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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 780-786
RAPID COMMUNICATION
Dendritic Cells Derived In Vitro From Acute Myelogenous Leukemia Cells
Stimulate Autologous, Antileukemic T-Cell Responses
By
A. Choudhury,
J.C. Liang,
E.K. Thomas,
L. Flores-Romo,
Q.S. Xie,
K. Agusala,
S. Sutaria,
I. Sinha,
R.E. Champlin, and
D.F. Claxton
From The University of Texas M.D. Anderson Cancer Center, Houston,
TX; and Immunex Corporation, Seattle, WA.
 |
ABSTRACT |
We have previously reported that leukemic dendritic cells (DC) can
be generated ex vivo from myelomonocytic precursors in chronic
myelogenous leukemia. In this study we report the generation of DC from
acute myelogenous leukemia (AML) cells and their potent ability to
stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML
patients with a variety of chromosomal abnormalities were studied for
their ability to generate DC. In all but 1 case, cells with the
morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels
of major histocompatibility complex class I and class II antigens as
well as the costimulatory molecules B7-2 and ICAM-1. In three
cases these cells were determined to be of leukemic origin by
fluorescence in situ hybridization for chromosomal abnormalities or
Western blotting for the inv(16) fusion gene product.
Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able
to lyse autologous leukemia targets, whereas little cytotoxicity was
noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful
for immunotherapy of many AML patients.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE MYELOGENOUS leukemia (AML)
encompasses a group of disorders characterized by clonal accumulation
of myeloid blasts in the blood and marrow. The malignant cells exhibit
variable degrees of myeloid differentiation and are associated with a
number of well-characterized cytogenetic abnormalities and gene
rearrangements. Specific cytogenetic changes are often associated with
specific disease phenotypes and clinical outcomes.1 Normal
pluripotent hematopoietic stem cells coexist in the marrow with the
leukemic clone. After successful antileukemic therapy, the normal
hematopoietic elements repopulate the marrow. In most cases, the
remission is only transient. The therapeutic challenge for most
patients with these disorders thus lies in achieving long-term remission.
There is considerable data indicating immune activity against human
leukemias. The therapeutic efficacy of allogeneic marrow or stem cell
transplant is at least in part due to the graft versus leukemia
effect.2 Autologous immune responses have also been suggested. Interleukin-2 (IL-2) infusion has been used for AML both at
the time of relapse3 and in remission.4
Dendritic cells derived from the myeloid lineage have been
characterized as the primary professional antigen-presenting cells (APC) responsible for eliciting T-cell responses.5,6
Considerable interest has developed in the potential use of dendritic
cells (DC) for the generation of effective cancer immunotherapy. We recently reported that human chronic myelogenous leukemia (CML) will
differentiate into DC under the influence of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and
tumor necrosis factor- (TNF-) and that these DC elicit
leukemia-specific cytotoxicity in autologous lymphocytes.7
We speculated that this approach to the development of antileukemic
immunoreactivity might be extended to other diseases. Santiago-Schwartz
et al8 showed in one case that AML cells were able to give
rise to cells of dendritic phenotype and function in culture.
We present here results of AML cultured for the ex vivo development of
DC and the cytotoxic activity of autologous lymphocytes stimulated by
the AML-derived DC.
| |
MATERIALS AND METHODS |
Patient samples.
Blood and bone marrow samples for this study were collected from
patients with informed consent at the time of sample collection for
diagnostic tests under guidelines and procedures approved by the
institutional review board of the M.D. Anderson Cancer Center. Relevant
clinical and diagnostic laboratory data for the 19 cases are shown in
Table 1. Remission samples were collected during morphological remission (<5% marrow blasts) after therapy for
the disease. Patients 1 and 11 were the only ones with any history of
antecedant hematological disorder.
Generation of DC from AML cells.
