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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2048-2055
CD34+ Acute Myeloid and Lymphoid Leukemic Blasts Can
Be Induced to Differentiate Into Dendritic Cells
By
A. Cignetti,
E. Bryant,
B. Allione,
A. Vitale,
R. Foa, and
M.A. Cheever
From Corixa Corp, Seattle, WA; the Fred Hutchinson Cancer Research
Center, Seattle, WA; the Divisione Ospedaliera di Ematologia, Azienda
Ospedaliera S. Giovanni Battista, Torino, Italy; the Dipartimento di
Biotecnologie Cellulari ed Ematologia, University "La Sapienza,"
Rome, Italy; and the Dipartimento di Scienze Biomediche ed Oncologia
Umana, University of Torino, Torino, Italy.
 |
ABSTRACT |
CD34+ hematopoietic stem cells from normal individuals
and from patients with chronic myelogenous leukemia can be induced to differentiate into dendritic cells (DC). The aim of the current study
was to determine whether acute myeloid leukemia (AML) and acute
lymphoblastic leukemia (ALL) cells could be induced to
differentiate into DC. CD34+ AML-M2 cells with chromosome
7 monosomy were cultured in the presence of granulocyte-macrophage
colony-stimulating factor (GM-CSF), tumor necrosis factor  (TNF),
and interleukin-4 (IL-4). After 3 weeks of culture, 35% of the AML-M2
cells showed DC morphology and phenotype. The DC phenotype was defined
as upmodulation of the costimulatory molecules CD80 and CD86 and the
expression of CD1a or CD83. The leukemic nature of the DC was validated
by detection of chromosome 7 monosomy in sorted DC populations by
fluorescence in situ hybridization (FISH). CD34+ leukemic
cells from 2 B-ALL patients with the Philadelphia chromosome were
similarly cultured, but in the presence of CD40-ligand and IL-4. After
4 days of culture, more than 58% of the ALL cells showed DC morphology
and phenotype. The leukemic nature of the DC was validated by detection
of the bcr-abl fusion gene in sorted DC populations by FISH. In
functional studies, the leukemic DC were highly superior to the
parental leukemic blasts for inducing allogeneic T-cell responses.
Thus, CD34+ AML and ALL cells can be induced to
differentiate into leukemic DC with morphologic, phenotypic, and
functional similarities to normal DC.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DC) are potent
initiators of T-cell-mediated immune responses.1 DC pulsed
with tumor antigens can effectively prime T-cell responses in vitro and
in vivo and are being developed as cancer vaccines in
humans.2,3 Human DC can be generated in vitro from
CD34+ stem cells present in bone marrow (BM), umbilical
cord blood, and peripheral blood cells.4-8 It has recently
been reported that chronic myelogenous leukemia (CML) cells can be
induced to differentiate into DC.9-12 DC derived from CML
cells retain the bcr-abl genotype. The implication is that CML cells
with both a leukemic genotype and a DC phenotype might be potent
inducers of T-cell responses to leukemia antigens.
The current study examined whether CD34+ acute leukemic
cells can similarly be induced to differentiate into leukemic DC. The expectation of being able to induce differentiation of acute leukemia cells into DC is different than the expectation for CML
cells. CML is a disorder of pluripotent hematopoietic stem
cells. It is known that CML cells can maturate along normal lines of
hematopoietic development into normal functioning myeloid and lymphoid
cells carrying the Philadelphia (Ph) chromosome. By contrast, in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), the malignant hematopoietic cells fail to undergo
extensive maturation, resulting in the expansion of large numbers of
leukemic blasts blocked at specific phenotypic and functional stages of differentiation.
