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Prepublished online as a Blood First Edition Paper on November 7, 2002; DOI 10.1182/blood-2002-02-0378.
GENE THERAPY
From the Institute of Immunology, Vienna International
Research Cooperation Center (VIRCC), University of Vienna,
Vienna, Austria; Novartis Forschunsginstitut, Vienna,
Austria; CCRI St Anna Children's Hospital, Vienna, Austria;
Division of Hematology, Internal Medicine I University of Vienna,
Austria; and Intercell AG, Vienna,
Austria.
Myeloid lineage-derived dendritic cells (DCs) are considered the
professional antigen-presenting cell type responsible for eliciting
T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is
a disease in which tumor antigens are expressed by the malignant clone
that also has the potential to differentiate into DC-like cells
(leukemic DCs) with antigen-presenting capacity. This study
investigated whether the constitutive expression of the cytokine
interleukin-7 (IL-7) in primary AML cells during their differentiation
toward leukemic DCs results in superior antigen-presenting
cells. A bicistronic retroviral vector encoding the IL-7
cytokine and the surface immunoselectable low-affinity nerve
growth factor receptor (LNGFr) gene was constructed and used for
transduction experiments. A serum-free system was used to transduce and
differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the
cytokine vector varied among patients, ranging from 5% to 30% as
judged by LNGFr expression. The leukemic origin of the transduced cells
was confirmed in a patient with a chromosomal translocation t(9:11) by
fluorescence in situ hybridization analysis. Cytokine
modified-cells consistently secreted IL-7 (mean, 415 pg
± 190/106 cells/48 hours; n = 5). We demonstrate that
IL-7-transduced cells are included in the differentiated
leukemic DC subset, and, as shown in a particular case, that about half
of the mature CD80+ and CD83+ populations
coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and
generate cytotoxic T lymphocytes against autologous leukemic blasts.
(Blood. 2003;101:2184-2190) Genetic modification of malignant cells with
cytokine and/or costimulatory molecule genes yields potent tumor
vaccines in several animal models, including leukemia
models.1-7 This approach also leads to promising results
in humans (reviewed in Pardoll8).
Dendritic cells (DCs) act as natural adjuvants and are thus of special
importance for immunotherapy of cancer.9 The central role
of DCs in the priming of T-cell-mediated immune responses has prompted
studies to further potentiate DC function by gene transfer. Besides
genes coding for tumor antigens, DCs have been engineered to express
immunomodulatory cytokines, such as interleukin-7 (IL-7),
granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and
Primary acute myelogenous leukemia (AML) cells are the malignant
counterpart to myeloid lineage-committed hemopoietic progenitor cells.
The recent demonstration that a proportion of primary AML cells can be
differentiated in vitro into a DC-like cell type14-17 has
provided the rationale for another form of immunotherapy: the use of in
vitro-generated DC-like AML (hereafter referred to as leukemic DCs),
naturally expressing tumor antigens, to induce antigen-specific T cells
leading to the eradication of unmodified cancer cells.
Herein, we investigated whether cytokine gene transfer could be applied
to these leukemias during their differentiation toward leukemic DCs,
thus further potentiating their function. IL-7 was selected because of
its known effects on survival, activation, and development of T
cells,18,19 as well as its regulatory effects on T-cell
cytokine production.20,21 Our results demonstrate the
feasibility of introducing the IL-7 cytokine gene in these DC-like leukemia cells. Furthermore, IL-7-transduced
leukemic DCs cells proved to be superior antigen-presenting cells
enhancing T-cell responses.
