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Leukemia-Derived Immature Dendritic Cells
Differentiate into Functionally Competent
Mature Dendritic Cells That Efficiently
Stimulate T Cell Responses
Alessandro Cignetti, Antonella Vallario, Ilaria Roato, Paola
Circosta, Bernardino Allione, Laura Casorzo, Paolo Ghia
and Federico Caligaris-Cappio
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The Journal of Immunology is published twice each month by
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1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:2855-2865; ;
doi: 10.4049/jimmunol.173.4.2855
http://www.jimmunol.org/content/173/4/2855
The Journal of Immunology
Leukemia-Derived Immature Dendritic Cells Differentiate into
Functionally Competent Mature Dendritic Cells That
Efficiently Stimulate T Cell Responses1
Alessandro Cignetti,2* Antonella Vallario,*† Ilaria Roato,* Paola Circosta,*
Bernardino Allione,§ Laura Casorzo,‡ Paolo Ghia,*† and Federico Caligaris-Cappio3*†
R
elapse is the main reason of treatment failure in acute
myeloid leukemia (AML).4 Though ⬎80% of AML patients achieve complete remission after conventional chemotherapy, ⬎50% invariably relapse (1), underscoring the need of
novel treatment strategies (2). One such strategy entails the development of immunotherapy approaches designed to obtain AMLspecific CTLs potentially able to eradicate or control minimal residual disease (3).
Priming of effective CTL responses requires the presentation of
relevant Ags by a professional APC (4). In this context, AML
provides the unique opportunity to derive APC from leukemic
cells themselves, combining the expression of all available leukemic Ags with the presence of the several accessory and costimulatory signals which are necessary to prime naive T cells (5). The
simplest way to derive professional APC from myeloid malignancies is to induce the differentiation of leukemic cells into dendritic
*Laboratory of Cancer Immunology, Institute for Cancer Research and Treatment,
†
Department of Oncological Sciences, University of Torino Medical School, and
‡
Unit of Pathology, Institute for Cancer Research and Treatment, Candiolo, Italy; and
§
Division of Hematology, Azienda Ospedaliera Santissimi. Antonio e Biagio, Alessandria, Italy
Received for publication December 22, 2003. Accepted for publication June 4, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the “Associazione Italiana per la Ricerca sul Cancro”
(AIRC), Milano, and by Ministero dell’Università e Ricerca Scientifica (MIUR),
Roma, Italy.
2
Address correspondence and reprint requests to Dr. Alessandro Cignetti, Laboratory
of Cancer Immunology, Institute for Cancer Research and Treatment, Strada Provinciale 142, Candiolo (TO), 10060, Italy. E-mail address: [email protected]
3
Current address: Università Vita Salute San Raffaele, 20132 Milano, Italy.
4
Abbreviations used in this paper: AML, acute myeloid leukemia; DC, dendritic cell;
BM, bone marrow; PB, peripheral blood; i-DC, immature DC; m-DC, mature DC;
FISH, fluorescence in situ hybridization; MLTC, mixed lymphocyte-tumor culture;
MDC, macrophage-derived chemokine; TARC, thymus and activation-regulated chemokine; IP-10, IFN-␥-inducible protein 10; MIG, monokine induced by IFN-␥;
PARC, pulmonary and activation-regulated chemokine.
Copyright © 2004 by The American Association of Immunologists, Inc.
cells (DC) (6, 7), which can actually be obtained upon in vitro
culture of primary AML cells in the presence of various cytokine
combinations (8 –11). Leukemic DC have proven to be potent
stimulators of allogeneic T lymphocytes in mixed leukemia-lymphocyte reactions, and a few reports also suggest that autologous
CTL can be generated in vitro by stimulation with leukemic DC
(9, 11–14).
In chronic myeloid leukemia and in AML, leukemic DC have
been initially obtained in vitro by stimulation with GM-CSF together with IL-4 and/or TNF-␣ (8 –11, 15–17). With the latter
cytokine combination, the leukemia-derived DC do not acquire a
fully mature phenotype but rather an immature one, displaying low
or intermediate levels of costimulatory molecules and low to undetectable levels of the DC maturation marker CD83 (8, 12, 13,
18 –24); they also produce low to undetectable amounts of IL-12,
another hallmark of DC maturation (11, 25, 26). Recent studies
have suggested that normal immature DC (i-DC) or semimature
DC (induced by TNF-␣) injected in humans might have an inhibitory effect on T cell function (27). On the contrary, normal mature
DC (m-DC) are the most potent APC for efficient T cell priming in
vivo (28, 29). So far, the stepwise differentiation of leukemic blasts
to i-DC and then to m-DC has never been studied and characterized from a preclinical point of view (26, 30).
If DC of leukemic origin are planned to be used in vivo as a
cancer vaccine, it becomes crucial to determine whether they can
differentiate to m-DC not only in terms of phenotype but, most
importantly, of function. To this end, we have studied 20 AML
patients analyzing whether leukemic i-DC could fully differentiate
to m-DC in response to CD40L and whether leukemic m-DC were
functionally competent in terms of: 1) production of cytokines that
support Ag-specific T cell activation, proliferation, and Th1 differentiation; 2) generation of autologous T cell effectors; 3) migration from tissues to lymph nodes; 4) amplification of Ag presentation by attraction and recruitment of monocytes and other
i-DC; 5) attraction of resting and activated T cells. We here show
0022-1767/04/$02.00
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Primary acute myeloid leukemia cells can be induced to differentiate into dendritic cells (DC). In the presence of GM-CSF, TNF-␣,
and/or IL-4, leukemia-derived DC are obtained that display features of immature DC (i-DC). The aim of this study was to
determine whether i-DC of leukemic origin could be further differentiated into mature DC (m-DC) and to evaluate the possibility
that leukemic m-DC could be effective in vivo as a tumor vaccine. Using CD40L as maturating agent, we show that leukemic i-DC
can differentiate into cells that fulfill the phenotypic criteria of m-DC and, compared with normal counterparts, are functionally
competent in vitro in terms of: 1) production of cytokines that support T cell activation and proliferation and drive Th1 polarization; 2) generation of autologous CD8ⴙ CTLs and CD4ⴙ T cells that are MHC-restricted and leukemia-specific; 3) migration
from tissues to lymph nodes; 4) amplification of Ag presentation by monocyte attraction; 5) attraction of naive/resting and
activated T cells. Irradiation of leukemic i-DC after CD40L stimulation did not affect their differentiating and functional capacity.
