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IMMUNOBIOLOGY
From the First Department of Internal Medicine, Ehime
University School of Medicine, Ehime, Japan; the Second Department of
Internal Medicine, Okayama University School of Medicine, Okayama,
Japan; and Department of Hematology and Oncology, Tokyo Graduate
Medical School, University of Tokyo, Tokyo, Japan.
Bcr-abl fusion peptide-specific CD4+ T-lymphocyte
clones have recently been shown to augment colony formation by chronic
myelogenous leukemia (CML) cells in a bcr-abl type-specific and HLA
class II-restricted manner without addition of exogenous antigen.
These findings suggest that CML cells can naturally process and present endogenous bcr-abl fusion protein to CD4+ T lymphocytes in
the context of HLA class II molecules. To verify this possibility, the
ability of CML-derived dendritic cells (DCs) to present endogenous
bcr-abl fusion protein to bcr-abl fusion peptide-specific
CD4+ T-lymphocyte clones was investigated. The bcr-abl b3a2
peptide-specific and HLA-DRB1*0901-restricted CD4+
T-lymphocyte clones produced interferon- Chronic myelogenous leukemia (CML) is a hematologic
malignancy characterized by an initial chronic phase of clonal
hematopoiesis with continued differentiation into mature
granulocytes.1 Most cases of CML develop to blast crisis,
a terminal stage resembling acute leukemia. Because the prognosis of
CML blast crisis is very poor, treatment of CML in the chronic phase is
essential to achieve cure of this disease. Although treatment with
interferon (IFN) is effective for CML, the rate of complete remission
achieved by IFN therapy is not sufficient. At present, only bone marrow transplantation can cure this disease, although graft versus host disease and various opportunistic infections are serious complications in the posttransplantation stage. Recently, donor lymphocyte infusion has been found to be effective treatment in patients with CML who
relapse after allogeneic bone marrow transplantation.2 Although this finding suggests that CML cells are relatively sensitive to cell-mediated immunotherapy, donor lymphocyte infusion is frequently associated with severe graft versus host disease. Therefore, the development of novel cell-mediated immunotherapy for CML that induces
graft versus leukemia without graft versus host disease is required. To
develop effective immunotherapy for CML, identification of the
CML-specific tumor antigens is required.
Translocation between chromosomes 9 and 22 resulting in formation of a
chimeric protein of bcr and abl has been detected in more than 95% of
cases of CML. One of the 2 major fusion types, b2a2 and b3a2, is
produced in most cases of CML. Because bcr-abl chimeric protein is
expressed only in CML cells but not in normal cells, the fusion
sequence may act as a potential target for a T lymphocyte-mediated
immune response to CML. Indeed, it has been possible to generate
bcr-abl fusion peptide-specific CD4+ and CD8+
T lymphocytes in vitro.3-15 It has been demonstrated that
dendritic cells (DCs), which are professional antigen-presenting cells
(APCs), are generated from CML cells after treatment with a combination of cytokines.16-18 The generation of cytotoxic T
lymphocytes (CTLs) directed against CML cells has also been achieved by
stimulation of CD8+ T lymphocytes with CML-derived DCs as
APCs.19
A recent report by ten Bosch et al demonstrated that b3a2
peptide-specific and HLA-DRB1*0401-restricted CD4+
T-lymphocyte cell lines showed a proliferative response to
HLA-DRB1*0401-bearing b3a2+ CML blasts.4 On
the other hand, these CD4+ T-lymphocyte cell lines did not
respond to HLA-DRB1*0401-bearing b3a2 Generation of bcr-abl fusion peptide-specific CD4+
T-lymphocyte clones
Generation of immature and mature DCs from CML cells
Flow cytometry of DCs The surface phenotype of monocytes, immature DCs, and mature DCs generated from CML cells was examined by flow cytometry using the following mAbs: anti-CD1a mAb (Immunotech, Marseilles, France), anti-CD80 mAb (Immunotech), anti-CD83 mAb (Immunotech), anti-CD86 mAb (Immunotech), anti-HLA class I mAb (Pharmingen, San Diego, CA), and anti-HLA-DR mAb (Pharmingen).Fluorescence in situ hybridization The percentage of bcr-abl fusion gene-positive cells was determined by fluorescence in situ hybridization (FISH) using Vysis LSI bcr-abl translocation probe (Vysis, Downers Grove, IL) containing a bcr-specific probe labeled with SpectrumGreen and an abl-specific probe labeled with SpectrumOrange in accordance with the manufacturer's protocol. A total of 200 nuclei were counted for the reciprocal translocation between chromosomes 9q34 and 22q11.2.IFN-  production, 2 × 106