Patient samples
Generation of bicistronic retroviral vector and packaging cell
lines
These plasmid constructs were transfected into the Phoenix packaging cell line (kindly provided by Dr Nolan, Stanford, CA) using a calcium phosphate system (Stratagene, Amsterdam, The Netherlands). Cell-sorting selection was used to generate stable producer cell lines strongly expressing the LNGFr gene. Virus supernatants were prepared in serum-free medium (X-VIVO 15; Bio Whittaker, Walkersville, MD) supplemented with L-glutamine at 32°C and 5% CO2, collected between 36 to 48 hours and followed by cryopreservation. The producer cell line expressing IL-7LN secreted up to 23 ng/106 cells of IL-7 in 48 hours, measured by standard enzyme-linked immunosorbent assay (ELISA). The generated retroviral vectors were tested for their ability to infect different cell types (CD34+ progenitor cells, leukemia cell lines, PBMCs isolated from healthy volunteers, and monocyte-derived DCs) under optimal culture conditions. As it could be expected, PBMCs and monocyte-derived DCs were not susceptible to viral infection since they are not cycling cells (unpublished observations, C.B.-F., April 1999). Generation of leukemic DCs and retroviral infection Cryopreserved leukemic cells were thawed, washed, and resuspended in serum-free medium (X-VIVO 15) supplemented with L-glutamine (2.5 mM), penicillin (125 TU/mL), and streptomycin (125 mg/mL). A total of 0.5 to 1 × 106 blast cells/mL were plated in fibronectin-coated (RetroNectin 10 µg/cm2; Takara, Shiga, Japan) 6-well plates (Costar, Vienna, Austria) and cultured in serum-free medium in the presence of the following proliferative cytokine cocktail: stem cell factor (SCF; 20 ng/mL, Peprotech, London, United Kingdom), granulocyte-macrophage colony stimulating factor (GM-CSF; 100 ng/mL, Novartis Research Institute, Vienna, Austria), flt3 ligand (FL; 100 ng/mL kindly provided by Immunex), tumor necrosis factor (TNF; 50 U/mL, Bender,
Vienna, Austria) and IL-3 (100 ng/mL, Novartis Research Institute).
About one third of samples showed a viability less than 70%
(trypan blue exclusion) after 3 to 4 days in culture and were excluded. After 48 to 72 hours of stimulation, leukemia samples were infected in
3 consecutive rounds with viral supernatants in the presence of 4 µg/mL polybrene (Sigma) and cytokines as previously
described.22 The transduction efficiency was monitored by
immunofluorescence staining using a LNGFr monoclonal antibody, 48 to 72 hours after the last infection.22 Comparative studies were
initially performed to define a proper cytokine cocktail allowing
viability and differentiation of transduced leukemic cells. A
serum-free differentiation cytokine cocktail containing lower
concentrations of SCF and FL (10 ng/mL and 50 ng/mL, respectively) plus
GM-CSF (100 ng/mL), IL-4 (200 U/mL, Novartis), CD40 ligand (500 ng/mL,
Immunex), and TNF (50 U/mL) was selected. Transduced cells were
further differentiated for 7 to 10 days prior to being tested in
functional assays.
Immunophenotype and cell-sorting analysis Unmanipulated and transduced AML samples were stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mouse monoclonal antibodies (mAbs) against CD86, human leukocyte antigen (HLA)-DR, CD54, CD58, CD4, CD8 (Pharmingen, Heidelberg, Germany), CD80, HLA-DQ (Becton Dickinson, Erembodegem, Belgium), CD83 (Immunotech, Marseille, France) CD1a (clone VIT 6, produced in our institute), and LNGFr (mAb 20.4 purchased from American Type Culture Collection [ATCC]), or with appropriate isotype control mAb. Cells were incubated at 4°C for 20 minutes, washed, and analyzed in a FACScan flow cytometer with CellQuest version 3.3 and analysis software (Becton Dickinson). Forward- and side-scatter gates or propidium iodide exclusion (Sigma) was used to exclude cell debris.Cell sorting for LNGFr-positive and -negative subsets was performed 48 to 72 hours after transduction. To this end, AML cells were stained with the LNGFr mAb, sorted based on LNGFr+ surface expression (FACS Vantage flow cytometer [Becton Dickinson]), and replated in a differentiation cytokine cocktail for further studies. Fluorescence in situ hybridization analysis (FISH) Dual-color FISH was performed on cytospin preparations of