+ CTLs and natural killer cells,
namely perforin, granzyme B, Fas ligand, TNF-, and lymphotoxin,
using the RT-PCR. As shown in Fig 1, mRNAs
for all cytolytic mediators examined were expressed in both MY-1 and
TO-1. Flow cytometric analysis showed that TNF- and lymphotoxin-
and - were expressed on MY-1 and TO-1 as membrane-bound forms (data
not shown).
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| Fig 1.
Expression of cytolytic mediator mRNAs in
bcr-abl-specific CD4+ T-cell clones. Expression of
perforin, granzyme B, Fas ligand, TNF-, and lymphotoxin- mRNAs
was investigated by RT-PCR, as detailed in Materials and Methods. The
mRNAs were extracted from MY-1 (lane 1) and TO-1 (lane 2) after
stimulation with the b3a2 peptide and autologous APCs for 5 days. Lane
M shows 100-bp ladder markers.
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Augmentation of CML cell colony formation by bcr-abl-specific
CD4+ T-cell clones.
We investigated whether CD4+ CTLs can inhibit CML cell
growth by examining the effects of b3a2-specific CD4+ CTL
clones on colony formation by CML cells. Unexpectedly, as shown in
Fig 2A, the numbers of CFU-GM
and BFU-E generated from bone marrow cells of two patients with b3a2
CML who were HLA-DRB1*0901 positive were augmented significantly after
coculture with MY-1 and TO-1. The augmentative effects of MY-1 and TO-1
on CML cell colony formation were antigen specific and HLA-DRB1*0901
restricted, as these clones had no effect on colony formation by b2a2
type or HLA-DRB1*0901-negative CML cells and HLA-DRB1*0901-positive normal bone marrow cells. In addition, augmentation of CML cell colony
formation by MY-1 and TO-1 was inhibited by anti-HLA-DR MoAb (Fig 2B).
The numbers of colonies formed by b2a2 as well as b3a2 CML cells
increased when they were cultured in the presence of MY-1 and TO-1
culture supernatants, in comparison with those of CML cells grown in
the absence of the culture supernatants (Fig 2C). These data strongly
suggest that CD4+ CTLs are not cytotoxic against CML cells,
but in fact augment CML cell growth by producing myelostimulatory
cytokines, such as IL-3 and GM-CSF, through recognition of leukemia
cells in an antigen-specific and HLA-restricted manner.
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| Fig 2.
Augmentation of CML cell colony formation by
bcr-abl-specific CD4+ T-cell clones. (A) The numbers of
CFU-GM and BFU-E generated from bone marrow cells of various patients
in the absence ( ) and presence of b3a2-specific CD4+
T-cell clones, MY-1 ( ), and TO-1 ( ) are shown. (B) The numbers of
CFU-GM and BFU-E generated from bone marrow cells of a patient with
HLA-DRB1*0901-positive b3a2 CML which had been untreated or treated
with anti-HLA-DR MoAb and cultured without () or with MY-1 ()
and TO-1 () are shown. (C) The numbers of CFU-GM and BFU-E generated
from bone marrow cells of various patients with CML in the absence
() and presence of MY-1 () and TO-1 () culture supernatant are
shown. Cells in triplicate wells were cultured and the data are
expressed as mean colony counts ± standard deviation. *, P < .05; **, P < .01.
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DISCUSSION |
Recently, reports describing the immunogenicity of synthetic bcr-abl
fusion peptide to CD4+ as well as CD8+ T
lymphocytes of healthy individuals have been accumulating. Binding of
b3a2 fusion peptides to HLA class I alleles A3, A11, B8, and B44 has
been reported and these peptides have been shown to prime
CD8+ CTLs in vitro.4-8 Furthermore, b3a2
peptides have been shown to induce HLA-DR1(DRB1*0101)-, DR2
(DRB1*1501)-, DR4 (DRB1*0401)-, and DR11 (DRB1*1101)-restricted
proliferative responses of CD4+ T
lymphocytes.9-12 In this study, we showed for the first
time that b3a2 fusion peptide can also elicit a proliferative response of CD4+ T lymphocytes restricted by HLA-DR9 (DRB1*0901),
the most frequent HLA-DR allele in the Japanese population. We also
believe this study is the first in which b3a2-specific CD4+
CTL clones have been established.