clone cells and 5 × 105 DCs were suspended in 2 mL RPMI
1640 supplemented with 10% FCS and cultured in a 16 mm well. Before
coculture, cell debris was removed from a suspension of DCs by
Ficoll-Conray gradient centrifugation; more than 95% DCs were viable
as determined by the trypan blue exclusion test. In some experiments,
sodium azide-free anti-HLA class I mAb, w6/32 (American Type Culture
Collection, Rockville, MD), or anti-HLA-DR mAb, L243 (American Type
Culture Collection), was added to the culture medium at an optimal
concentration (10 µg/mL). After 72 hours, the supernatants were
collected and assayed for IFN- production by enzyme-linked
immunosorbent assay (ELISA) (Endogen, Woburn, MA). For statistical
analysis of IFN- production, one-way analysis of variance was used
to test for overall differences among the groups, followed by the
Bonferroni test to compare the separate group means.
Cytotoxicity assays 51Cr release assays were performed as described previously.22 Briefly, 1 × 104 51Cr (Na  cpm
spontaneous release)/(cpm maximal release cpm spontaneous release) × 100. To examine the Ca++ dependency of the
cytotoxicity, cytotoxicity assays were performed in the presence of
ethyleneglycotetraacetic acid (EGTA) (Sigma, St Louis, MO) at various
concentrations and 2 mM MgCl2. To evaluate the role of
perforin in CTL-mediated cytotoxicity, effector T lymphocytes were
pretreated with an inhibitor of vacuolar type H+-adenosine
triphosphatase, concanamycin A (CMA) (Wako Pure Chemical Industries,
Osaka, Japan), at various concentrations for 2 hours and then incubated
with target cells in the presence of CMA.23
Dependency of peptide concentration on cytotoxicity and
IFN- production by b3a2-specific CD4+ CTL clones was
examined as follows. Mature DCs generated from autologous monocytes
suspended in 1 mL RPMI 1640 medium supplemented with 10% FCS were
incubated with or without b3a2 peptide at various concentrations for 4 hours. After extensive washing, peptide-pulsed and -unpulsed DCs were
used as target cells and stimulator cells for cytotoxicity assays and
IFN- production, respectively, as described above.
IFN- production was more
sensitive than measuring 3H-thymidine uptake to determine
CD4+ T-lymphocyte response to antigen stimulation (data not
shown), IFN- production was measured to determine the T-lymphocyte
response to DCs in the present study. As shown in Table
1, MY-1 and TO-1 produced IFN- in the
presence of autologous PBMCs as APCs when these cell lines were
stimulated with the b3a2 peptide but not with b2a2 or its physiologic
counterpart peptide, bcr b3b4 or abl 1A-a2. The production of IFN-
by MY-1 and TO-1 was inhibited by adding anti-HLA-DR mAb to the
culture medium but not by adding anti-HLA class I mAb. These data
indicate that MY-1 and TO-1 produce IFN- in a b3a2-specific and
HLA-DR (HLA-DRB1*0901)-restricted manner as in the
proliferative response.
Flow cytometric analysis of CML-derived monocytes, immature DCs, and mature DCs Monocytes isolated from PBMCs of patients with CML in the chronic phase were cultured without cytokine, with IL-4 and GM-CSF, or with IL-4, GM-CSF, and TNF- . The cells generated were considered as
monocytes, immature DCs, and mature DC, respectively. Flow cytometric
analysis of CML-derived monocytes, immature DCs, and mature DCs is
shown in Figure 1. Monocytes expressed
HLA class I and HLA-DR at relatively high levels, whereas expression of DC-associated markers, CD1a, CD80, CD83, and CD86, was undetectable or
very low. When monocytes were cultured with IL-4 and GM-CSF for 8 days,
expression of CD1a and CD80 was induced and the expression level of HLA
class I increased slightly, whereas expression of CD83 and CD86 was
unchanged. On the other hand, mature DCs expressed DC-associated
markers including CD1a, CD80, CD83, and CD86 at high levels. In
addition, expression of HLA class I on mature DCs was higher than that
on monocytes.