naive leukemia blasts and sorted leukemia-derived DCs using the 3' (labeled with CY3, red) and 5' (labeled with FITC, green) probes that flank the breakpoint of the MLL gene (kindly provided by Ed Schuuring, the Netherlands) as described.23 Slides were mounted in Vectashield with the fluorescence dye 4,6-diamino-2-phenyl indole (DAPI). Cells were visualized using a fluorescence microscope (Axioplan; Zeiss, Goettiengen, Germany). In each sample, 100 cells were analyzed.Allogeneic mixed leukocyte reaction (MLR) Responder cells for the allogeneic MLR were highly purified T cells isolated from the PBMCs of healthy donors as previously described.22 Freshly isolated or 10- to 14-day cultured/transduced leukemia cells were irradiated with 30 Gy and used as stimulators. A constant number of 5 × 104 T cells were cultured in triplicates and incubated with graded number of irradiated leukemia cells. After 5 days in culture, stimulation of responding T cells was monitored by measuring methyl-3H-thymidine incorporation (Amersham, Buckinghamshire, United Kingdom) for an additional 18 hours. Radioactivity was determined using a 1450 microbeta Wallac-Trilux instrument (Life Science, Vienna, Austria).Measurement of cytokine production Virus supernatants as well as supernatants from fresh leukemia cells, cytokine-modified leukemia cells, or cells modified with control vector were analyzed for IL-7 secretion using the specific IL-7 capture and detection mAbs (Pharmingen), following the manufacturer's instructions. The IL-7 concentration was extrapolated to 106 cells in 48 hours.The cytokine production by T cells was determined as follows: 4 to
5 × 104 of fresh leukemias, cytokine-modified cells,
cells transduced with control vector, or untransduced cells were
cultured (in RPMI medium supplemented with 10% fetal calf serum
[FCS]) alone or in the presence of 1 × 105
allogeneic T cells in 96-well plates (Costar). After 48 to 72 hours of
culture, supernatants were harvested and measured for IL-2, IL-4, and
Induction of autologous CTL AML blasts transduced either with the IL-7LN or control LXLN vector were sorted, differentiated to leukemic DCs, irradiated (30 Gy), and used to stimulate autologous PBMCs obtained after complete remission. About 106 nonadherent PBMCs were cocultured with 2.5 × 105 irradiated leukemic DCs in 1.5 mL Yssel medium (Gibco Lofer, Austria) containing 8% FCS, L-glutamine, and penicillin/streptomycin. Recombinant IL-2 (rIL-2; 20 ng/mL, Novartis) was added after 48 hours and then every 2 to 3 days. On day 7, a second restimulation with autologous irradiated IL-7LN leukemic DCs or LXLN leukemic DCs was performed in medium containing rIL-2. CTL activity was measured 5 to 7 days after the second stimulation.T-cell cytotoxicity assay The described nonradioactive Europium (Eu3+)-release assay25 was used to monitor the cytotoxic potential of the in vitro-expanded peripheral blood lymphocytes, used as effector cells. Thawed leukemia cells were cultured for 3 to 5 days in X-VIVO 15 medium containing rGM-CSF (50 ng/mL), allowing a more efficient labeling with Eu3+, and used as target cells. Briefly, 5 × 106 leukemic cells were treated with the Eu3+-DTPA-containing buffer (following the manufacturer's instructions; Wallac Oy, Turku, Finland), washed, incubated for one hour in RPMI phenol red-free medium at 37°C, and used in the assay. Labeled target cells (5 × 103) and serial dilutions of effector cells were set up in triplicates in 96-well plates in RPMI phenol red-free medium containing 10% FCS and incubated for 4 to 7 hours at 37°C and 5% CO2. Finally, 25 µL of supernatant and 200 µL of enhancement solution (Wallac) were mixed in 96-well flat-bottom plates and measured in the 1234 DELFIA Research fluorometer (Pharmacia). The percentage of cytotoxicity was calculated as follows: Percent specific lysis = [(Experimental Eu3+ release Spontaneous
Eu3+ release)/(Maximum Eu3+ release Spontaneous Eu3+ release)] × 100.
Spontaneous Eu3+ release was less than 12% of the maximun release obtained by 2% Triton X-100 (Sigma) lysis. Statistics Data are presented as mean values ± SDs. For comparison of 2 groups, the paired Student t test was used. P values of less than .05 were considered statistically significant.