Recombinant human IL-4 was kindly provided by Schering-Plough,
Kenilworth, NJ. Recombinant human TNF- was kindly provided by The
National Institutes of Health, Bethesda, MD. Recombinant trimeric human
CD40L was kindly provided by Immunex Corp, Seattle, WA. This is a
trimeric, soluble molecule capable of binding to and activating the
CD40 receptor. Peripheral blood mononuclear cells (PBMNC)were separated
from AML patients and normal donors using a Histopaque-1077 density
gradient (Sigma, St Louis, MO) and cultured immediately or
cryopreserved. To generate DC, mononuclear cells at 2 to 4 × 106 cells/mL were cultured in 75-mm2 tissue
culture flasks (Corning Inc, Corning, NY) in Aim-V serum-free medium
(GIBCO Laboratories, Grand Island, NY) with various combinations of
human cytokines. Recombinant GM-CSF (Immunex) and IL-4 were added at a
final concentration of 1,000 U/mL. Recombinant TNF- was used at a
final concentration of 500 U/mL. Recombinant CD40L was added at a
concentration of 1 µg/mL. The medium was changed every 3 to 4 days,
and the unfractionated AML cell cultures were used for phenotypic and
functional studies.
Phenotypic analysis of cells by flow cytometry.
Fluorescein- or phycoerythrin-conjugated mouse monoclonal antibodies
against CD3, CD14, CD19, as well as fluorochrome-labeled isotype
controls were purchased commercially (Becton Dickinson, Duarte, CA).
Fluorescein- or phycoerythrin-labeled antibodies against HLA-D, HLA-A,
HLA-B, HLA-C, CD1a, CD40, CD54, CD80, and CD86 were purchased from
Pharmingen (San Diego, CA). Freshly isolated or day-14 cells were
dispersed to single-cell suspension by gentle trituration through a
pitpette. They were then incubated with various combinations of
antibodies, washed with phosphate-buffered saline (PBS)-hIgG and
analyzed using a FACSscan II flow cytometer and the LYSIS software
(Becton Dickinson). Forward scatter versus side scatter gates were set
up during analysis to eliminate debris and clumps, and the remaining
cell populations were analyzed for expression of phenotypic markers.
Genetic analysis of cells by fluorescence in situ hybridization
(FISH).
To determine if the DC generated in culture were derived from the
leukemic clone or the normal population of the patients' peripheral
blood cells, we selected 2 patients whose leukemic cells were known to
exhibit a specific chromosomal abnormility, ie, trisomy of chromosome
8, that could be easily identified in the interphase nuclei by FISH.
Freshly isolated PBMNC, as well as dendritic cells generated in the
culture following 14 days of incubation with GM-CSF, IL-4, and TNF-
or CD40 ligand were analyzed. Fluorescein-labeled chromosome
8-specific alpha satellite DNA probe (Oncor, Gaithesburg, MD) was used
according to the manufacturer's instructions. At least 400 interphase
nucleii were examined in each sample to quantitate the percentage of
cells with three hybridization signals for chromosome 8.
Western blotting for CBFB-MYH11.
This was performed as previously described9 using
polyclonal antipeptide antibodies. Antisera included C2 and an
antiserum to an MYH11 peptide developed as described.9
Allogeneic mixed leukocyte response.
PBMNC from normal volunteers were used as responder cells. Freshly
isolated or 10- to 14-day cultured AML cells irradiated with 30 Gy were
used as stimulator cells. A total of 2 × 105
allogeneic PBMNC were added as responders to varying numbers of
accessory cells. Lymphocyte proliferation was measured after 5 days of
culture with addition of 3H-thymidine for 18 hours.
Expansion of T cells from AML patients and stimulation with
autologous DC.
PBMNC of AML patients were suspended at a concentration of 5 × 106 cells/mL and cultured in Aim-V containing 1 µg/mL
anti-CD3 antibody (Ortho Biotech, Raritan, NJ) and 100 U/mL of IL-2. Nonadherent cells were resuspended in fresh medium
containing 100 U/mL IL-2 every 3 days. On day 7 the cells were washed
and cocultured at a ratio of 3:1 with washed autologous AML-DC or
freshly thawed washed autologous leukemia cells in Aim-V Medium without
additives for 3 days. The dendritic cell activated lymphocytes (DC-AL)
or fresh leukemia-stimulated lymphocytes were expanded for an
additional 4 to 7 days in IL-2 containing medium before being assayed
for antileukemic activity.
T-cell cytotoxicity assay.