It is now well known that endogenous and exogenous antigens are
processed and expressed on MHC class I and class II, respectively. However, recent studies have shown that endogenous proteins can also be
expressed on MHC class II molecules and recognized by CD4+
T lymphocytes.24-29 These reports suggest that
bcr-abl-specific CD4+ T lymphocytes can distinguish CML
and normal cells directly via recognition of the bcr-abl fusion peptide
in the context of HLA class II expressed on leukemic cells. The recent
study of ten Bosch et al10 showed that a b3a2-specific
CD4+ T-cell line proliferated in response to stimulation
with b3a2-positive CML blasts in an HLA-DR-restricted manner, which
strongly suggests that CD4+ T lymphocytes can directly
recognize bcr-abl fusion peptide that is naturally processed and
expressed on CML cells. Although they observed a proliferative response
of CD4+ T lymphocytes to CML cells, they did not show
cytotoxic activity against CML blasts. In our study, although
b3a2-specific CD4+ CTL clones exerted strong cytotoxicity
against b3a2 peptide-loaded B-LCL, these clones did not inhibit, but
actually augmented, colony formation by HLA-DR-matched CML cells.
There are several possible explanations for the failure of
CD4+ CTLs to inhibit CML cell growth. The first hypothesis
is that the complexes of b3a2 peptide and HLA-DR molecules that
CD4+ CTLs recognize are expressed only on some populations
of chronic phase CML cells that have reached a certain differentiation
level and that the CML progenitor cells that proliferate in in vitro assays do not express the bcr-abl fusion protein and/or HLA-DR. The second hypothesis is that CD4+ CTLs recognize bcr-abl
fusion peptide in the context of the HLA class II expressed on CML
cells, but the CML cells are resistant to CD4+ CTL-mediated
cytotoxicity. MY-1 and TO-1 both seemed to express cytolytic mediators
reported to be important for CTL- and natural killer cell-mediated
cytotoxicity, namely perforin, granzyme B, Fas ligand, TNF-, and
lymphotoxin,30 but the main cytotoxic pathway of these
CD4+ CTL clones is unknown. The Fas/Fas ligand system has
been reported to account for the main pathway of CD4+
CTL-mediated cytotoxicity in a murine system31-33 and CML
cells have been reported to be sensitive to Fas-mediated
apoptosis.34 We found that colony formation by CML cells
was inhibited markedly by adding the agonistic anti-Fas MoAb to the
assay medium (data not shown). In the light of these findings, the
second hypothesis seems unlikely, but it cannot be excluded, because
recently, we found that peptide-specific human CD4+ CTL
clones lysed peptide-loaded Fas-deficient mutant target cells (manuscript in preparation). Therefore, the details of the cytotoxic mechanism mediated by bcr-abl fusion peptide-specific CD4+
CTL clones and the sensitivities of CML cells to various cytolytic mediators need to be elucidated to resolve this issue. The third hypothesis, which we think is the most important, is that bcr-abl peptide cannot be expressed on CML cells as suggested by Pawelec et
al,11 who showed that bcr-abl peptide-specific
CD4+ T lymphocytes did not recognize CML cells. At present,
the definitive reason for the lack of inhibition of CML cell growth by
CD4+ CTLs is unknown and further studies to explore the
above hypotheses are necessary.
Coculture of CML cells and bcr-abl peptide-specific CD4+
T-cell clones resulted in increased numbers of CML cell colonies in comparison with those formed by CML cells cultured alone. These augmentative effects of CD4+ T-cell clones on CML cell
growth were b3a2 specific and HLA-DRB1*0901 restricted, suggesting that
bcr-abl-specific CD4+ T lymphocytes can recognize bcr-abl
fusion protein in the context of HLA-DR molecules. There are two
possible mechanisms whereby bcr-abl protein is recognized by
CD4+ T lymphocytes. One is that CD4+ T
lymphocytes may directly recognize the bcr-abl peptide and HLA-DR
molecule complexes expressed on CML cells. The report by ten Bosch et
al10 supports this hypothesis, as described above, but
further studies are necessary to confirm this issue, as conflicting data have been also reported.11 The other mechanism is that bcr-abl protein released from CML cells as a result of cell death was
processed and expressed on live CML cells or residual normal APCs.