Our data indicate that acute myeloid leukemia cells can fully differentiate into functionally competent m-DC and lay the ground
for testing their efficacy as a tumor vaccine. The Journal of Immunology, 2004, 173: 2855–2865.
2856
AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC
that leukemic blasts could be induced to differentiate into cells
fulfilling the phenotypic and functional criteria of m-DC in 10 of
20 cases. Irradiation of leukemic i-DC after CD40 stimulation did
not affect their differentiation to m-DC in terms of phenotype, cytokine/chemokine production, and migration properties. These
data lay the ground for testing the efficacy of leukemic m-DC as a
vaccine aimed at eradicating minimal residual disease in a relevant
number of AML patients.
CD40, HLA class I, HLA-DR (BD Pharmingen, San Diego, CA), CD13,
CD14 (BD Biosciences, San Jose, CA), and CD83 (Immunotech, Westbrook, ME). Unconjugated mAb to CCR7 (IgM) was obtained from BD
Pharmingen. PE-conjugated secondary goat polyclonal Ab against murine
IgM was purchased from Southern Biotechnology Associates (Birmingham, AL). For other chemokine receptor mAbs, see Ref. 31. Isotypematched mAbs were used as negative controls. Samples were analyzed
with a FACSCalibur cytometer (BD Biosciences).
Materials and Methods
Patients
After informed consent, samples from either bone marrow (BM) or peripheral blood (PB) were obtained at diagnosis from 20 AML patients. In
some cases, samples were also collected during morphological remission
(⬍5% BM blasts) after chemotherapy. Table I shows the main clinical and
diagnostic laboratory data of all cases.
Generation of leukemic and normal DC
Flow cytometric analysis
Unmanipulated and cultured AML samples were stained with FITC, PE, or
PECy5-conjugated mouse mAbs using standard multicolor methodology,
and analyzed by flow cytometry (FACS). Blasts and DC were gated based
on side light scatter profile vs CD45, as previously described (31). Mouse
mAbs to the following Ags were used: CD1a, CD33, CD34, CD80, CD86,
Interphase FISH was performed on CD83⫹ purified leukemic m-DC and on
naive leukemic blasts (10). Three patients were selected according to their
cytogenetic abnormality and interphase nuclei were analyzed using the
probes of interest. For 7q⫺ and 5q⫺ deletion (patients GE and VA), dual
color FISH was performed, using probes labeled either with spectrum
green or spectrum orange. A centromeric probe for chromosome 7 (7p11.1q11.1) and a probe for 7q31 were used for the 7q⫺ deletion, while a probe
for 5p15.2 and a probe for 5q33-34 were used for the 5q⫺ deletion (VYSIS,
Downers Grove, IL). For monosomy 7 (patient CA), only the centromeric
probe for chromosome 7 was used. Fixation, denaturation, and hybridization were conducted following manufacturers’ instruction. At least 200
nuclei were examined under fluorescence microscopy.
RNA preparation and RT-PCR
RNA was extracted from 1 to 2 ⫻ 106 cells using RNAzol (Biotecx Laboratories, Houston, TX) following the manufacturer’s instructions. cDNA
was prepared at 42°C using a reverse-transcription mix containing Superscript II (Invitrogen Life Technologies, Grand Island, NY). PCR amplification of cDNA samples was performed for 30 or 35 cycles. For chemokines and ␤-actin primer sequences, see Ref. 32. For each cytokine and
chemokine, proper positive controls were used. All reactions were conducted in a PerkinElmer thermocycler (Foster City, CA).
Cytokine and chemokine production
Supernatants were obtained from cultures that, after washing, were seeded
at 2 ⫻ 106cells/ml and kept in serum-free medium for 24 or 72 h. Supernatants from i-DC were obtained after stimulation of i-DC cultures with
GM-CSF, TNF-␣ and IL-4; supernatants from m-DC were obtained after
stimulation of i-DC cultures with CD40L. Cytokine and chemokine production was measured by ELISA using the following commercially available kits: IL-12, IL-12 HS (high sensitivity), IL-15, IL-18, macrophagederived chemokine (MDC), thymus and activation-regulated chemokine
(TARC), MCP-1, MIP-1␣, MIP-3␣, RANTES (R&D Systems, Minneapolis, MN), IFN-␥-inducible protein 10 (IP-10) (CytImmune Sciences, College Park, MD), IL-6, IL-8, IL-10 (Bender MedSystems, Vienna, Austria),
and IL-16 (Technogenetics, Milano, Italy).
Table I. Patients’ characteristics
Differentiation
a
b
Patient
Sex
FAB
Sample
% Blasts
Cytogenetic
i-DC
m-DC
% Positive
CD14
Blasts
CD86
GA
CER
FE
BO
CA
IE
BE
DA
PE
RA
GI
ZA
VA
BI
GE
RO
FE
FR
CR
SU
M
F
M
F
F
F
F
M
F
M
F
F
F
M
M
M
M
F
F
M
M0
M0
M1
M1
M1
M1
M2
M2
M2
M2
M2
M4
M4
M4
M4
M4
M4
M5
M5
M5
PB
BM
PB
PB
BM,b PB
BM,b PB
PB
BM
PB
PB
PB
PB
BM,b PB
PB
BM
PB
PB
BM, PB
PB
PB
78
81a
44
88
95a
95
92
70
77
85
87
95
86
77
72a
95
68
89
87a
94
⫺7
t (7;8)
ND
ND
⫺4, ⫺5, ⫺7
46 XX
46 XX
5q⫺, 7⫹, 8⫹, 22⫹
ND
46 XY
46 XX
46 XX
5q⫺
ND
7q⫺, 11q⫺
ND
ND
46 XX
46 XX
46 XY
No
Yes
No
No
Yes
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
⬍5
20
46
36
10
23
6
81
80
82
⬍5
79
⬍5
⬍5
76
⬍5
⬍5
88
⬍5
⬍5
ND
19
53
90
95
55
⬍5
77
92
98
Purified cells were used to generate leukemic DC.