T-cell cytotoxicity was measured in vitro using the lactate
dehydrogenase-release assay.10 Cryopreserved, thawed target cells were cocultured with T cells at effector target ratios of 1:1,
1:10, and 1:25 ratios for 6 hours in 96-well round-bottomed plates in
phenol red-free RPMI + 0.1% human albumin (Miles Inc, Elkhart, IN).
Spontaneous release of effector and target cells was controlled by
separate incubation of the respective populations. Maximal
LDH enzyme release was measured after lysis of the target cells with 0.5% triton X-100 (Sigma). Cell-free supernatants were incubated in a separate 96-well plate with lactate dehydrogenase (LDH)
substrate10 for 10 minutes before measuring absorbance using a microplate reader (Dynatech MR5000) at 490 nm with a 650-nm reference. Percentage cytotoxicity was calculated according to the
formula:
where
E is the LDH release by effector-target coculture, St
is the spontaneous release by target cells, Se is the
spontaneous release by effector cells, and M is the maximal
release by target cells.
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RESULTS |
Characteristics of AML PBMNC cultured in various cytokine combinations.
PBMNC from AML patients of a variety of FAB types, clinical histories,
and cytogenetics were used to generate DC. Clinical details of the AML
patients whose cells were used are listed in Table 1. As may be seen,
patients had a variety of FAB types and cytogenetic changes. Two
patients (patients 1 and 11) had histories of antecedant hematological
disorders (anemia or myelodysplastic syndromes) before AML diagnosis.
Nearly all cultures for this work were initiated from samples
cryopreserved at a previous date. Because of the limited material
available from any given patient, not all studies shown could be done
for every patient's leukemia.
Cultures of AML cells were replenished with fresh medium and cytokines
every 3 to 4 days. Cells cultured in various cytokine combinations were
assessed for total number of viable cells and cells with dendritic
morphology after various culture periods. Approximately 25% to 45% of
the initial cell population was recovered after 9 days of culture. No
significant change in numbers of cells was observed between day 9 and
day 20 of culture (not shown). Similar yields of viable cells were
noted for AML cell cultures from other patients. Freshly isolated AML
PBMNC displayed a spherical morphology without discernable cells
exhibiting dendritic processes (Fig 1A).
Between days 10 and 14, cultures with GM-CSF, IL-4, and TNF- (Fig
1B) or GM-CSF, IL-4, and CD40L (Fig 1C) consistently displayed an
increase in cell size and showed cell clusters with dendritic
morphology in all but one AML patient (patient 16).
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| Fig 1.
Morphology of freshly isolated and cultured AML cells.
Phase contrast micrographs of freshly isolated AML PBMNC (A; 100×
origonal magnification); AML cells cultured for 14 days in GM-CSF,
IL-4, and TNF (B; 250× original magnification); or GM-CSF, IL-4,
and CD40L (C; 250× origianl magnification). (A and B) Obtained with
cells from patient 13; (C) Obtained with cells from patient 18.
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Phenotype of cultured AML cells.
Flow cytometry has been performed on various cases of AML cultured for
11 to 14 days in GM-CSF, IL-4, and TNF- or GM-CSF, IL-4, and CD40L.
In each of these cases the flow cytometric phenotype of the
cytokine-cultured cells has been compared with autologous leukemia,
either freshly thawed cells or cells cultured for equivalent periods in
medium without cytokines. Figure 2 shows
the phenotype of AML cells of one representative patient (patient 18),
cultured in medium alone (Fig 2A); GM-CSF, IL-4, and TNF (Fig 2B);
or GM-CSF, IL-4, and CD40L (Fig 2C). Consistent with the increase in
cells of dendritic morphology, the triple cytokine cultures showed an
upregulation of the dendritic cell lineage marker CD1a as well as the
costimulatory molecules B7-1 (CD80), B7-2 (CD86), CD40, and ICAM-1
(CD54). Cells from patients 7 and 10 through 13 were also cultured and
analyzed by flow cytometry. In all of these other five cases examined,
culture with either cytokine combination generated increases in
expression of CD1a, CD80, CD86, and ICAM-1. The cultured cells were
negative for CD3 and CD19 (data not shown). Morphological and
phenotypic changes of cultured AML cells similar to those described in
the current study were also noted with other AML patients in a previous
report from our laboratory.11
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| Fig 2.