Evidence that bcr-abl peptide-specific CD4+ T lymphocytes
can respond to peptides derived from purified whole bcr-abl fusion
protein and cell lysates containing bcr-abl protein is accumulating.
Murine CD4+ T lymphocytes specific for b3a2 peptide have
been shown to proliferate in response to whole-fusion protein purified
from a cell extract,35 and recently, human b3a2
peptide-specific CD4+ T lymphocytes were reported to
respond to APCs exposed to b3a2-containing cell lysates.12
Furthermore, the findings of Jiang et al36 that CML cells
can process and present exogenous antigens suggests that CML cells
themselves can present antigens to CD4+ T lymphocytes.
These recent reports lend strong support to our observation that
bcr-abl-specific CD4+ T lymphocytes responded to bcr-abl
fusion peptide derived from CML cells.
In summary, we have reported the establishment and functional
characterization of bcr-abl-specific CD4+ CTL clones. It
should be noted that the present data do not mean that
bcr-abl-specific CD4+ T lymphocytes are ineffective for
immunotherapy of CML. In animal models, the adoptive transfer of immune
tumor antigen-specific CD8+ T lymphocytes alone had an
antitumor effect, but the combination of antigen-specific
CD4+ and CD8+ T lymphocytes was generally more
effective.37 Therefore, the development of combination
adoptive therapy involving various types of immunocompetent cells,
including bcr-abl-specific CD4+ and CD8+ T
lymphocytes, and effective APCs, such as dendritic cells, is expected
to lead to the development of new effective immunotherapy for CML.
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FOOTNOTES |
Submitted March 3, 1998;
accepted June 29, 1998.
Supported by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports and Culture of Japan; the Mochida
Foundation for Medical and Pharmaceutical Research; the Inamoro
Foundation; and the Suzuken Memorial Foundation.
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.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
REFERENCES |
1.
Shtivelman E,
Lifshitz B,
Gale RP,
Canaani E:
Fused transcript of abl and bcr genes in chronic myelogenous leukaemia.
Nature
315:550,
1985[Medline]
[Order article via Infotrieve]
2.
Heisterkamp N,
Stam K,
Groffen J:
Structural organization of the bcr gene and its role in the Ph translocation.
Nature
315:758,
1985[Medline]
[Order article via Infotrieve]
3.
Melo JV:
The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype.
Blood
88:2375,
1996[Free Full Text]
4.
Cullis JO,
Barrett AJ,
Goldman JM,
Lechler RI:
Binding of BCR/ABL junctional peptides to major histocompatibility complex (MHC) class I molecules: Studies in antigen-processing defective cell lines.
Leukemia
8:165,
1994[Medline]
[Order article via Infotrieve]
5.
Bocchia M,
Wentworth PA,
Southwood S,
Sidney J,
McGraw K,
Scheinberg DA,
Sette A:
Specific binding of leukemia oncogene fusion protein peptides to HLA class I molecules.
Blood
85:2680,
1995[Abstract/Free Full Text]
6.
Bocchia M,
Korontsvit T,
Xu Q,
Mackinnon S,
Yang SY,
Sette A,
Scheinberg DA:
Specific human cellular immunity to bcr-abl oncogene-derived peptides.
Blood
87:3587,
1996[Abstract/Free Full Text]
7.
Greco G,
Fruci D,
Accapezzato D,
Barnaba V,
Nisini R,
Alimena G,
Montefusco E,
Vigneti E,
Butler R,
Tanigaki N,
Tosi R:
Two bcr-abl junction peptides bind HLA-A3 molecules and allow specific induction of human cytotoxic T lymphocytes.