BM was used to generate leukemic DC.
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BM or PBMCs were isolated after centrifugation over a density gradient
(Lymphoprep; Nycomed, Oslo, Norway) and used immediately or cryopreserved. Fresh or thawed primary AML cells were seeded into 25 or 75
cm2 flasks at 1 ⫻ 106/ml and cultured in serum-free medium (X-Vivo15;
BioWhittaker, Walkersville, MD). In four selected cases, leukemic cells
were obtained by depleting PBMC with a mixture of magnetically labeled
mAbs against CD3, CD19, CD16, and CD56 (Miltenyi Biotec, Auburn,
CA), resulting in a purity of ⬎98% leukemic cells (Table I). To generate
leukemic i-DC, purified or unfractionated AML cells were stimulated with
GM-CSF, IL-4 (both at 80 ng/ml), and TNF-␣ (10 ng/ml) (BioSource International, Camarillo, CA). Cytokines and fresh medium were added every 4 –5 days.
Normal i-DC were obtained from purified CD14⫹ cells using GM-CSF
and IL-4 as described elsewhere (31). To generate leukemic and normal
m-DC, cells from i-DC cultures were CD40 cross-linked using 300 ng/ml
of a rCD40L-FLAG-tag fusion protein supplemented with 300 ng/ml
“CD40L enhancer” (Alexis Biochemicals, San Diego, CA). After 24, 48, or
72 h, depending on the assay, cells were used for functional analysis.
Fluorescence in situ hybridization (FISH) analysis
The Journal of Immunology
Stimulatory function of the leukemic DC
Allogeneic mixed lymphocyte-tumor cultures (MLTC) were set up with
PBMC from healthy donors as responders and naive leukemic cells, leukemic, and normal DC as irradiated stimulators, as previously described
(10). All cultures were performed in serum-free medium (AIM-V; Invitrogen Life Technologies). Normal DC were derived from CD14⫹ purified
PBMC of healthy sibling donors that were HLA-matched with the patient.
Autologous MLTC were similarly performed, but responder T cells used
for proliferation assay were previously cocultured with different stimulators, i.e., autologous blasts, i-DC or m-DC. Briefly, patients’ PBMC were
primed with autologous blasts, i-DC, or m-DC and similarly restimulated
after 7 days. Proliferation was then measured by pulsing cells with
[3H]TdR 4 days after a last stimulation of all differently cultured T cells
with autologous blasts.
Induction of autologous T cell effectors
ELISPOT assay
The ELISPOT assay was performed using cells from MLTC as effectors
(4 ⫻ 104cells/well) and the following autologous targets (1 ⫻ 104/well):
naive leukemic blasts, CD14⫹ purified monocytes, and EBV-transformed
B cells. ELISPOT assays were conducted according to manufacturer’s instruction (Mabtech, Stockholm, Sweden), with few modifications (33). Before developing the assay, T cells were routinely transferred from the
ELISPOT plate to a normal 96-well plate and viability of recovered cells
was verified after 2 additional days of culture (without cytokines) by
checking all wells under the microscope and also by randomly counting
some wells with trypan blue exclusion. This allowed us to rule out cell loss
or cell death due to technical inaccuracy and also to check correspondence
between cell viability and IFN-␥ activity in individual wells. By performing this control of cell viability, in fact, we could exclude that a remarkable
T cell death had occurred in those same wells where IFN-␥ activity was
detected. Blocking experiments were performed by preincubating target
cells for 20⬘ with 25 ␮g/ml of an anti-HLA class I (clone W6.32) or an
anti-HLA class II (clone L243) and an isotype-matched irrelevant mAb (all
from BD Pharmingen). Spots were counted by a computer-assisted ELISPOT reader (AID; Bioline, Torino, Italy). Indicated spot numbers per
seeded PBMC represent mean values of duplicates.
Chromium release assay
Target cells (primary AML cells) were incubated with 50 ␮Ci of
Na251CrO4 (Amersham Biosciences, Cologno Monzese, Italy) for 2 h at
37°C and washed. Thereafter, 5 ⫻ 103 viable target cells were incubated
with CD8⫹ effector cells from MLTC at different E:T ratios for 4 h at 37°C.
After incubation, the supernatants were collected and radioactivity was
measured on a gamma counter. Spontaneous release was determined by
harvesting the supernatant of target cells incubated in the absence of effector cells and maximum release was determined by resuspending target
cells incubated in the absence of effector cells. The percent of specific lysis
was calculated as follows: (experimental release ⫺ spontaneous release/
maximum release ⫺ spontaneous release) ⫻ 100%. The spontaneous release always ranged between 5 and 20% of the total label incorporated by
the cells. To determine class I restriction in the recognition of target cells,
blocking studies were performed by adding 25 ␮g/ml anti-HLA class I
mAb (clone W6.32) to 51Cr-labeled targets 30 min before the assay. An Ab
of the same isotype was used as negative control. The Abs were not washed
out before mixing target and effector cells.
Generation of T cell subsets and migration assay
CD4⫹ and CD8⫹ T cells were purified from PBMC by depleting CD19⫹,
CD56⫹, CD16⫹, CD14⫹, and CD8⫹ or CD4⫹ cells with magnetic microbeads (Miltenyi Biotec), and activated by stimulation with anti-CD3 and
FIGURE 1. Maturation of leukemic DC up-modulates CD83 and CD80 expression. Blasts, leukemic, and normal i-DC and m-DC cultures were analyzed
by flow cytometry for the expression of CD83 (A) and CD80 (B). Data are expressed as percent of positive cells and each line represents data obtained
with leukemic (n ⫽ 10) or normal CD14⫹ (n ⫽ 8) samples.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
AML blasts were differentiated to leukemic m-DC, irradiated at 30 Gy and
used to stimulate autologous PBMC obtained after complete remission.