AML cells cultured in the triple cytokine combinations
upregulate expression of costimulatory molecules. PBMNC from AML
patients were cultured in the triple cytokine cocktail for 14 days.
Cells were labeled with fluorescent monoclonal antibodies and analyzed
by flow cytometry. (A) Results obtained with the cells of patient 18 cultured for 14 days in Aim-V alone. (B) Cells cultured in GM-CSF,
IL-4, and TNF-. (C) Cells cultured in GM-CSF, IL-4, and CD40L. Cells
from patients 7 and 10 through 13 were also studied in similar
experiments and comparable increases in CD1a and costimulatory molecule
expression were observed.
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Demonstration of leukemic origin of cultured DC.
To confirm that the DC arising in culture are in fact derived from the
leukemic cells we selected cases with identifiable clonal markers.
Cells from patient 7, with trisomy 8, were cultured in the triple
cytokine cocktails and analyzed by interphase FISH for +8 genotype. The
results obtained with cells cultured in GM-CSF + IL-4 + TNF- are
shown in Fig 3. Freshly isolated PBMNC from patient 7 displayed trisomy 8 in 93% of the cells. After 14 days of
culture in GM-CSF + IL-4 + TNF-, 92% of the cells showed trisomy 8. Eighty-eight percent of the cells cultured in GM-CSF + IL-4 + CD40L
were positive for +8. Given that 44% to 87% of the cells in these
cultures expressed the morphology and immunophenotype of DC in these
cultures (data not shown), the leukemic clones would have to include
the dendritic cells in these cultures. Cells from patient 19 cultured
in a similar manner with GM + IL-4 + TNF- showed trisomy 8 in 88%
of the cells.
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| Fig 3.
DC-rich AML cell cultures from a trisomy 8 patient show
+8 chromosome in their nuclei. Fluorescein-labeled chromosome
8-specific alpha satellite DNA probe was used to perform FISH analysis
on freshly isolated PBMNC or cells cultured for 14 days in GM-CSF + IL-4 + TNF-, or GM-CSF + IL-4 + CD40L. Results
depicted were obtained with cells cultured in GM-CSF + IL-4 + TNF-. At least 400 interphase nuclei were examined in each sample to
quantitate the percentage of cells with three hybridization signals for
chromosome 8. Cells from patient 7 and patient 14 were used for this
study. The number of +8 positive cells in the three groups of cells
were 93%, 92%, and 88%, respectively. With patient 14, only DC
generated with GM-CSF + IL-4 + TNF- were analyzed. Eighty-eight
percent of the cells were observed to express trisomy 8.
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Patient 13, whose leukemic karyotype showed inv(16), yielded cells that
on culture were 63% DC. These cells were submitted for Western
blotting using antisera directed at the two constituent moieties of the
CBFB-MYH11 fusion protein. Results are shown in Fig 4. Signals for both CBFB and MYH11
corresponding to the 70-kD signal expected for the type A
variant of this protein12 were seen in both the cultured DC
and the thawed native leukemic cells. These bands are similar in
intensity, indicating that the cultured cell population continued to
express the leukemia associated protein.
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| Fig 4.
AML cells cultured in GM-CSF, IL-4, and TNF- express
the inv(16) gene product. AML cells from patient 14 were cultured for
14 days in GM-CSF + IL-4 + TNF-. Triton X-100 lysates of freshly
isolated and cultured AML cells were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then analyzed
by Western blotting using antibodies against the N-termini (C2) and
C-termini (AH107) of the CBFB-MYH11 inv(16) gene product. Lysates from
K562 cell line served as a negative control.
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Allostimulatory activity of the cultured DC.
Freshly isolated and cultured AML cells were examined for their ability
to stimulate allogeneic T cells in a mixed leukocyte response (MLR)
assay. Figure 5 compares the
allostimulatory ability of fresh TNF- combination and CD40L
combination cultured PBMNC from five AML patients. In each case GM-CSF + IL-4 together with TNF- or CD40L showed a higher allostimulatory
potential than freshly isolated AML cells.
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| Fig 5.