Feasibility of using bicistronic retroviral vectors to transduce primary AML cells during their differentiation toward leukemic DCs We and others have previously described that retroviral vectors are suitable vehicles to transfer genes into DCs' progenitors, and per se do not influence the functional properties of these cells.22,26-28 This observation led us to investigate whether primary AMLs, on their differentiation to leukemic DCs, could be transduced with retroviral vectors encoding cytokine genes. IL-7 was selected based on its ability to enhance T-cell properties.We initially performed a set of experiments to define the optimal
culture conditions of these leukemia cells for retroviral infection,
since these vectors only stably integrate into dividing cells. A
serum-free system containing SCF, GM-CSF, Flt3 ligand, TNF A bicistronic retroviral vector encoding the IL-7 and the LNGFr reporter gene and a control vector encoding the reporter gene alone were generated in our laboratory and used to infect primary AML cells ("Patients, materials, and methods"). The proportion of transduced leukemic cells expressing the LNGFr was determined by flow cytometry 48 to 72 hours after the last infection. The transduction efficiency obtained with both cytokine and control vectors in 8 patients, as well as patients' details are displayed in Table 1. Transduction rates with the cytokine vector, as judged by LNGFr expression, ranged from 5% (patient no. 8) to 30% (patient no. 4) (mean, 12.7% ± 8.3% SD; n = 8), whereas with the control vector gene transfer efficiencies ranged from 6% to 35% (mean, 19.8% ± 9% SD; n = 8). Higher transduction efficiency with the control vector versus the bicistronic vector was consistently observed in most cases, as it has been described.29 These results demonstrate that primary AMLs can be efficiently infected with retroviral vectors encoding cytokines and a selectable marker. Cytokine-transduced cells are included in the leukemic DC population It is generally accepted that a proportion of AMLs can be differentiated in vitro to leukemic DCs.14-17 We defined leukemic DC phenotype as those culture samples with at least one de novo expression of the CD80, CD86, CD83, or CD1a molecules and up-regulation of 2 or more of the following molecules: HLA-DR, HLA-DQ, CD54, or CD58. Of 8 patients tested, 6 fulfilled these criteria (patient nos. 1 to 6) and were selected for further studies. Transduced AML cells were cultured for 7 to 10 days in the serum-free differentiation cytokine medium (described in "Patients, materials, and methods") and subsequently double stained using the LNGFr antibody, which only detects retroviral infected cells, and a DC marker. Figure 1 shows the results from a patient with AML subtype M5b (patient no. 5). In this case, about one third of the IL-7LN+-transduced cells have a DC phenotype coexpressing CD86, CD80, and CD83 DC markers. This implies that in the mature leukemic DC population expressing CD83+ (10%) and CD80+ (11%), about 50% of these cells also contain the LNGFr transgene. Likewise, the transduced LNGFr+ population also coexpresses different levels of MHC class II, DQ, CD40, CD54, CD58, and CD1a (Figure 1). These results demonstrate that bicistronic retroviral vectors can target leukemic DCs during their in vitro generation. In addition, the proportion of leukemic DCs generated in the cytokine-transduced versus control population was not significantly different (data not shown).
To further prove that the IL-7 retroviral vector targets
predominantly leukemic cells and not residual normal cells contained in
peripheral blood samples (blast cells source), we performed dual-color
FISH analysis in a patient (no. 2) whose leukemic cells harbored a
translocation t(9:11) (p22;q23) and the respective MLL/AF9 gene fusion
("Patients, materials, and methods). The separation of the 3' and 5'
probes in one allele confirmed the presence of a translocation
involving the MLL gene in 98% of the IL-7LN+ sorted
population (Figure 2), in 95% of the
LNGFr
Cytokine-modified leukemia cells secrete IL-7 After 48 to 72 hours of retroviral infection with control and cytokine vectors, cells were sorted based on the surface expression of the LNGFr reporter gene. Supernatants from sorted IL-7LN+ and LXLN+ cells as well as from untransduced cells or fresh leukemias were tested for IL-7 secretion using specific ELISA. Table 2 shows the results obtained in 5 different patients. An average of 415.5 pg ± 190 of IL-7 is secreted by 106 transduced leukemia cells in 48 hours. The cytokine was never detected in supernatants from fresh leukemia cells, unmodified cells, or cells transduced with the control vector.
IL-7-transduced leukemic DCs are superior stimulators in allogeneic MLRs Primary AMLs often have low immunogenicity due in part to lack of costimulatory molecules, whereas differentiated leukemic DCs acquire the ability to activate T cells.14-17 We investigated whether the constitutive expression of IL-7 into leukemic DCs could improve their T-cell stimulatory capacity. Primary AML cells transduced with either control or IL-7 vector were replated in the described differentiation cytokine cocktail ("Patients, materials, and methods") for an additional 7 to 10 days and used at several stimulator-responder ratios in a classical allogeneic MLR. Figure 3A shows the results of 6 patients at the ratio 2 × 104 stimulators: 5 × 104 T cells. Although there was variability with overall stimulatory capacity among different patients, the same pattern was observed in all cases. Cytokine-modified leukemia cells induced consistently higher (up to 2.3-fold) stimulation of T cells compared with cells transduced only with the control vector. Interestingly, a patient with low (< 10%) IL-7LN transduction efficiency (patient no. 6) displayed the lowest T-cell stimulatory capacity compared with cells transduced with the control vector. Differences between control and IL-7-transduced samples showed a significance of P = .048 (Student t test). In titration experiments, the strongest T-cell proliferation capacity (2.3-fold) was observed at a higher number of stimulators (patient no. 4: transduction efficiency 35% LXLN and 29% with the IL-7LN vector) as depicted in Figure 3B. In all 6 cases, fresh leukemia cells did not induce T-cell activation (data not shown).