Autologous MLTC were set up with patients’ PBMC (107 nonadherent
PBMC) cocultured with 2 ⫻ 106 leukemic m-DC in AIM-V serum-free
medium. On days 7, 14, and 21, PBMC were restimulated with autologous
irradiated leukemic m-DC. rIL-2 (20 IU/ml) was added 72 h after the
second stimulation and then every 3– 4 days. At day 28, PBMC were separated into CD4⫹ and CD4⫺ cells using CD4⫹ microbeads (Miltenyi Biotec) and purified fractions were further restimulated with autologous leukemic m-DC. Assay for functional activity was performed 5–7 days after
the fourth or fifth restimulation.
2857
2858
AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC
anti-CD28 (100 ng/ml and 1 ␮g/ml, respectively) (BD Pharmingen). Purity
of the CD4⫹ or CD8⫹ fraction was always ⬎95%. Activated T cells were
cultured with IL-2 (2 ng/ml; Chiron, Emeryville, CA) and IL-7 (5 ng/ml;
R&D Systems) in the presence of irradiated allogeneic feeder cells
(PBMC) for 7 to 14 days. Naive/resting CD4⫹ and CD8⫹ T cells were
prepared from nonadherent PBMC negatively depleted of CD14⫹, CD19⫹,
CD56⫹, CD45RO⫹, CD25⫹, HLA-DR⫹, and CD8⫹ or CD4⫹ cells using
goat anti-mouse Ig-coated magnetic microbeads (Miltenyi Biotec). Purity
of the resulting CD45RA⫹/CD4⫹ or CD8⫹ cells was always ⬎95% as
controlled by FACS.
Chemotaxis assays were performed using the Transwell system (5 ␮m
pores; Costar, Cambridge, MA), as previously described (31, 32). For chemotaxis of leukemic m-DC, escalating doses of rMIP-3␤ (R&D Systems)
were added to X-Vivo15 and distributed on the lower compartment of the
chemotaxis system; CCR7⫹ leukemic m-DC were placed into the Transwell inserts. Plates were incubated for 4 h. A control condition with no
recombinant chemokine was also included. For normal monocytes and T
cell subset migration, supernatants were collected from leukemic and normal DC cultures and used at 100% v/v or with serial 1/2 dilutions and
distributed on the lower compartment of the chemotaxis system. Monocytes or T cells were then placed into the Transwell inserts. T cells were
obtained as described above, while normal monocytes were obtained from
healthy donors with the monocyte isolation kit as described for DC generation. In selected cases, monocytes or T cells were incubated before the
migration assay with saturating concentrations of MCP-1 (2 ␮g/ml), MDC,
or IP-10 (4 ␮g/ml) for 30 min to down-modulate CCR2, CCR4, and
CXCR3 expression, respectively, as evaluated by FACS analysis. Plates
were incubated for 2, 4, and 12 h (activated T cells, monocytes, and naive
T cells, respectively). A negative control condition with fresh medium was
always included.
In all migration assays, the liquid accumulated in the lower compartment of the chemotaxis system was carefully recovered and migrated cells
were counted by flow cytometry. Results are expressed as the percentage
of migrating cells, i.e., number of migrated cells/number of input
cells ⫻ 100.
The statistical significance ( p values) between i-DC and m-DC or between
leukemic and normal i-DC and m-DC was analyzed by two-tailed Student
t test for paired data.
Results
Generation of leukemic and normal i-DC and m-DC
We analyzed 20 AML samples to determine whether AML blasts
could be induced to differentiate first to i-DC and then to m-DC.
When a significant proportion of cells cultured in the presence of
GM-CSF, TNF-␣, and IL-4 started to show evidence of DC morphology, their phenotype was analyzed by flow cytometry (usually
FIGURE 3. Leukemic m-DC are better T cell stimulators than i-DC in
allogeneic and autologous MLTC. A, Allogeneic MLTC was performed
with 105 allogeneic PBMCs as responder cells and different numbers of
thawed AML blasts or leukemic and normal i-DC and m-DC as stimulator
cells at responder/stimulator ratios (R/S) ranging from 3:1 to 30:1. Figure
shows data obtained at a R/S of 30:1. B, Autologous MLTC was performed
similarly to allogeneic MLTC, but responder cells were patients’ T cells
that had been differently stimulated at week intervals with autologous
blasts, i-DC, or m-DC. Data in A and B are presented as mean ⫾ SE of
three cases in each panel for a total of six patients analyzed. ⴱ, p ⬍ 0.001
between i-DC and m-DC groups (both leukemic and normal) analyzed with
a t test for paired data. All cultures were conducted in serum-free medium.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Maturation of leukemic DC induces IL-12 and IL-15 production and down-modulates IL-10 production. A, Supernatants from i-DC
and m-DC cultures were analyzed for IL-12p70 production by ELISA.
Cells were seeded at 2 ⫻ 106 cells/ml and harvested after 24 h. Data
represent the mean ⫾ SD of 10 leukemic samples and of 4 normal samples
(CD14-derived DC). ⴱ, p ⬍ 0.01; §, p ⬍ 0.05. B, Supernatants from blasts,
i-DC, and m-DC cultures were analyzed for IL-15 and IL-10 production by
ELISA. Cells were seeded at 2 ⫻ 106 cells/ml and harvested after 72 h.
Data represent the mean ⫾ SD of 10 leukemic samples. For blasts and
i-DC, IL-10 samples were divided in two subgroups, according to their
different pattern of IL-10 production. ⴱ, p ⬍ 0.01; §, p ⬍ 0.05.
Statistical analysis
The Journal of Immunology
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FIGURE 4. Leukemic
m-DC
generate
MHC-restricted CD8⫹ and CD4⫹ T cells in autologous MLTC. CD4⫹ (A) and CD8⫹ (B and
C) T cells from autologous MLTC were tested
for their activity against naive autologous blasts,
monocytes and EBV-transformed B cells. A and
B, T cell reactivity was quantified using an
ELISPOT assay for IFN-␥. Results shown are
referred to a T cell-target ratio of 4:1, i.e., 4 ⫻
104 CD4⫹ or CD8⫹ T cells and 1 ⫻ 104 blasts,
monocytes, or EBV-transformed B cells. Figure
shows mean ⫾ SE of four experiments for
CD4⫹ T cells and three experiments for CD8⫹
T cells. C, CTL activity was determined by 51Cr
release assay. Figure shows mean ⫾ SE of two
independent experiments.