Leukemia cells present in patients are often commingled with normal
hematopoietic cells. When studying mixed populations, determination of
whether CD34+ leukemic cells can be induced to
differentiate into DC requires a leukemia marker that persists
throughout the differentiation process. The presence of normal
CD34+ stem cells with leukemic CD34+ blasts can
confound the interpretation of results, because some of the normal
CD34+ stem cells will differentiate into DC. In addition,
the presence of normal monocytes in peripheral blood should also be
taken into account. To determine whether acute leukemia cells
differentiate into DC, it is necessary to document the leukemic origin
of the DC. A leukemia-specific marker is required. Cell surface
phenotype markers could be used, but are less satisfactory, because
most myeloid leukemic cells express many of the same markers as normal DC.13
In the current study, AML and ALL cells with defined genotypic and/or
phenotypic abnormalities were chosen to monitor the differentiation
process of the leukemic clone. CD34+ leukemias were chosen
for this initial study on the presumption that the behavior of leukemic
cells arrested at a more immature stage of differentiation might more
closely resemble that of pluripotent stem cells in response to
differentiating growth factors and cytokines. The results show that
cells with DC morphology and phenotype similar to those described for
normal DC can be derived from the malignant clone in AML and ALL
patients. The derived leukemic DC are more efficient in the stimulation
of allogeneic T lymphocytes than are the original unprocessed leukemic
blasts. Thus, leukemia derived DC can now be tested as stimulators of
antileukemia T-cell responses in vitro and as leukemia vaccines in vivo.
| |
MATERIALS AND METHODS |
Isolation of peripheral blood mononuclear cells (PBMC) from acute
leukemia patients and generation of DC.
PBMC or BM from acute leukemia patients were collected at diagnosis
from the Division of Hematology, Ospedale S. Giovanni Battista (Torino,
Italy) and from the Division of Hematology, Universita' "La
Sapienza" (Rome, Italy). PBMC were isolated after centrifugation
over a Ficoll density gradient (Sigma, St Louis, MO) and cryopreserved
in medium consisting of 90% fetal bovine serum (FBS; Hyclone
Laboratories Inc, Logan, UT) and 10% dimethyl sulfoxide (DMSO; Sigma).
Before use, cells were thawed, washed, resuspended in serum-free medium
(X Vivo-10; BioWhittaker, Walkersville, MD), and seeded into
25-cm2 flasks at 2 × 105/mL. Cells from
AML patients were stimulated with 80 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 80 ng/mL interleukin-4 (IL-4; a
gift of Immunex, Seattle, WA), and 10 ng/mL tumor necrosis
factor  (TNF; Biosource International, Camarillo, CA). Cells from
ALL patients were stimulated with 3 µg/mL CD40-Ligand (CD40L; trimer; a gift of Immunex) and 80 ng/mL IL-4. Cytokines and fresh medium were
added every 5 to 7 days to the cultures.
Phenotypic analysis.
When a significant proportion of cells in culture started to show DC
morphology, ie, being larger in size, associated in clusters, and
having typical cytoplasmic motile processes, they were analyzed for
phenotype. Fluorescein- or phycoerythrin-conjugated mouse monoclonal
antibodies (MoAbs) to the following antigens were used: CD1a, CD7,
CD10, CD19, CD33, CD34, CD40, CD86, HLA class I, HLA-DR (Pharmingen,
San Diego, CA), CD13 CD14, CD80 (Becton Dickinson, San Jose, CA), and
CD83 (Immunotech, Westbrook, ME). Fluorescein- or
phycoerythrin-conjugated isotype control mouse MoAbs were purchased from Pharmingen. CD34 is a marker of hematopoietic stem cells. CD13 and
CD33 are myeloid antigens. CD19 is expressed by both mature and
immature B cells and CD10 is expressed during the early stages of
B-cell differentiation. CD80 (also known as BB1/B7-1), CD86 (also known
as B70/B7-2), and CD40 are accessory and costimulatory molecules. CD1a
and CD83 (also known as HB15) are considered to be DC
markers.1,13,14 CD14 is expressed by monocytes. CD7 is a
lymphoid marker that can be aberrantly expressed by some myeloid
leukemias. Thawed blasts or cultured leukemic cells were incubated with
fluorescein- or phycoerythrin-conjugated MoAbs, alone or in
combination, for 20 minutes, washed with a phosphate-buffered saline
(PBS) buffer containing 1% human serum, and analyzed
using a FACSCalibur flow cytometer (Becton Dickinson). To exclude dead cells and debris, the viable cells were gated based on propidium iodide
staining (Sigma). DC were defined by the expression of either CD1a or
CD83 coupled with the expression of the costimulatory molecules CD80
and CD86. When a significant upmodulation of these molecules was
observed, cells were used for fluorescence in situ hybridization (FISH)
analysis and allogeneic mixed lymphocyte reactions (allo-MLR; see below).