GM-CSF, IL-4 with TNF- or CD40L are the most effective
combinations for augmenting allostimulatory activy of AML cells.
Allogeneic MLR assays were performed as described in Materials and
Methods. Freshly isolated ( ), GM + IL-4 + TNF--cultured
( ), and GM + IL-4 + CD40L-cultured ( ) AML cells from five
patients were compared for their ability to stimulate PBMNC of a normal
allogeneic donor. Results represent the mean ± SD of triplicate
cultures.
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Stimulation of autologous antileukemic activity by AML derived DC.
Autologous T cells were cocultured with freshly isolated or cultured
DC-rich AML cells and tested for cytotoxicity against autologous
leukemic or autologous PBMNC obtained in remission. Figure 6 represents the autologous
antileukemic cytotoxicity of T cells stimulated with AML-DC, fresh AML
cells, or IL-2 alone. In each of these patients, stimulation of
autologous lymphocytes with AML-DC markedly increased their
cytotoxicity against autologous leukemia targets. Fresh AML cells could
induce a minor level of cytotoxicity in 1 of 4 patients. IL-2-expanded
lymphocytes failed to show antileukemic cytotoxicity at the
target:effector ratio tested. Figure 7A
through C represents the cytotoxicity of DC-AL against autologous
leukemic and remission cells. In Fig 7A, GM-CSF + IL-4 + TNF- was used to generate the DC from five additional patients,
whereas GM-CSF + IL-4 + CD40L was used to generate DC in Fig 7B. Figure
7C represents the reactivity of DC-AL used in Fig 7B, against
autologous remission cells. Results depicted in Fig 7B and C comprise a
single experiment with the same patient samples, whereas those depicted
in Fig 7A represent a different experiment.
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| Fig 6.
Stimulation of autologous, antileukemic cytotoxicity by
fresh AML cells, AML-DC cultures, and IL-2 alone. Autologous T cells
were cocultured with fresh AML PBMNC, AML-DC-rich cultures, or IL-2
alone as described in Materials and Methods. They were then tested for
cytotoxicity against autologous leukemia targets by LDH-release assay.
Results represent the mean cytotoxicity of triplicate cultures. ()
AML-DC-stimulated T cells, ( ) fresh AML PBMNC stimulated T cells,
( ) T cells cultured in IL-2 alone. Cytotoxicity of DC-AL compared
with IL-2-stimulated T cells at 25:1 ratio is significant (P < .005, Student's t-test) for each patient. Cytotoxicity of
fresh AML cell-stimulated T cells compared with IL-2-stimulated T
cells at 25:1 ratio is significant (P < .010, Student's
t-test) for patient 13.
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| Fig 7.
Autologous AML-DC-stimulated T cells lyse leukemic
targets but not autologous remission PBMNC. Autologous T cells were
cultured with AML-DC cultures and tested as cytotoxic effector cells in
an LDH-release assay. Results represent the mean cytotoxicity of
triplicate cultures. (A) The antileukemic cytotoxicity of autologous
lymphocytes stimulated with DC generated with GM + IL-4 + TNF-;
(B) DC generated with CD40L. The effector cells described in (B) were
tested for cytotoxicity against remission PBMNC, rather than leukemic
cells in (C). (B and C) represent a single experiment, whereas (A) was
a separate experiment with a different series of patients. Each symbol
represents one patient. Results represent the mean cytotoxicity of
triplicate cultures. Cytotoxicity of DC-AL at a ratio of 25:1 against
leukemic targets (B) compared with remission targets (C) is significant
(P < .005, Student's t-test) for every patient.
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| |
DISCUSSION |
These findings show that cells of DC phenoytpe and function can be
derived from the majority of human AML samples. Given the known
biological heterogeneity of AML, it not surprising that the results are
variable and that occasional AML cell samples would fail to show
differentiation toward DC. The leukemias show highly variable degrees
of maturation arrest in vivo and highly variable degrees of monocytoid
differentiation. The ontogeny of DC is thought to proceed directly
through CD34 progenitors or through the monocyte lineage, and in fact
cells of dendritic cell phenotype may be derived from normal
monocytes.6 The ability of a given leukemia to
differentiate toward DC may be tied to the level of maturation block in
the monocytoid lineage.