2859
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AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC
Maturation of leukemic DC induces the production of IL-12 and
IL-15 and down-modulates the production of IL-10
To further evaluate the differentiation stage of leukemic DC, we
measured by ELISA the production of IL-12p70 by leukemic i-DC
and m-DC and compared it to CD14-derived normal counterparts.
As shown in Fig. 2A, supernatants from leukemic and normal i-DC
cultures showed negligible IL-12 production. On the contrary, supernatants from both normal and leukemic m-DC cultures showed
significant amounts of IL-12 production. Similarly to IL-12, also
levels of IL-15 production in both leukemic and normal DC subpopulations were significantly higher in m-DC than in i-DC (Fig.
2B and not shown).
As for IL-10, we found two different patterns of production (Fig.
2B): 1) IL-10 was produced by leukemic blasts but not by leukemic i-DC and m-DC in 5 of 10 cases; 2) IL-10 was not secreted by
leukemic blasts but its production was induced in leukemic i-DC
and down-regulated in leukemic m-DC in the remaining 5 of 10
cases. Altogether, the data indicate that IL-10 can be expressed and
produced by leukemic blasts or leukemic blast-derived i-DC, but
not by leukemic blast-derived m-DC.
FIGURE 5. Mature leukemic DC express CCR7 and migrate in response to MIP-3␤. A, Blasts and leukemic i-DC and m-DC cultures were
analyzed by flow cytometry for the expression of CCR7. A value of p of
the m-DC group compared with the i-DC group is ⬍0.01 when analyzed
with a t test for paired data. B, Migration assay of CCR7⫹ leukemic m-DC
in response to rMIP-3␤. Migration of four leukemic and three normal
m-DC independent cases pooled together are presented.
after 7–14 days) (10). As CD1a and CD83 are considered markers
of i-DC and m-DC, respectively, (34, 35), AML cells were considered to have differentiated into i-DC when ⬎50% of cells were
positive for CD1a and/or CD80. Samples were then stimulated
with CD40L and analyzed for CD83 expression after 24, 48, and
72 h of culture. When at least 50% of the CD80⫹ cells coexpressed
CD83, cultured AML cells were considered to have become leu-
Table II. Chemokine expression by leukemic and normal DC
Chemokines
MCP-1
a
PCR
Leukemic
i-DC
m-DC
10/10
10/10
1,451 (142–2,378)
750 (585–1,829)
10/10
10/10
CD14
i-DC
m-DC
4/4
4/4
1,419 (648–2,411)
995 (304–1,546)
4/4
4/4
b
PCR
a
DC Type
a
ELISA
MDC
b
Number of positive samples per total tested.
Median (range) of picograms per milliliter.
ELISA
MIP-1␣
TARC
b
6,610 (719–8,661)
7,859 (352–8,573)
8,731 (1,451–9,145)
8,968 (8,849–9,929)
PCR
a
ELISA
b
PCR
a
10/10
10/10
6,732 (3,154–9,576)
7,588 (3,559–12,145)
10/10
10/10
4/4
4/4
7,242 (273–1,884)
9,957 (3,709–12,024)
4/4
4/4
ELISA
MIP-3␣
b
PCR
a
ELISAb
609 (13–3,884) 5/9
181 (152–1,201) 7/9
7 (0–72)
8 (0–102)
2,006 (415–3,597) 4/4
ND
4/4
0
19 (0–204)
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
kemic m-DC. According to these criteria, AML blasts were able to
differentiate to m-DC in 10 of 20 cases (see Table I). Before differentiation, all 10 cases were CD86-positive, while only 8 of 10
cases were CD14-positive. On the contrary, cells were CD86 and
CD14 positive only in 1 of 10 cases in which DC differentiation
was not achieved (Table I). Fig. 1 shows how the up-modulation of
CD83 and CD80 is associated with maturation of leukemic as well
as of normal DC. The difference in CD83 and CD80 expression
between i-DC and m-DC (both leukemic and normal) is statistically significant, when data are analyzed with a t test for paired
data ( p ⬍ 0.05 and p ⬍ 0.005 for CD80 and CD83, respectively).
We also analyzed the expression of CD86, CD40, HLA class I, and
HLA-DR in leukemic i-DC and m-DC, without finding a significant difference between the two subsets (data not shown).
To prove that CD40L-induced maturation does not cause enrichment of residual normal cells over the original leukemic cells,
we performed cytogenetic FISH analysis in three selected cases.
Cells were analyzed for the presence of 7q⫺ (patient GE) and 5q⫺
(patient VA) deletion or for monosomy 7 (patient CA). FISH analysis revealed that, before culture, 89 and 76% of unfractionated
blasts were positive for 7q⫺ and 5q⫺ deletion, respectively, and
72% of blasts were positive for monosomy 7. Following culture,
79, 70, and 73% of CD83⫹ purified cells displayed the same cytogenetic abnormality, respectively (not shown). These data provide proof of principle that the m-DC obtained in our culture conditions are of leukemic origin.
The Journal of Immunology
2861
Leukemic m-DC stimulate T cell proliferation better than i-DC
in allogeneic and autologous MLTC
To prove that leukemic m-DC are capable of supporting T cell
activation and proliferation better than i-DC counterparts, we investigated their ability to stimulate allogeneic T cells in primary
MLTC. In three cases analyzed, we found that m-DC elicited a
proliferation significantly higher than i-DC and that the stimulatory activity of leukemic i-DC and m-DC is superimposable to that
of normal counterparts. CD14-derived normal i-DC and m-DC
were obtained from HLA-matched sibling donors (Fig. 3A). We
also compared the stimulatory activity of leukemic m-DC with that
of i-DC in tertiary MLTC in three additional cases. Patients’
PBMC were repeatedly stimulated with autologous blasts, i-DC, or
m-DC and their proliferation in response to naive autologous blasts
was measured after 3 wk. Again, we found that m-DC elicited
significantly higher proliferation of autologous T cells than i-DC
counterparts (Fig. 3B).