Magnetic cell sorting.
When a DC phenotype was obtained, the cells were magnetically sorted
into CD80+ or CD83+ populations using the MACS
system (Miltenyi Biotec, Auburn, CA). Briefly, cells were labeled with
MoAbs against CD80 or CD83 for 20 minutes, washed twice, and
resuspended in 80 µL of MACS buffer (PBS without Ca2+ and
Mg2+, supplemented with 1% bovine serum albumin [BSA]
and 5 mmol/L EDTA). Twenty microliters of MACS superparamagnetic beads
conjugated with a rat antimouse IgG1 MoAb (for CD80 sorting) or with a
mouse antifluorescein MoAb (for CD83 sorting) was added to the cell suspension and incubated for 15 minutes at 6°C to 12°C. After washing twice in MACS buffer, cells were separated on a magnetic stainless steel wool column according to the manufacturer's
recommendations. Negative cells were collected from the column eluate,
and positive cells remained attached to the magnetized matrix. To
obtain the positive fraction, the column was removed from the magnet
and flushed with MACS buffer into another tube. The purity of the positive fraction obtained with this technique was always greater than
95%.
FISH.
FISH was performed on cytospin preparations of original naive leukemic
blasts and unsorted and sorted leukemia-derived DC. Cells were cytospun
into glass slides and fixed in 4% buffered formalin. Fixed slides were
hybridized with commercially obtained probes for a chromosome
7-specific satellite (Oncor Inc, Gaithersburg, MD) or the bcr-abl
gene rearrangement (Vysis Inc, Downers Grove, IL) following standard
techniques for FISH. Briefly, slides were aged in 2× SSC at
73°C, denaturated in 70% formamide/2× SSC, and dehydrated in
an ethanol series. Probe was denatured according to the manufacturer's
recommendations, applied to slides, and hybridized overnight in a
humidified chamber at 37°C. Hybridized slides were washed in
0.4× SSC/0.3% NP-40 at 73°C for 2 minutes and rinsed in
2× SSC/0.1% NP-40. Slides hybridized with the 7 probe were
detected with anti-digoxigenin-fluorescein. The bcr-abl probe is
directly labeled and required no detection step. Slides were
counterstained in Vectashield with the fluorescent dye 4,6-diamino-2 phenyl-indole (DAPI). Cells were analyzed using a Zeiss Axioscope equipped with epifluorescence and appropriate filter combinations for
viewing the fluorophores (Carl Zeiss, Thornwood, NY). One hundred cells
were analyzed in each sample to quantitate the signal pattern.15
Stimulatory function of the leukemic DC.
Allo-MLR were set up with PBMC from healthy donors as responders and
naive leukemic cells or leukemia-derived DC as stimulators. A total of
105 responder cells were seeded in 96-well plates in the
presence of varying concentration of stimulator cells, which had been
irradiated at 3,000 rads. Cultures were incubated at 37°C in AIM V
medium (GIBCO BRL, Grand Island, NY) in 5% CO2 for 5 days.
Cells were then pulsed with 1 µCi of tritiated thymidine
([3H]-thymidine; Amersham-Life Science, Boston, MA) 18 hours before harvesting into glass fiber filters.
[3H]-thymidine incorporation was measured on a
 -counter and expressed as mean counts per minutes (cpm) of triplicates.
| |
RESULTS |
In vitro generation of leukemic DC from AML-M2 blasts.