IFN- , but no
cytogenetic response was detected. As shown in Table
2, IFN- was not produced by monocytes,
immature DCs, or mature DCs alone. Little IFN-gamma was
produced by MY-1 and TO-1 when these clones were cocultured with
monocytes, and somewhat more IFN-gamma was produced by the clones when
cocultured with immature DCs. But mature DCs exerted an apparent
ability to stimulate bcr-abl peptide-specific CD4+
T-lymphocyte clones to produce IFN-.
Antigen specificity and HLA restriction of IFN- production
by b3a2 peptide-specific CD4+ T-lymphocyte clones in
response to CML-derived mature DCs was then determined. Table
3 shows IFN- production by MY-1 and
TO-1 cocultured with mature DCs generated from autologous monocytes and
monocytes of b2a2 or b3a2 type CML bearing various HLA-DRB1 types. FISH
analysis demonstrated that formation of the fusion gene between
bcr and abl was detected in more than 70% of DCs generated from all CML patients except CML patient no. 3, who was in
cytogenetic remission achieved by IFN- treatment. In DCs generated
from CML patient no. 3, fewer than 3% of cells appeared to be positive
for the bcr-abl fusion gene. Apparent production of IFN-
by MY-1 and TO-1 was detected when these clones were cocultured with
DCs generated from b3a2 type CML patients who were
HLA-DRB1*0901+. On the other hand, the degree of IFN-
production by MY-1 and TO-1, which were cocultured with DCs generated
from the b3a2 type CML patient in cytogenetic remission, b2a2 type CML
patients, or HLA-DRB1*0901 CML patients, was very low.
Production of IFN- by MY-1 and TO-1 cocultured with b3a2 type and
HLA-DRB1*0901+ DCs was inhibited by adding anti-HLA-DR but
not anti-HLA class I mAb to the culture medium. During culture, most
DCs remained alive as determined by microscopic observation and the
trypan blue exclusion test. These data strongly suggest that endogenous bcr-abl fusion protein is processed naturally in mature DCs and is
presented to bcr-abl peptide-specific CD4+ T lymphocytes
in the context of HLA class II molecules.
Lack of cytotoxicity mediated by bcr-abl peptide-specific CD4+ T-lymphocyte clones against CML-derived DCs Both MY-1 and TO-1 have previously been shown to be cytotoxic to a b3a2 peptide-loaded autologous B-lymphoblastoid cell line in an HLA-DRB1*0901-restricted manner. On the basis of these findings, the question of whether these CD4+ CTL clones lyse CML-derived DCs was addressed. As shown in Table 4, MY-1 and TO-1 did not exert cytotoxicity against b3a2 CML-derived HLA-DRB1*0901+ mature DCs, whereas these clones did lyse b3a2 peptide-loaded HLA-DRB1*0901+ DCs derived from CML cells, as well as peptide-loaded autologous DCs. The possibility that the non-DC population exerted inhibitory effect on CD4+ CTL-mediated cytotoxicity against peptide-unloaded DCs seems unlikely, because the purity of DCs generated from the CML patient used for the experiment shown in Table 4 was about 90% and the contaminating cells were normal lymphocytes.
Peptide concentration-dependent IFN- in response to stimulation with CML-derived
DCs but did not lyse DCs led us to examine the difference between the optimal concentrations of bcr-abl peptide for IFN- production and
cytotoxicity. As shown in Figure 2, the
minimal concentrations of bcr-abl peptide required for IFN-
production and cytotoxicity mediated by MY-1 and TO-1 were 10 pM/L and
1 nM/L, respectively. These data suggest that the minimum number of
HLA-peptide complexes needed for production of IFN- by
CD4+ CTLs is lower than that for CD4+
CTL-mediated cytotoxicity and that this difference may be one of the
reasons for the evidence that bcr-abl peptide-specific CD4+ CTL clones produced IFN- in response to CML-derived
DCs but did not lyse them.