PBMC from patients were repeatedly stimulated in vitro by autologous leukemic m-DC and analyzed for their ability to recognize
naive leukemic blasts. Cells were purified according to their
CD4 and CD8 expression (⬎94% purity after selection), restimulated once more with leukemic m-DC and tested after 7 days by
ELISPOT. We found that stimulation with leukemic m-DC generates CD8⫹ and CD4⫹ T cells that release IFN-␥ (Fig. 4, A and
B) in response to naive leukemia blasts in three of four and four of
four cases, respectively. In one AML patient, the initial number of
CD8⫹ cells was very low and the final yield of CD8⫹ T cells was
not enough to perform functional assays. CD4⫹ T cells released
IFN-␥ but not IL-4 in response to naive leukemic blasts (not
shown). MHC restriction of CD8⫹ and CD4⫹ effectors was proved
by the inhibition of IFN-␥ release observed with specific anti
class-I or class-II mAb, respectively, but not with an irrelevant
mAb of the same isotype (IgG2a). Leukemia specificity was further confirmed by the lack of recognition of autologous myeloid
(PB monocytes) and lymphoid (EBV-transformed B cells) targets
(Fig. 4, A and B). Finally, we also determined whether CD8⫹ T
cells from MLTC could mediate effective killing of unmanipulated
blasts in a traditional cytotoxicity assay. In two of two cases analyzed, CD8⫹ T cells efficiently lysed both autologous blasts and
leukemic m-DC (Fig. 4C). Killing of leukemic m-DC was significantly higher than that of primary blasts. Lysis of AML blasts was
blocked by a mAb to HLA-class I, but not by an irrelevant, isotype-matched, control mAb (Fig. 4C).
Leukemic m-DC but not i-DC express CCR7 and migrate in
response to MIP-3␤
Maturing DC up-regulate CCR7 expression to acquire the ability
to migrate from inflamed tissue to draining lymph nodes in re-
Leukemic i-DC and m-DC produce inflammatory chemokines
that induce the migration of PB monocytes
To amplify Ag presentation, normal DC also produce chemokines
to attract monocytes and other DC to inflamed tissue and to secondary lymphoid organs. We analyzed the expression of MCP-1,
MIP-1␣, RANTES, IL-8, MIP-1␤, and MCP-4 by RT-PCR in both
leukemic and normal DC subsets. mRNA for all these chemokines
was detected in the vast majority of samples (Table II). Among
these chemokines, we analyzed by ELISA the actual production of
MCP-1 (Fig. 6A), MIP-1␣, and IL-8 (Table II) and found that they
are also released at significant levels by both leukemic i-DC and
m-DC. We next tested whether supernatants from DC cultures
could attract in vitro normal monocytes in a chemotactic assay.
Fig. 6B shows that supernatants from both leukemic i-DC and
m-DC similarly induce the migration of freshly isolated PB
monocytes.
Considering the higher levels of CCR2 expression in normal
monocytes compared with the other chemokine receptors analyzed
(not shown), we postulated that their migration in response to leukemic DC supernatants was mainly CCR2 mediated. We performed “desensitization” experiments where, before chemotactic
assay, CCR2 expression was down-regulated by incubating target
cells with high doses of rMCP-1. CCR2 desensitization induced a
significant inhibition of monocyte migration in response to leukemic DC supernatants close to that obtained with rMCP-1 alone
(Fig. 6C).
Leukemic i-DC and m-DC produce chemokines that induce the
migration of naive and activated T cells
Normal DC also produce chemokines that attract T cells to the
sites of Ag presentation. In particular, MDC/TARC and IP-10/
monokine induced by IFN-␥ (MIG) are known to promote the
interaction of DC with recently activated T cells that respectively
express CCR4 and CXCR3. We found that both i-DC and m-DC
of leukemic origin produce considerable amounts of MDC and
TARC, similarly to CD14-derived normal DC (Fig. 7A and Table
II). A trend toward a higher level of MDC and TARC production
by m-DC than by i-DC was observed, which did not reach statistical significance. We also found that, in most cases, leukemic and
Table II. Continued
Chemokines
IP-10
RANTES
IL-8
PCRa
ELISAb
PCRa
ELISAb
PCRa
ELISAb
6/10
7/10
2/4
3/4
35 (0–73)
50 (0–674)
47 (0–257)
27 (0–110)
10/10
10/10
4/4
ND
29 (0–633)
ND
33 (7–215)
ND
10/10
10/10
4/4
ND
1468 (144–2,485)
ND
2,086 (255–2,464)
ND
IL-16
PCRa
ELISAb
10/10
973 (504–1,886)
10/10 1,182 (431–2,399)
4/4
642 (285–1,203)
4/4
409 (375–1,658)
MIP-1␤
MCP-4
MIP-3␤
PARC
MIG
PCRa
PCRa
PCRa
PCRa
PCRa
10/10
10/10
4/4
ND
10/10
10/10
4/4
4/4
5/10
10/10
2/4
4/4
3/10
5/10
3/4
4/4
6/10
7/10
3/4
4/4
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Leukemic m-DC generate autologous T cell effectors that are
MHC-restricted and leukemia-specific
sponse to CCR7 ligands (MIP-3␤, secondary lymphoid tissue chemokine). CCR7 was not expressed by leukemic i-DC while a statistically significant induction of CCR7 expression was observed
in leukemic m-DC (Fig. 5A). No significant difference in CCR7
expression was observed between leukemic and normal m-DC (not
shown). To prove that leukemic m-DC can effectively migrate
through CCR7 engagement, we performed a chemotactic assay in
vitro, where leukemic m-DC were exposed to rMIP-3␤. In all
four samples analyzed, leukemic m-DC migrated in response to
MIP-3␤, similarly to normal m-DC (Fig. 5B).
AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC
FIGURE 6. Leukemic i-DC and m-DC produce inflammatory chemokines that induce the migration of PB monocytes. A, Leukemic i-DC and
m-DC produce MCP-1. Supernatants from blasts, i-DC, and m-DC cultures
were analyzed for MCP-1 production by ELISA. Cells were seeded at 2 ⫻
106 cells/ml and harvested after 72 h. Data represent the mean ⫾ SD of 10
leukemic samples and of 4 normal samples (CD14-derived DC). B, Supernatants from leukemic DC cultures induce migration of PB monocytes.