To further prove that the effects observed were mainly due to the
IL-7 transgene, we performed sorting experiments based on LNGFr expression. The IL-7LN+ and LXLN+ subsets
as well as the untransduced (LXLN
The stimulatory capacity of the LXLN Finally, addition of recombinant IL-7 to the proliferation and/or differentiation cytokine cocktail prior to MLR assays did not significantly increase the stimulatory capacity of unmodified leukemic DCs. Furthermore, the phenotypic features of these cells were, by contrast to previous observations,31 not significantly affected (not shown). IL-7-transduced leukemic DCs induce higher levels of IL-2 in T cells We next studied whether the constitutive expression of the IL-7 cytokine gene into leukemic DCs influences the cytokine secretion profile in T cells. Unsorted and sorted leukemic cells transduced either with control or cytokine vector were cultured alone or with allogeneic T cells for 48 to 72 hours. Supernatants were harvested and tested for IL-2, IL-4, and IFN production using specific
ELISA assays. Figure 5A shows
the results with the unsorted population in 3 different
patients (patient nos. 3, 4, and 5). IL-7 modified-leukemia
cells induced consistently higher levels of IL-2 secretion (mean, 7 ng/mL ± 2 SDs; n = 3) compared with the control LXLN group (mean, 4.8 ng/mL ± 2 SDs; n = 3) with a significance of P = .002
(paired t test). No IL-4 or IFN production was detected
in the unsorted population (not shown). In sorted cells of patient no.
2, cytokine-modified cells induced up to 3.5-fold higher IL-2 secretion
than cells transduced with vector control or untransduced cells. In
addition IFN production was 2.5-fold higher in IL-7-transduced
leukemic cells versus control vector (280 pg/mL vs 110 pg/mL,
respectively; Figure 5B), and again no IL-4 was measured in any of the
analyzed subsets. Fresh leukemia cells do not produce any of these
mentioned cytokines (not shown). These findings further confirm the
ability of IL-7-modified leukemic DCs to enhance T-cell response and
potentiate the secretion of cytokines considered to be of Th1 prototype
and involved in tumor immunity.
Generation of autologous antileukemic activity by IL-7LN-transduced leukemic DCs It has been reported that leukemic DCs stimulate autologous lymphocytes that can destroy unmodified leukemia cells.15,17 We investigated whether the constitutive expression of IL-7 in leukemic DCs results in the generation of superior CTL. In Figure 6A, the antileukemic cytotoxicity of autologous T cells primed with irradiated leukemic DCs transduced either with IL-7LN or with the control LXLN vector obtained in patient no. 2 is displayed. The percentage of lysis (means of triplicates ± SDs) was determined by measuring the Eu3+ release after 7 hours of coculture ("Patients, materials, and methods"). T cells stimulated with cytokine-modified leukemic DCs are consistently able to generate, at different effector-target ratios, superior antileukemic cytotoxicity compared with T cells stimulated with the control vector. This functional effect might be due, in part, to differences in the expanded T-cell population. Although most of the T cells are CD4+ (Figure 6B), the proportion of CD8+ T cells generated with primed IL-7LN leukemic DCs was 2-fold higher (9%) compared with the percentage of CD8+ T cells obtained with control LXLN leukemic DCs (4.5%) (Figure 6B). No quantitative difference in T-cell expansion was observed between IL-7LN and LXLN groups (not shown).