Leukemia
10:693,
1996[Medline]
[Order article via Infotrieve]
8.
Buzyn A,
Ostankovitch M,
Zerbib A,
Kemula M,
Connan F,
Varet B,
Guillet J-G,
Choppin J:
Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes.
Eur J Immunol
27:2066,
1997[Medline]
[Order article via Infotrieve]
9.
ten Bosch GJA,
Toornvliet AC,
Friede T,
Melief CJM,
Leeksma OC:
Recognition of peptides corresponding to the joining region of p210BCR-ABL protein by human T cells.
Leukemia
9:1344,
1995[Medline]
[Order article via Infotrieve]
10.
ten Bosch GJA,
Joosten AM,
Kessler JH,
Melief CJM,
Leeksma OC:
Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide.
Blood
88:3522,
1996[Abstract/Free Full Text]
11.
Pawelec G,
Max H,
Halder T,
Bruserud Ø,
Merl A,
da Silva P,
Kalbacher H:
BCR/ABL leukemia oncogene fusion peptides selectively bind to certain HLA-DR alleles and can be recognized by T cells found at low frequency in the repertoire of normal donors.
Blood
88:2118,
1996[Abstract/Free Full Text]
12.
Mannering SI,
McKenzie JL,
Fearnley DB,
Hart DNJ:
HLA-DR1-restricted bcr-abl (b3a2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates.
Blood
90:290,
1997[Abstract/Free Full Text]
13.
Krensky AM,
Reiss CS,
Mier JW,
Strominger JL,
Burakoff SJ:
Long-term human cytotoxic T-cell lines allospecific for HLA-DR6 antigen are OKT4+.
Proc Natl Acad Sci USA
79:2365,
1982[Abstract/Free Full Text]
14.
Yasukawa M,
Zarling JM:
Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens.
J Immunol
133:422,
1984[Abstract]
15.
Tite JP,
Janeway CA Jr:
Cloned helper T cells can kill B lymphoma cells in the presence of specific antigen: Ia restriction and cognate vs. noncognate interactions in cytolysis.
Eur J Immunol
14:878,
1984[Medline]
[Order article via Infotrieve]
16.
Jacobson SJ,
Richert R,
Biddison WE,
Satinsky A,
Hartzman RJ,
McFarland HF:
Measles virus-specific T4+ human cytotoxic T cell clones are restricted by class II HLA antigens.
J Immunol
133:754,
1984[Abstract]
17.
Giralt S,
Hester J,
Huh Y,
Hirsch-Ginsberg C,
Rondón G,
Seong D,
Lee M,
Gajewski J,
Van Besien K,
Khouri I,
Mehra R,
Przepiorka D,
Körbling M,
Talpaz M,
Kantarjian H,
Fischer H,
Deisseroth A,
Champlin R:
CD8-depleted donor lymphocyte infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation.
Blood
86:4337,
1995[Abstract/Free Full Text]
18.
Yasukawa M,
Yakushijin Y,
Furukawa M,
Fujita S:
Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus.
J Virol
67:6259,
1993[Abstract/Free Full Text]
19.
Yasukawa M,
Inatsuki A,
Horiuchi T,
Kobayashi Y:
Functional heterogeneity among herpes simplex virus-specific human CD4+ T cells.
J Immunol
146:1341,
1991[Abstract]
20.
Yasukawa M,
Inatsuki A,
Kobayashi Y:
Differential in vitro activation of CD4+CD8 and CD8+CD4 herpes simplex virus-specific human cytotoxic T cells.
J Immunol
143:2051,
1989[Abstract]
21.
Carding SR,
Lu D,
Bottomly K:
A polymerase chain reaction assay for the detection and quantitation of cytokine gene expression in small numbers of cells.
J Immunol Methods
151:277,
1992[Medline]
[Order article via Infotrieve]
22.