Santiago-Schwarz8 and colleagues described the generation
of DC-like cells from the PBMNC of a single AML patient. In their studies, the terminal differentiation of an adherent DC-like population was achieved using a combination of GM-CSF, TNF-, and IL-6. In the
present study IL-6 failed to show any significant effect in generating
DC-like cells from AML PBMNC or increasing allostimulatory ability of
cultured AML-PBMNC (data not shown). Classically, DC are nonadherent
cells. Moreover, in our experiments, cells with DC morphology,
phenotype, and functional activity were obtained in the nonadherent
fraction of the cultured AML PBMNC. The significance and reasons behind
the observed differences in our results and those of Santiago-Schwarz
are not clear. We speculate that differences in results may arise from
differences in culture conditions and intrinsic variations in ability
and default pathways of differentiation of leukemic cells obtained from
various patients.
Our data are most compatible with derivation of the cultured dendritic
cells from the malignant clone. In two forms of AML with very different
cytogenetic and phenotypic characteristics, we show evidence for
prominent representation of the malignant clone in the cultured cell
populations. The Western blotting demonstration of CBFB-MYH11 in the
culture from an inv(16) patient proves that AML cells continue to
express the leukemic associated proteins even after undergoing
cytokine-induced differentiation in vitro. The finding of trisomy 8 by
FISH conclusively shows derivation of the DC from the malignant clone.
These findings are consistent with those of Robinson et
al,13 who found DC derived from the malignant clone in a
single case of acute leukemia with a deletion of chromosome 5q.
It has been recently proposed that helper T cells contribute to the
development of cytotoxic T cells by providing a priming signal to the
APC, which then becomes fully competent to prime cognate cytotoxic T
cells. The helper lymphocyte delivers the signal to the APC via the
CD40-CD40L pathway. Stimulation of CD40 on the APC by anti-CD40
antibody or exogenous multimeric CD40 ligand can replace the
requirement of helper T cells.14-16 The result of our study
suggests that the addition of CD40L to GM-CSF and IL-4 may serve not
only to facilitate generation of DC from AML myelomonocytic cells, but
to activate the DC, enabling the generation of cytotoxic T cells with
antileukemic reactivity.
The antileukemic cytotoxicity elicited from autologous lymphocytes
after stimulation with leukemia-derived DC is similar to what we have
previously observed for CML.7 Despite the heterogeneous nature of AML and the varied cytogenetic abnormalities of the patients
tested in this study, the majority of the AML cells were able to
generate DC and stimulate antileukemic cytotoxicity. Further study of
these cells is warranted to determine the target antigens responsible
for the observed immune activity. These results support the speculation
that normal myeloid antigens expressed as aberrant forms or in aberrant
levels maybe of greater utility in targeting immunotherapeutic
approaches than a putative "leukemia-specific" epitope defined by
a unique chromosomal abnormality associated with a particular form of
leukemia.11
The potential therapeutic implications of this work are substantial.
Leukemic DC can potentially be used as a cellular leukemia vaccine in
vivo or to generate antileukemic T cells in vitro for adoptive
immunotherapy. Their use in immunotherapy may eliminate minimal
residual disease and convert transient responses into durable
remissions in this normally fatal disease.
| |
ACKNOWLEDGMENT |
The authors thank Drs Mary Ellen Rybak (Schering-Plough) for providing
IL-4 and Mario Sznol (CTEP-NCI) for TNF-. We also thank Dr Margaret
Kripke for useful discussions and suggestions.
| |
FOOTNOTES |
Submitted August 26, 1998; accepted November 11, 1998.
Supported by funds from the Leukemia Society of America (Grant Ref No
6210-98), The National Institutes of Health (CA55164), and the Adler 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 A. Choudhury, PhD, The University of Texas
M.D. Anderson Cancer Center, Box 24, 1515 Holcombe Blvd, Houston, TX
77030.
| |
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393:480, 1998[Medline]
[Order article via Infotrieve]
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Abstract
Introduction![% Age Cytotoxicity = [(E − St − Se)/M − St)] × 100](/content/vol93/issue3/fulltext/780/img001.gif)