Chemotaxis of PB monocyte cells in response to leukemic and normal
i-DC and m-DC supernatants. Fresh PB monocytes were isolated by depleting T, B, and NK cells from PBMC. Data represent mean ⫾ SD of four
leukemic i-DC and m-DC samples and of three normal i-DC and m-DC
samples (CD14-derived DC). C, Chemotaxis of PB monocytes in response
to leukemic i-DC (n ⫽ 3) and m-DC-derived supernatants (n ⫽ 3) or
rMCP-1, before (f) or after (u) CCR2 down-regulation by receptor desensitization with high dose rMCP-1 (2 ␮g/ml). The difference in migration
obtained before and after desensitization is statistically significant in all
three groups (p ⬍ 0.01).
FIGURE 7. Leukemic i-DC and m-DC produce lymphoid chemokines that
induce the migration of resting and activated CD4⫹ and CD8⫹ T cells. A,
Supernatants from blasts, i-DC, and m-DC cultures were analyzed for MDC
production by ELISA. Cells were seeded at 2 ⫻ 106 cells/ml and harvested
after 72 h. Data represent the mean ⫾ SD of 10 leukemic samples and of 4
normal samples (CD14-derived DC). B, Chemotaxis of resting/naive and activated CD8⫹ T cells in response to leukemic and normal i-DC and m-DC
supernatants. Purified CD8⫹/CD45RA⫹ T cells were obtained from fresh
PBMC by negative selection. Activated T cells were obtained by stimulation
of purified CD8⫹ cells with anti-CD3 and anti-CD28 (see Materials and Methods). Five independent experiments are pooled together. Data represent
mean ⫾ SD of four leukemic i-DC and m-DC samples and of three normal
i-DC and m-DC samples (CD14-derived DC). The percent of cells migrating
in the presence of medium alone was subtracted from each data point. C,
Chemotaxis of CD8⫹-activated lymphocytes in response to leukemic i-DC and
m-DC-derived supernatants (n ⫽ 3) or rIP-10 and rMDC, before and after
CXCR3 or CCR4 down-regulation by receptor desensitization with rIP-10 (4
␮g/ml) and MDC (4 ␮g/ml), respectively. Three independent sets of experiments are pooled together. The percent of cells migrating in the presence of
medium alone was subtracted from each data point. The difference in migration obtained before and after desensitization is statistically significant in all
three groups (p ⬍ 0.05).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
2862
The Journal of Immunology
2863
Irradiation of leukemic i-DC after CD40L stimulation does not
affect their differentiation to m-DC
To be used as a vaccine in vivo, leukemic DC should be irradiated
to prevent proliferation of residual leukemic cells. To evaluate how
irradiation affects maturation and function of leukemic DC, we
stimulated i-DC cultures from three different patients with CD40L
and after 4 h we exposed them to escalating doses of gamma rays.
The following parameters were then assessed at different time
points: viability, expression of maturation markers, cytokine/chemokine production, and chemotactic activity. As shown in Fig. 8,
a progressive decrease in cell viability was observed, which was
more pronounced after the third day of treatment, and complete
cell death was seen at day 4. Despite cell death, remaining living
cells showed induction of CD83 and CCR7 expression similar to
FIGURE 8. Irradiation of leukemic i-DC after CD40 stimulation does
not affect their differentiation to m-DC. i-DC cultures were stimulated with
CD40L (day 0) and, after 4 h, were gamma-irradiated with three different
doses (10, 30, and 60 Gy). Irradiated and nonirradiated cells were then
analyzed at different time points as indicated. A, Viability as determined by
counting cells on a Bauer chamber after staining with a 0.4% trypan blue
solution to exclude dead cells; B and C, phenotype as determined by FACS
analysis of residual living cells (gate on living DC was set as described in
Materials and Methods); D, migration in response to rMIP-3␤ (200 ng/ml);
E, chemokine and cytokine production as determined by testing culture
supernatants harvested 24 h after irradiation. All data are referred to one
representative case of three analyzed.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
normal DC subsets express mRNA for IP-10 and/or MIG. As documented by ELISA, IP-10 was detected at the protein level as well,
without a significant difference between leukemic i-DC and m-DC.
Finally, also MIP-3␤ and pulmonary and activation-regulated
chemokine (PARC) were expressed at the mRNA level by leukemic and normal DC subsets (Table II). Only MIP-3␤ expression
was significantly up-regulated in m-DC as compared with i-DC.
MIP-3␤ and PARC are known to mediate the attraction of naive/
resting T cells to the T cell area of secondary lymphoid organs. To
prove that the chemokines expressed and/or released by leukemic
DC can attract naive/resting T cells, we purified CD8⫹/CD45RA⫹
cells from fresh PBMC and tested them in a chemotactic assay.
CD8⫹/CD45RA⫹ T cells migrated in response to both i-DC and
m-DC supernatants, without significant differences between the
two subsets (Fig. 7B). We also found that supernatants from leukemic i-DC and m-DC were equally capable of inducing migration
of activated CD8⫹ T lymphocytes (Fig. 7B). Migration of naive/
resting and activated CD4⫹ cells in response to leukemic DC supernatants was slightly (but not significantly) higher than that of
CD8⫹ counterparts (not shown), possibly due to the additional
effect of IL-16 on CD4⫹ cells (36). In fact, IL-16 was detected by
ELISA in the leukemic DC supernatants (Table II). Regardless of
the T cell subtype analyzed, the levels of migration induced by
leukemic DC supernatants were not different from those induced
by normal DC supernatants (Fig. 7B).
The analysis of the chemokine secretion pattern suggests that,
among the several chemokines detected at the protein level in leukemic DC supernatants, at least two chemokine/chemokine receptor pairs might be involved in the attraction of activated T cells:
IP-10-MIG/CXCR3 and MDC-TARC/CCR4. To investigate this
issue, we performed again desensitization experiments as described in the previous paragraph. We found that CXCR3- and
CCR4-desensitized T cells migrated significantly less than normal
counterpart cells in response to leukemic DC supernatants.