Molldrem JJ,
Clave E,
Jiang YZ,
Mavroudis D,
Raptis A,
Hensel N,
Agarwala V,
Barrett AJ:
Cytotoxic T lymphocytes specific for a nonpolymorphic proteinase 3 peptide preferentially inhibit chronic myeloid leukemia colony-forming units.
Blood
90:2529,
1997[Abstract/Free Full Text]
23.
Fujisao S,
Matsushita S,
Nishi T,
Nishimura Y:
Identification of HLA-DR9 (DRB1*0901)-binding peptide motifs using a phage fUSE5 random peptide library.
Hum Immunol
45:131,
1996[Medline]
[Order article via Infotrieve]
24.
Nuchtern JG,
Biddison WE,
Klausner RD:
Class II MHC molecules can use the endogenous pathway of antigen presentation.
Nature
343:74,
1990[Medline]
[Order article via Infotrieve]
25.
Jaraquemada D,
Marti M,
Long EO:
An endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class II-restricted T cells.
J Exp Med
172:947,
1990[Abstract/Free Full Text]
26.
Weiss S,
Bogen B:
MHC class II-restricted presentation of intracellular antigen.
Cell
64:767,
1991[Medline]
[Order article via Infotrieve]
27.
Takahashi T,
Chapman PB,
Yang SY,
Hara I,
Vijayasaradhi S,
Houghton AN:
Reactivity of autologous CD4+ T lymphocytes against human melanoma: Evidence for a shared melanoma antigen presented by HLA-DR15.
J Immunol
154:772,
1995[Abstract]
28.
Oxenius A,
Bachmann MF,
Mathis D,
Benoist C,
Zinkernagel RM,
Hengartner H:
Functional in vivo MHC class II loading by endogenously synthesized glycoprotein during viral infection.
J Immunol
158:5717,
1997[Abstract]
29.
Armstrong TD,
Clements VK,
Martin BK,
Ting JPY,
Ostrand-Rosenberg S:
Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.
Proc Natl Acad Sci USA
94:6886,
1997[Abstract/Free Full Text]
30.
Kägi D,
Ledermann B,
Bürki K,
Zinkernagel RM,
Hengartner H:
Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo.
Annu Rev Immunol
14:207,
1996[Medline]
[Order article via Infotrieve]
31.
Stalder T,
Hahn S,
Erb P:
Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity.
J Immunol
152:1127,
1994[Abstract]
32.
Hanabuchi S,
Koyanagi M,
Kawasaki A,
Shinohara N,
Matsuzawa A,
Nishimura Y,
Kobayashi Y,
Yonehara S,
Yagita H,
Okumura K:
Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.
Proc Natl Acad Sci USA
91:4930,
1994[Abstract/Free Full Text]
33.
Ju S-T,
Cui H,
Panka DJ,
Ettinger R,
Marshak-Rothstein A:
Participation of target Fas protein in apoptosis pathway induced by CD4+ Th1 and CD8+ cytotoxic T cells.
Proc Natl Acad Sci USA
91:4185,
1994[Abstract/Free Full Text]
34.
Selleri C,
Sato T,
Del Vecchio L,
Luciano L,
Barrett AJ,
Rotoli B,
Young NS,
Maciejewski JP:
Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon-a in chronic myelogenous leukemia.
Blood
89:957,
1997[Abstract/Free Full Text]
35.
Chen W,
Peace DJ,
Rovira DK,
You S-G,
Cheever MA:
T-cell immunity to the joining region of p210BCR-ABL protein.
Proc Natl Acad Sci USA
89:1468,
1992[Abstract/Free Full Text]
36.
Jiang Y-Z,
Mavroudis D,
Dermime S,
Hensel N,
Couriel D,
Molldrem J,
Barrett AJ:
Alloreactive CD4+ T lymphocytes can exert cytotoxicity to chronic myeloid leukaemia cells processing and presenting exogenous antigen.
Br J Haematol
93:606,
1996[Medline]
[Order article via Infotrieve]
37.
Kern DE,
Klarnet JP,
Jensen MCV,
Greenberg PD:
Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL.
J Immunol
136:4303,
1986[Abstract]
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Abstract
Introduction
Abstract