Perforin-mediated cytotoxicity of bcr-abl peptide-specific CD4+ T-lymphocyte clones The cytolytic mechanisms of MY-1 and TO-1 against peptide-loaded DCs were also investigated. Two major pathways (granule exocytosis, which involves the secretion of perforin and granzymes, and the Fas/Fas ligand system), have been shown to be responsible for CTL-mediated cytotoxicity. Among them, the perforin/granzyme pathway is known to be Ca++ dependent. On the basis of this finding, the cytotoxic activity of bcr-abl peptide-specific CD4+ CTL clones was determined in the absence of extracellular Ca++. No cytotoxicity against peptide-loaded DCs was observed in the presence of the Ca++-chelating agent EGTA at concentrations of more than 1 mM (Figure 3A), indicating that bcr-abl peptide-specific CD4+ CTL-mediated cytotoxicity against DCs is completely Ca++ dependent.
It has been reported that treatment of CTLs with CMA results in the complete inhibition of perforin-based cytotoxic activity.23 In light of this finding, the significance of granule exocytosis in bcr-abl peptide-specific CD4+ CTL-mediated cytotoxicity was examined using CMA. As shown in Figure 3B, the cytotoxicity of MY-1 and TO-1 against peptide-loaded DCs was almost completely inhibited by pretreatment of CTL clones with CMA at concentrations of more than 10 nM/L, as has been reported for perforin-dependent CD8+ CTL-mediated cytotoxicity. Taken together with previous results using reverse transcriptase-polymerase chain reaction, which demonstrated expression of perforin and granzyme B messenger RNAs in MY-1 and TO-1,9 these data strongly suggest that granule exocytosis is the main pathway of cytotoxicity mediated by bcr-abl peptide-specific CD4+ CTLs.
In the present study, the ability of DCs generated from monocytes
of patients with CML to present endogenous bcr-abl fusion protein to
bcr-abl peptide-specific CD4+ T-lymphocyte clones was
investigated. The findings obtained from the present series of
experiments are as follows. First, mature DCs derived from monocytes of
patients with CML expressed DC-associated cell surface antigens,
including CD1a, CD80, CD83, and CD86, at high levels compared with
monocytes and immature DCs, in the same manner as DCs from healthy
individuals.24,25 Second, the b3a2-specific and
HLA-DRB1*0901-restricted CD4+ T-lymphocyte clones produced
IFN- DCs can develop from peripheral blood monocytes stimulated with GM-CSF
and IL-4.26,27 Under these culture conditions, monocytes develop into cells with characteristics of immature DCs. Immature DCs
have a high level of endocytic activity but low T lymphocyte stimulatory capacity.28 Immature DCs also have relatively
low levels of surface CD80 and CD86, which are important costimulatory molecules for T-lymphocyte activation, compared with mature DCs, and
are characterized by the absence of CD83 expression. Addition of
TNF- It is noteworthy from the present study that mature DCs derived from monocytes of patients with CML can naturally process and present endogenous bcr-abl fusion protein to CD4+ T lymphocytes in the context of HLA class II molecules. The possibility that DCs captured exogenous bcr-abl fusion protein that had been released from dead CML cells seems unlikely because most DCs were alive during culture and bcr-abl-specific CD4+ T-lymphocyte clones did not respond to the culture supernatant of DCs derived from CML cells (data not shown). In general, endogenous antigens are degraded in the cytoplasm into oligopeptides, a fraction of which is transported into the endocytoplasmic reticulum by the transporter associated with antigen presentation. In the endoplasmic reticulum, these peptides bind to newly synthesized MHC class I molecules and the resulting complexes are transported to the cell surface where they are recognized by CD8+ T lymphocytes. On the other hand, exogenous antigens are processed into peptides capable of binding to MHC class II molecules in an endocytic compartment and are presented to CD4+ T lymphocytes. However, it has recently been shown that the MHC class II pathway can process and present endogenous antigens as well as exogenous antigens.29-31 For example, virus-infected cells are recognized by CD4+ T lymphocytes in a viral antigen-specific and MHC class