Normal CD34+ BM cells and CD34+ CML BM cells
can be induced to differentiate into DC by GM-CSF, TNF, and IL-4. To
assess whether CD34+ AML cells can be similarly induced to
differentiate into DC, PBMC containing 95% CD34+ leukemic
blasts from a patient with AML-M2 (chromosome 7 monosomy) were cultured
in the presence of 80 ng/mL GM-CSF, 10 ng/mL TNF, and
80 ng/mL IL-4 for 3 weeks. Before culture, the AML blasts were
essentially negative for CD80, CD86, CD1a, and CD40 but positive for
HLA-DR and HLA class I (Fig
1A). After 20 days of culture, approximately 35% of the cells
exhibited the typical morphology of DC with thin cytoplasmic processes
and increased size. The morphologic DC showed upmodulation of CD80,
CD86, CD40 and CD1a (Fig 1A). HLA-class I expression was not modified
and CD14 was negative (not shown). In the overall population, HLA-DR
expression was decreased (Fig 1A); however, when tested in a subsequent
experiment, the majority of CD80+ cells were
HLA-DR+ (Fig 1B). Normal CD34+ stem cells lose
CD34 expression when induced to differentiate into DC. By marked
contrast, CD34 continued to be expressed by the majority of AML cells
with DC morphology (Fig 1A).
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| Fig 1.
(A) Phenotype of naive AML blasts and
leukemia-derived DC (patient TA). Freshly thawed PBMC from an AML-M2
patient and the same cells after 20 days of culture in the presence of
GM-CSF, TNF, and IL-4 were stained with fluorescein-or
phycoerythrin-conjugated antibodies and analyzed by flow cytometry. To
exclude debris, the viable cells were gated based on propidium iodide
staining. Histograms represent the log of fluorescence (horizontal
axis) versus the relative cell number (vertical axis). Thin lines
represent the isotype-matched indifferent murine MoAb control. The
number in each box represents the percent of positive cells. (B)
CD80/HLA-DR coexpression on leukemia-derived DC (patient TA). Cells at
day 20 of culture were double-stained with phycoerythrin-conjugated
anti-CD80 MoAb (vertical axis) and anti-HLA-DR fluorescein-conjugated
MoAb (horizontal axis). Fluorochrome-conjugated isotype-matched murine
MoAbs (IgG1) were used as a negative control. The number in each
quadrant represents the percentage of positive cells.
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The initial PBMC population contained 95% leukemia cells. The
maintenance of expression of CD34 by the cytokine-induced DC provided
substantial evidence that the DC were of leukemic origin rather than
from a minor population of normal precursors in the PBMC. To further
assess whether the DC were of leukemic origin, the induced DC
population was analyzed for chromosome 7 monosomy by FISH. To obtain a
more homogeneous DC population for analysis, CD80+ cultured
cells were positively selected by immunomagnetic sorting. Before
sorting, 35% of cultured cells expressed CD80. All of the original
blasts and 94% of sorted CD80+ cells showed chromosome 7 monosomy (Fig 2). Interphase nuclei from
normal PBMC, which were used as a control, showed a false-positive background of 5%.
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| Fig 2.
Genotype of CD80-sorted, leukemia-derived DC (patient
TA). The figure shows DAPI counterstained nuclei from a CD80-sorted
positive culture of leukemia-derived DC hybridized with a chromosome
7-satellite (green signal) probe. Cells with 1 signal indicating
monosomy 7 or 2 signals indicating the normal pattern are shown.
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To investigate the function of the leukemic DC as stimulators of T-cell
responses, their ability to stimulate an allo-MLR was compared with
that of the original leukemic population. Unfractionated cell
populations from 20-day cultures containing DC and thawed blasts from
peripheral blood were used to stimulate PBMC from an unrelated normal
donor at stimulator:responder ratios ranging from 0.5:1 to 0.06:1. The
leukemic DC elicited significantly higher proliferation than did the
original leukemic population (Fig 3).