This study shows that bicistronic retroviral vectors can be used to introduce genes, like the IL-7 cytokine gene, into primary myeloid leukemic cells during their differentiation toward leukemic DCs. We also demonstrate that IL-7 increases the T-cell stimulatory capacity of these cells, enhances the Th1 cytokine profile in T cells, and potentiates the generation in vitro of autologous antileukemic CTL. Retroviral vectors can transduce a wide range of hematopoietic cells
including DCs' progenitors of myeloid origin22,27,28 and
hematopoietic malignancies.32,33 AML is a malignant
disease of myeloid precursors that can be driven to a
DC-type;14-17 thus, we postulated that retroviral vectors
might also deliver genes into these leukemic DCs. Because these vectors
infect only cycling cells, a serum-free system used to target DC
progenitors22 was further optimized for primary AMLs,
which are known to have low proliferation rates in many cases. In
general the combination of 5 cytokines, namely SCF, GM-CSF, IL-3, FL,
and TNF In preclinical models, transfection with costimulatory molecules of undifferentiated leukemic cells (which per se are not expressed in these cells) and/or cytokine genes resulted in good therapeutic effects.5,7,36-38 Herein, we demonstrate, that following culture and transduction with the IL-7 vector in the serum-free medium described, leukemic DCs display CD86, CD80, and CD83 DCs markers as well as IL-7, as judged by the identification of the immunoselectable LNGFr transgene and the detection of the IL-7 protein only in the supernatants of cytokine-modified cells. The proportion of leukemic DCs transduced varied considerably among patients. For example, a great majority (> 70%) of the IL-7-modified cells of patient no. 1 displayed phenotypic DC features, whereas in patient no. 6 only a low proportion (<20%) of the LNGFr+ cells showed DC markers (not shown); and in patient no. 5 half of the transduced cells coexpress CD80+ and CD83+ (Figure 1). In addition, the percentage of leukemic cells expressing CD80 and CD83 DC markers was in most cases less than 25%, in the lower range compared with published data.16,17 This may be explained by the known heterogeneity of AMLs.17 The main advantage of cytokine-modified DCs relies on their
functional properties. DCs migrate and home to T-cell areas in lymphatic tissues where they encounter and prime naive T cells. The
local and continuous secretion of the cytokine in this environment probably could not be achieved by its systemic in vivo
administration. In addition, the side effects often observed
under systemic cytokine treatment would be avoided. The transgene
expression of IL-7 in leukemic DCs could be of particular relevance
since IL-7 induces proliferation of naive T cells,39
regulates T-cell survival, enhances cytolytic T-cell
function,40 and contributes to the generation of memory T
cells.41 Thus, it is tempting to speculate that the local
secretion of IL-7 during the leukemic DCs T-cell interaction occurring
in secondary lymphoid organs might contribute to the generation of a
more efficient T-cell response, and it could also influence humoral
immunity by acting on the B cells present in these areas. Indeed,
Westermann et al10 reported the in vitro modulation of DC
function by IL-7 using monocyte-derived DCs. They observed up to a
2.7-fold increase in T-cell activation, findings that are in line with
our leukemic DCs study (Figures 3-4). We also noticed stronger T-cell
stimulation with increasing number of cytokine-modified cells. In
addition, IL-7-modified leukemic cells induced consistently higher
levels of the Th1 prototype cytokines IL-2 and Any immunotherapeutic approach to leukemia/cancer treatment will be most effective at the stage of minimal residual disease. The immunosupppression related to the disease or derived from the treatment might limit its success. Recent studies described IL-7 as a regulator of naive CD8+ and CD4+ T-cell expansion in lymphopenic hosts, restoring immunity;41,43 whether this effect could also take place in the context of cancer vaccines remains to be explored. To our knowledge this is the first study that shows the possibility of introducing genes into primary leukemic DCs. The transgene expression of IL-7 in these cells results in superior antigen-presenting cells. Cytokine transduction of leukemic DCs may thus further be exploited as a potentially powerful form of immunotherapy of AML.
We thank Manfred Berger for his excellent technical advice. We are grateful to Dr Ornella Parolini for the critical reading of the manuscript. We also thank Drs Andreas Szekeres and Karel Drbal for the useful discussions and suggestions.
Submitted February 5, 2002; accepted October 29, 2002.
Prepublished online as Blood First Edition Paper, November 7, 2002; DOI 10.1182/blood-2002-02-0378.
Supported by the ICP program of the Austrian Federal Ministry for Education, Science and Culture.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Concha Bello-Fernández, Institute of Immunology, Vienna International Research Cooperation Center (VIRCC), Brunnerstrasse 59, A-1235 Vienna, Austria; e-mail: concha{at}kabsi.at or concha.bello{at}univie.ac.at.
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Abstract
Introduction
cells (untransduced), and in 92% of the
naive leukemic blasts (data not shown). These data indicate that almost
all cytokine-modified cells are of leukemic origin and therefore, also
the transduced leukemic DCs. Normal mononuclear cells that could be
present in the peripheral blood of these patients and that could
eventually generate DCs, are not susceptible to retroviral infection
using our type of vectors ("Patients, materials, and methods") as
it is also described.
) and control group (
, LXLN) at different E/T
ratio is indicated as: *P < .03 and
**P < .005. Panel B shows the phenotype of the effector T
cells used in the cytotoxic assay. Cells were stained with CD8-FITC and
CD4-PE mAbs and analyzed by flow cytometry. Makers were set according
to an isotype-control mAb. The number in each box represents the
percent of positive cells.