II-restricted manner.32-36 It has also been reported that tumor cells transfected with syngeneic MHC class II genes could present endogenously synthesized tumor-associated protein-derived peptides in the context of MHC class II molecules to CD4+ T lymphocytes.37,38 Colony formation of CML cells has been shown to be augmented by b3a2 peptide-specific CD4+ T-lymphocyte clones in a b3a2-specific and HLA-DR-restricted manner.9 ten Bosch and colleagues have reported that bcr-abl (b3a2) peptide-specific CD4+ T lymphocytes could recognize b3a2 fusion protein expressing blasts from a patient with CML blast crisis in an HLA-DRB1*0401-restricted manner.4 These authors also reported that CD4+ T-lymphocyte clones specific for the bcr-abl (b2a2) fusion peptide responded to an autologous B-lymphoblastoid cell line transfected with invariant chain complementary DNA in which the HLA class II-associated invariant chain peptide was replaced by a b2a2 fusion oligonucleotide sequence.39 Taken together with previous data, our present findings strongly suggest that DCs generated from monocytes from patients with CML can process and present endogenously synthesized bcr-abl fusion protein to CD4+ T lymphocytes. Although HLA-DRB1*0901-restriced CD4+ T-lymphocyte clones specific for b3a2 peptide, MY-1 and TO-1, lysed b3a2 peptide-loaded autologous DCs and HLA class II-matched allogeneic DCs generated from CML cells, peptide-unloaded DCs derived from HLA-DRB1*0901+ patients with b3a2 type CML were not lysed by these CTL clones. Because cytotoxicity mediated by MY-1 and TO-1 appeared to be Ca++ dependent and was inhibited by CMA, an inhibitor of perforin-dependent cytotoxicity, the granule exocytosis mechanism seemed to be the main pathway of cytotoxicity mediated by MY-1 and TO-1. In a murine system using various mutant and knockout mice, it has been demonstrated that granule exocytosis is dominant in CD8+ CTL-mediated cytotoxicity, whereas the main pathway of cytotoxicity mediated by CD4+ CTLs is the Fas/Fas ligand system.40-43 In contrast, we have clearly demonstrated that human CD4+ CTLs specific for herpes simplex virus as well as allogeneic antigens exerted cytotoxic activity via the perforin-dependent pathway.44,45 Taken together, granule exocytosis seems to be an important pathway for CD4+ CTL-mediated cytotoxicity to CML cells. IFN- The present data increase our understanding of the role of CD4+ T lymphocytes and DCs in the anti-CML immune response. Our previous report showing that bcr-abl peptide-specific CD4+ T lymphocytes augmented colony formation by CML cells9 suggests that stimulation of bcr-abl-specific CD4+ T lymphocytes may exacerbate the disease. On the other hand, the critical role of CD4+ T lymphocytes in induction of antitumor immunity has been demonstrated in human in vitro as well as murine in vivo and in vitro systems.47,48 CD4+ T lymphocytes activated by antigen stimulation provide a helper function for induction of CD8+ CTLs through the release of cytokines. In addition, interactions between CD40 ligand and CD40 on the CD4+ T lymphocytes and DCs, respectively, appear critical in activating the DCs to present antigens to and costimulate the priming of CD8+ CTL precursors.49 Taken together, DCs generated from CML cells, which can present endogenous bcr-abl fusion protein to both CD4+ and CD8+ T lymphocytes in the context of HLA class II and class I, respectively, seem to play a crucial role in generating an anti-CML immune response. Accordingly, immunotherapy using DCs derived from CML cells might be an effective treatment for CML.
We thank Drs Junko Torii and Eiji Sada for their help with statistical analyses.
Submitted October 5, 2000; accepted March 27, 2001.
Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan; the Naito Foundation; and the Sagawa Cancer Research Foundation.
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: Masaki Yasukawa, 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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Top
Abstract
Introduction
) and
HLA-DRB1*0901
) mature DCs preincubated
with various concentrations of b3a2 peptide for 4 hours was determined
by 51Cr release assays. (B) IFN-
) in virus-specific CD4+ human cytotoxic T-cell clones.
Blood.
1993;81:1527-1534