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| Fig 3.
Proliferation of PBMC in response to allogeneic naive AML
blasts or leukemia-derived DC (patient TA). Allo-MLR was performed with
105 allogeneic PBMC as responder cells and different
numbers of thawed AML blasts or leukemia-derived DC at
stimulator:responder ratios ranging from 0.5:1 to 0.06:1. Leukemic DC
were derived from 20-day cultures. Proliferation was measured 5 days
after stimulation by [3H]-thymidine incorporation.
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We investigated whether CD34+ AML-M2 cells from a second
patient could be similarly induced to differentiate into DC. The
leukemic blasts did not have any detectable chromosomal abnormality.
Thus, an aberrantly expressed CD7 lymphoid marker on the cell surface was used to identify the leukemic origin of cultured DC. The AML cells
from the patient denoted SC were defined phenotypically as myeloid
blasts with aberrant CD7. The blasts expressed CD13, CD33, CD34, and
CD7 a phenotype that cannot be assigned to normal myeloid cells. After
7 days of culture in the presence of GM-CSF, TNF, and IL-4,
upmodulation of CD80, CD86, and CD1a was obtained (Fig 4A). Thirty-three
percent of CD80+ cells were CD7+ (ie, 28% of
86%; Fig 4B). In the peripheral blood of healthy donors, CD7 can
normally be expressed by a subset of T lymphocytes and natural killer
(NK) cells and also by monocytes very weakly. Given the fact that
monocyte (CD14), T-cell (CD3), and NK-cell (CD56) markers were negative
in 7-day cultures from this patient (data not shown), CD7 expression is
most likely attributable to leukemic cells. Data did not show whether
the CD80+/CD7 cells are leukemic cells
that have downmodulated CD7 or are normal DC that were induced from
normal precursor after cytokine exposure. Allogeneic MLR stimulatory
activity of the cultured DC population was also validated for this
patient (not shown), but this stimulatory activity might reside either
in the CD80+/CD7+ or in the
CD80+/CD7 population.
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| Fig 4.
(A) Phenotype of naive AML blasts and
leukemia-derived DC (patient SC). Freshly thawed PBMC from an AML-M2
patient and the same cells after 7 days of culture in the presence of
GM-CSF, TNF, and IL-4 were stained with fluorescein-or
phycoerythrin-conjugated antibodies and analyzed by flow cytometry. To
exclude debris, the viable cells were gated based on propidium iodide
staining. Histograms represent the log of fluorescence (horizontal
axis) versus the relative cell number (vertical axis). Thin lines
represent the isotype-matched indifferent murine MoAb control. The
number in each box represents the percentage of positive cells. (B)
CD7/CD80 coexpression on naive AML blasts and leukemia-derived DC
(patient SC). Cells at day 0 and day 7 of culture were double-stained
with fluorescein-conjugated anti-CD7 MoAb (horizontal axis) and
anti-CD80 phycoerythrin-conjugated MoAb (vertical axis).
Fluorochrome-conjugated isotype-matched murine MoAbs (IgG1) were used
as a negative control. The number in each quadrant represents the
percentage of positive cells.
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In vitro generation of leukemic DC from B-cell ALL blasts.
The data given above shows that CD34+ AML-M2 can be induced
to differentiate into DC. Some B-cell acute lymphocytic leukemias are
CD34+ and might also be inducible into DC. The growth and
differentiation factors for B cells are different than those for
myeloid cells. It is unknown whether human B cells can be induced to
differentiate into DC. To assess whether CD34+ B-cell ALL
cells can be induced to differentiate into DC, we studied the effect of
CD40L plus IL-4 on CD34+ ALL cells that contained
Ph+ as a cytogenetic marker. Two patients were studied.