(Fig. 7C).
AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC
nonirradiated cultures, which was maintained for up to 60 h. Moreover, supernatants harvested 24 h after irradiation showed no difference between treated and untreated cells in terms of chemokine
and cytokine production. Fig. 8 shows ELISA data for TARC and
IL-12; similar data were obtained for IL-15, IL-10, MDC, and
MCP-1. Finally, irradiated CCR7⫹ m-DC were able to migrate in
response MIP-3␤ and levels of migration were similar between
irradiated and nonirradiated cells up to 48 h. Thus, irradiated leukemic DC maintain their functional properties up to at least 2 days
after treatment.
ment. For vaccine design purposes, though, it has to be kept in
mind that a decrease in cell number occurs within the same time
frame. Dose, frequency, and route of DC delivery are crucial parameters for vaccine effectiveness, which have not been optimized
yet in clinical trials with normal nonirradiated DC (37). Nevertheless, based on our data, it is reasonable to anticipate that a higher
cell number should be used when vaccinating with leukemic m-DC
as compared with normal DC, also because cells with m-DC phenotype do not represent the totality of cells in bulk cultures of
leukemic DC. Tumor cell number is not a limitation in AML patients, though, considering the relative ease of tumor cell acquisition at disease presentation. The availability of large numbers of
leukemic cells also overcomes the issue of the low yield of viable
leukemic m-DC at the end of the differentiating culture (6, 7, 38).
In our series, cells with m-DC phenotype represented 10 –50% of
the initial number of input cells, which would enable (considering
500 ⫻ 106 as the minimum number of leukemic cells obtainable at
diagnosis), the manufacturing of at least 10 vaccine preparations of
5 ⫻ 106 cells/vaccine. The real limiting factor remains only the
fact that differentiation into leukemic m-DC could be achieved
only in half of the patients analyzed. On one hand, a possible
solution would be the prediction of which patients will generate
leukemic DC and which patients will not. Recently, it has been
shown that the expression of CD14 (30) and of CD86 (20) on
AML blasts correlates with their capacity of generating cells with
DC features. In agreement with these observations, our data confirm the predictive value of both markers and particularly of CD86,
which was expressed by AML blasts in all 10 cases that could
differentiate to m-DC. This marker (alone or in combination with
CD14) could be very helpful for determining which patients are
eligible for vaccination. On the other hand, the addition of other
cytokines to the GM-CSF/TNF-␣/IL-4 mixture (for instance, stem
cell factor and Flt-3) (13, 21–23) or the use of other compounds
(such as phorbol esters or bryostatin) (26, 39) might increase the
number of responsive cases.
To our knowledge this is the first report that studies the differentiating potential of leukemic i-DC into m-DC and provides full
functional characterization of leukemic m-DC from a preclinical
point of view. In addition, we show that leukemic m-DC efficiently
prime T cells from AML patients, generating MHC-restricted
CD4⫹ and CD8⫹ cells that are leukemia-specific. Our data suggest
that irradiated leukemic m-DC should be tested as a vaccine aimed
at eradicating minimal residual disease in a significant fraction of
AML patients.
Discussion
The aim of this study was to provide evidence that DC of leukemic
origin may be used in vivo as a tumor vaccine following induction
of maturation. For clinical efficacy, leukemic DC should not only
present endogenous tumor Ags to T cells, but also carry out several
activities that normal myeloid DC have in vivo, such as migration
from tissues to draining lymph nodes, attraction and recruitment of
new APC and T cells, and induction and sustenance of T cell
activation and proliferation. They should induce a Th1 rather than
a Th2 or T regulatory type of T cell response (27, 37) and, more
importantly, generate a leukemia-specific T cell response.
In this study, we show that leukemic i-DC can be induced to
fully differentiate into m-DC using CD40L as maturating agent,
and that leukemic m-DC fulfill the functional criteria listed above.
For instance, leukemic m-DC (but not i-DC) up-regulate CCR7
expression and are able to migrate in vitro in response to the CCR7
ligand MIP-3␤. Moreover, leukemic m-DC (but not i-DC) secrete
IL-12 and IL-15, two cytokines that support T cell activation, proliferation, and differentiation, and down-regulate the secretion of
IL-10, which has inhibitory activity on T cells and on DC as well.
This pattern of cytokine production finds functional correspondence in the superior capacity of leukemic m-DC to stimulate allogeneic as well as autologous T cell proliferation, when compared
with i-DC. More importantly, the ability of m-DC to generate both
CD8⫹ and CD4⫹ leukemia-specific T cells was also documented
in the autologous setting. Other groups have shown that leukemiaderived DC (that were obtained otherwise than inducing maturation of i-DC with CD40L) can stimulate T cell responses in vitro
(9, 11–14). However, this is the first study that provides concurrent
evidence that the antileukemic activity is MHC restricted, is directed against unmanipulated blasts, resides specifically in purified
CD8⫹ or CD4⫹ subsets, and can be obtained using serum-free
medium, which is mandatory for clinical purposes. In addition, we
have shown that normal myeloid (i.e., autologous monocytes from
PB) and lymphoid (EBV-transformed B cells) targets are not recognized by our T cell effectors, suggesting the existence of leukemia-associated or leukemia-specific Ags amenable to identification. In two cases, we also could provide proof of principle that our
CTL were really effective in the killing of naive AML cells, suggesting that Ag recognition is not accompanied by any impairment
of CTL functional properties due to tumor counterattack, such as
CTL apoptosis induced by FasL-expressing AML cells.
Finally, to be used in vivo, leukemic m-DC need to be irradiated
to prevent proliferation of residual undifferentiated leukemic cells.
Therefore, leukemic DC should maintain their functional properties also after irradiation. Notably, leukemia-specific autologous T
cells were obtained using irradiated leukemic DC as stimulators
and this observation already provides evidence for their capacity to
activate T cells in vitro. In addition, leukemic i-DC that were irradiated 4 h after CD40L stimulation could still differentiate into
functional competent m-DC, as shown by their capacity to upmodulate maturation markers such as CD83 and CCR7, to produce
cytokines/chemokines and to migrate in vitro up to 48 h after treat-
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