Blast cells from the first ALL patient (patient AB) were positive for
the B-cell markers CD10 and CD19 and negative for the myeloid markers
CD33 and CD13. Before culture, the leukemic BM contained 92% leukemic
blasts. Blasts were essentially 100% CD34+, HLA class
I+, and HLA-DR+; 28% CD40+, 11%
CD83+, 26% CD86+, and CD1a
and CD80. After 4 days of culture of BM cells with 3 µg/mL CD40L and 80 ng/mL IL-4, CD80, CD83, and CD86 increased from
3%, 11%, and 26% to 99%, 60%, and 76%, respectively
(Fig 5). CD19 decreased from 82% to 70%. CD1a expression remained negative, and HLA class I and DR
remained high (Fig 5). Double staining with fluorescein-conjugated anti-CD83 MoAb and phycoerythrin-conjugated MoAb anti-CD80, -CD34, -CD19, or -CD10 showed that 68%, 25%, 97%, and 26% of the
CD83+ cells were positive for CD80, CD34, CD19, and CD10,
respectively (not shown).
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| Fig 5.
Phenotype of ALL blasts and of leukemia-derived
DC (patient AB). Freshly thawed BM cells from a Ph+ ALL
patient and the same cells after 4 days of culture in the presence of
CD40L and IL-4 were stained with fluorescein- or
phycoerythrin-conjugated antibodies and analyzed by flow cytometry. To
exclude debris, the viable cells were gated based on propidium iodide
staining. Histograms represent the log of fluorescence (horizontal
axis) versus the relative cell number (vertical axis). Thin lines
represent the isotype-matched indifferent murine MoAb control. The
number in each box represents the percentage of positive cells.
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To examine the leukemic origin of the DC, cells from day 7 of culture
were positively selected according to CD83 expression and examined for
the presence of the bcr-abl fusion gene by FISH. Before culture, 74%
of unfractionated blasts were bcr-abl positive. After culture, 27% of
CD83-sorted positive cells were bcr-abl+
(Fig 6). Cells from a normal control PBMC
culture showed 4 of 100 interphase nuclei analyzed with a fusion
signal, giving a background false-positive frequency of 4%. Provided
that the bcr-abl blasts observed may not be leukemic
cells, the results show that ALL can be induced to differentiate into
cells with a DC phenotype, but imply that normal DC precursors may
coexist with the leukemic blasts.
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| Fig 6.
Genotype of CD83-sorted, Ph+
leukemia-derived DC (patient AB). The figure shows interphase nuclei
from a CD83-sorted positive culture of leukemia-derived DC. A normal
interphase nucleus shows 2 orange (bcr) and 2 green (abl) signals.
Nuclei with bcr-abl fusion show 1 orange signal (bcr), 1 green signal
(abl), and the fusion (bcr-abl) orange and green signal (arrow).
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The stimulatory capacity of cultured ALL BM cells was assessed by
allo-MLR. PBMC from an unrelated normal donor were stimulated to
proliferate in response to cultured ALL cells containing
leukemia-derived DC to a greater extent than in response to original
leukemic blasts (Fig 7). The difference was
more evident when a lower number of stimulator was used. Because many
of the cells with DC phenotype were bcr-abl, it
cannot yet be concluded that leukemic DC were responsible for the
stimulatory capacity. Unfortunately, it is not possible to sort cells
on the basis of bcr-abl expression to determine the stimulatory
capacity of ALL DC.
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| Fig 7.
Proliferation of PBMC in response to allogeneic naive ALL
blasts or leukemia-derived DC (patient AB). Allo-MLR was performed with
105 allogeneic PBMC from normal unrelated donor as
responder cells and different numbers of thawed ALL blasts or
leukemia-derived DC at stimulator:responder ratios ranging from 1:1 to
0.037:1. Leukemia DC were derived from 4-day cultures. Proliferation
was measured 5 days after stimulation by [3H]-thymidine
incorporation.
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Results from the second CD34+ ALL patient (denoted patient
NA) were superimposable on the above results (data not shown). At diagnosis, the leukemic BM contained 96% leukemic blasts that were
CD10+ and CD19+ and CD13 and
CD33. After 7 days of culture in the presence of
CD40L and IL-4, cells with DC morphology and phenotype were generated.
CD80, CD83, and CD86 increased from 2%, 13%, and 41%, respectively,
to 78%, 58%, and 98%, respectively. HLA class I and HLA-DR were
unchanged. Before culture, 85% of unfractionated blasts were
bcr-abl+. After culture, 59% of CD83-sorted positive cells
were bcr-abl+. In allo-MLR, the superior stimulatory
activity of the cultured DC population compared with the original
blasts was also validated for this patient.
| |
DISCUSSION |
Malignant cells often have low immunogenicity due in part to low
expression levels of requisite costimulatory molecules such as CD80 and
CD86. Transfection of costimulatory molecules can render nonimmunogenic
malignant cells immunogenic.16 An alternative ploy for
providing appropriate costimulatory molecules is to fuse malignant
cells with B cells or DC.17-19 The present study shows that
for some leukemias the same end result can be achieved by inducing
differentiation along the DC pathway.
DC are potent inducers of T-cell responses. The cytokine-derived
leukemic DC in the current study functioned well as inducers of
allogeneic T-cell responses. Although not proven, the assumption is
that the leukemic DC will better stimulate antileukemia T-cell responses than normal leukemic cells. Potential applications of leukemic DC for immunotherapy include (1) vaccination in vivo to induce
antileukemia T-cell immunity; (2) priming in vitro to induce
leukemia-specific T cells for identifying immunogenic leukemia antigens; and (3) activating antileukemia T cells in vitro for use in
allogeneic or autologous T-cell therapy regimens.
For using leukemic DC to induce antileukemia T-cell responses, it is
crucial to verify that the DC are derived from the malignant clone. DC
were generated from 3 leukemia patients with defined genetic
abnormalities: 1 AML-M2 with monosomy 7 and 2 ALL with t(9;22). FISH
analysis confirmed that the derived DC, sorted on the basis of DC
phenotypic markers, had the same genetic abnormalities expressed by
freshly isolated leukemic cells. This not only proves that the DC
originated from the malignant clone, but also suggests that the
leukemic DC continue to express at least some leukemia-related proteins
associated with the genetic abnormality. As an example, 27% and 59%
of CD83+ sorted DC derived from the 2 ALL studied contained
the bcr-abl fusion gene. The cytokine-induced DC are likely to present
peptides derived from the bcr-abl protein substantially more
efficiently and effectively to T cells than the parental leukemia
cells. It is likely that other aberrantly expressed downstream leukemia proteins will be more efficiently presented. Alternatively, important leukemia-associated proteins might be lost during the cytokine-induced DC differentiation. The decreased expression of surface markers related
to leukemic phenotype such as CD7 in AML and CD10 in ALL suggests that
the pattern of genes expressed by leukemic cells changes during the
differentiation process. We have not yet otherwise examined or
catalogued the expression by leukemic DC of proteins related to the
malignant status.
In initial experiments CD34+ AML-M2 cells were stimulated
with GM-CSF, TNF, and IL-4, because normal CD34+ stem
cells and CD34+ CML stem cells can be differentiated into
DC by the use of these cytokines.9-12 In contrast, the
CD34+ ALL cells were stimulated with CD40L plus IL-4. The
Ph+ ALL blasts belonged to the B-lymphoid lineage, being
positive for the B-cell markers CD10 and CD19 and negative for the
myeloid markers CD33 and CD13. CD40L is known to induce proliferation and upregulation of costimulatory molecules on normal and malignant B
cells.20-23 Lymphoid DC derived from normal thymic
precursors have been described,24,25 but it was not known
that B-cell precursors, either normal or leukemic, could maturate into
DC-like cells. Stimulation of the CD34+ ALL cells with
CD40L and IL-4 induced morphologic and phenotypic changes, including
the expression of the CD83 DC-associated marker along with concurrent
persistent expression of the CD19 B-cell marker. Persistent expression
of CD19 also validated that the leukemic DC were derived from the
B-cell malignancy.
Differentiating agents have been used for leukemia therapy with some
success.26,27 A very optimistic view of future biologic therapy of leukemia might include therapy with agents such as those
used herein to induce DC differentiation in vivo. Induction of DC
differentiation in vivo might both decrease the replication rate of
leukemic cells and stimulate an immune response to aberrantly expressed
leukemia antigens.
The cytokine-derived leukemic DC possessed morphologic, phenotypic, and
functional characteristics of DC. The recognized valuable characteristics of DC for specific immunotherapy are the ability to
present antigen and to stimulate specific T-cell responses. Stimulation
of T-cell responses requires stimulation by antigen in the context of
costimulatory molecules. The crucial role of costimulatory molecules,
especially CD80, in the generation of an antileukemic response has been
shown in murine leukemia models.28-31 For the current
study, the most important goal was to achieve increased expression of
the costimulatory molecules CD80 and CD86. For all 4 CD34+
leukemias in this study, CD80 was negative in the original blast populations and was increased in cytokine-derived DC cells, with the
degree of fluorescence intensity varying from case to case. The same
held true for CD86, which was weakly expressed in some of the original
blast populations and increased in the induced population. The
expression of costimulatory molecules was associated with the
expression of CD1a or CD83 on the leukemic DC, as demonstrated by
double florescence staining (not all data shown). The presence of these
markers helped to define the maturation pattern of the leukemic cells
towards the DC lineage.1,13
T cells recognize peptides in the binding cleft of major
histocompatibility complex (MHC) molecules. Thus,
efficient antigen presentation also requires expression of HLA
molecules. Class I MHC and class II MHC were expressed equivalently by
the parental leukemic blasts and the derived leukemic DC. Ability to
stimulate allogeneic T-cell responses in a standard MLR was used to
evaluate T-cell stimulatory capacity of the DC. For the 4 leukemic DC
lines generated, the ability to stimulate allogeneic T cells was
substantially superior to that of the naive blast population. The
presumption is that the ability to present proteins expressed
endogenously by the leukemia cells, including leukemia-associated
proteins, is also increased. For normal DC, the ability to present
exogenous antigens is highly dependent on the degree of maturation of
DC. The leukemic DC were not yet evaluated for ability to present endogenous or exogenous proteins.
This report demonstrated that CD34+ leukemic cells from 2 AML-M2 patients and 2 ALL patients could be induced to differentiate into DC. From the point of view of morphology and phenotype, acute leukemia encompasses multiple different diseases. The response to the
differentiating activity of growth factors and cytokines is likely to
differ substantially for leukemia subtypes according to the stage of
maturation of the various subtypes. It is not surprising, therefore,
that another group could not detect upmodulation of costimulatory
molecules in 1 case of AML after stimulation with GM-CSF, TNF, and
IL-4.31 Our most recent data from experiments not presented
are in agreement with this observation. We have studied a total of 8 AML patients, including the 2 patients here. Upmodulation of
costimulatory molecules and DC markers was obtained in 5 of 8 cases (in
3 of 4 CD34+ AML and in 2 of 4 CD34
AML). However, a leukemia marker was available in only the 2 cases
presented here. Thus, some leukemic cells cannot be induced to
differentiate into DC with the cytokine regimens used. Different sets
of cytokines and differentiating agents might be explored.
| |
ACKNOWLEDGMENT |
The authors thank Immunex Corp for the kind gift of CD40L trimer and
Kate Rupert for technical assistance for FISH.
| |
FOOTNOTES |
Submitted December 4, 1998; accepted May 20, 1999.
Supported by National Institutes of Health, National Cancer Institute
Grants No. 5 R37 CA30558 and CA18029, and by Associazione Italiana per
la Ricerca sul Cancro (AIRC), Milano, Italy.
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 M.A. Cheever, MD, Corixa Corp,
1124 Columbia St, Suite 200, Seattle, WA 98104; e-mail:
cheever{at}corixa.com.
| |
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ABSTRACT
INTRODUCTION