Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia

Abstract

Dendritic cells (DC) can facilitate immune responses that might help in the induction of effective antitumor T cell responses. We reported previously that leukemic blasts from selected patients with acute myeloid leukemia (AML) were able to differentiate in vitro into cells with mature DC features. However, despite the use of a wide variety of cytokine combinations, leukemic DC could not be obtained from all AML patients. In this study, we investigated in a wide range of AML patients (n = 30), the nature and functional characteristics of the blast compartment that can be induced to acquire DC features in vitro. Our results demonstrate that leukemic DC generated in the presence of GM-CSF, IL-4 and matured with CD40L, are composed of two major subsets: DC derived from CD14+ leukemic cells and leukemic DC derived from in vivo expanded circulating blood myeloid DC (MDC). Leukemic DC of both subsets exhibited DC morphology, had a phenotype of mature DC, and could induce a potent proliferative response of naive CD4+ T cells. Moreover, both subsets produced large amounts of IL-12p70 and leukemic CD14+-derived DC could induce a potent Th1 response. These results can be considered as a prerequisite before the design of vaccine immunotherapy protocols for the adjuvant treatment of AML patients.

Introduction

Allogeneic bone marrow transplantation has proved to be an efficient treatment for patients with acute myeloid leukemia (AML). This is mainly attributed to the so-called graft-versus-leukemia effect mediated by the donor-derived immune system, especially T cells.1 Thus, modulation of the immune system appears to be an attractive modality for the treatment of AML patients, especially those patients at high risk of relapse and who cannot benefit from allogeneic transplantation. More than 20 years ago, some patients with AML received pooled, irradiated allogeneic leukemic cells in order to enhance their immune system.2 At present, donor lymphocyte infusions (DLI) following allogeneic transplantation are the treatment of choice for relapse in chronic myeloid leukemia and AML patients.3 Unfortunately, DLI is less successful in the treatment of AML patients. One possible reason for this decreased efficiency of DLI in AML patients could be related to the fact that AML blasts are poor antigen-presenting cells and fail to induce a potent and sustained antileukemic immune response.4

Dendritic cells (DC) are bone marrow-derived leukocytes which are responsible for the initiation of immune responses and exert a sentinel-like function.5 We and others previously reported that myeloid leukemic blasts from selected patients were able to differentiate in vitro into cells with mature DC features.6,7,8,9,10,11,12,13,14 These leukemic-derived DC were shown in some cases to have a potent capacity to induce T cell proliferation, while still retaining the leukemic chromosomal abnormality of the original blasts. These observations constitute a unique model where the antigen-presenting cell (APC) necessary for tumor antigen presentation and the tumoral cells themselves correspond to the same cell. Therefore, DC generated from AML patients can facilitate an immune response that might help in the induction of effective antileukemic T cells responses. Unfortunately, in all published studies, including the one from our group, and despite the use of a wide variety of cytokine combinations, leukemic DC could not be obtained from all AML patients. Furthermore, yields of leukemic DC were very heterogeneous between patients, including patients having the same leukemia subtype. In all the aforementioned studies, it was not possible to postulate any characteristic of an AML predictive of generation of cells with DC features. Moreover, phenotypic and functional properties of these leukemic DC are still sparse.

In parallel with the in vitro generation of leukemic DC, we recently demonstrated that patients with AML, when compared to healthy individuals, can have in vivo a dramatic quantitative imbalance in their circulating blood DC subsets, with a great number of patients showing a tremendous expansion of either myeloid dendritic cells (MDC), plasmacytoid dendritic cells (PDC) or both subsets.15 Since leukemias are heterogeneous at the level of maturation stages or comprise cells of different lineages, all these data raise the question of the nature of the blast compartment that can be induced to acquire DC features in vitro. In this study, we investigated the relationship between leukemic blasts and leukemic DC in a wide range of AML FAB subtypes. We assessed the different leukemic DC fractions generated in vitro for their phenotypic and functional properties.

Patients and methods

Patient samples

AML peripheral blood samples were obtained after informed consent from 30 patients at diagnosis and before any chemotherapy. AML diagnosis was performed according to standard criteria. AML peripheral blood mononuclear cells (PBMC) analyzed in this study were derived from patients with ages ranging from 15 to 86 years treated at the Institut PaoliCalmettes (Marseille, France) between 1993 and 2001. AML were subtyped according to the FAB classification.16 The cells were separated on a Ficoll–Hypaque density gradient and the PBMC fraction containing the malignant cells was harvested and cryopreserved in liquid nitrogen using medium containing RPMI (Biowhittaker, Verviers, Belgium) supplemented with 20% fetal calf serum (FCS) (Biowhittaker) and 10% dimethylsulfoxide (Sigma, St Quentin Fallavier, France). At all times leukemic samples and normal controls were similarly handled to prevent functional differences resulting from differences in cryo-preservation and cell separation. Table 1 shows the relevant clinical and diagnostic laboratory data for all 30 cases.

Table 1 Patients characteristics

Detection and sorting of circulating blood DC subsets

The circulating leukemic myeloid blood DC (MDC) were identified as previously described elsewhere in details.15 Briefly, circulating MDC were identified by three-color staining using the following monoclonal antibodies: ZM3.8-PC5 (mAb against ILT3, an immunoglobulin-like transcript), BU15-PE (mAb against CD11c) and FITC-labeled mAbs against lineage markers CD3, CD14, CD16, CD19 and CD56 (Beckman-Coulter, Marseille, France). Cells that did not label with these lineage markers were designated as lin. With respect to the expression of CD11c, MDC were identified as lin/CD11c+/ILT3+. Leukemic MDC were sorted on a FACSVantage cytometer (Becton Dickinson, Le Pont de Claix, France) and matured for 3 days in RPMI 1640 medium containing 10% FCS (BioWhittaker) in the presence of 100 ng/ml GM-CSF (a kind gift of Novartis, Berne, Switzerland), 20 ng/ml IL-4 (a kind gift of Schering-Plough Research Institute, Kenilworth, NJ) and irradiated CD40L-transfected murine L-cells. Murine L-cells transfected with human CD40L were kindly provided by Schering-Plough (Laboratory for Immunological Research, Dardilly, France),17 and used after a 75 Gy irradiation.

In vitro DC generation

CD14+ monocytes from healthy donors and CD14+ cells (as determined by FACS analysis in peripheral blood) from AML patients were immunomagnetically purified with CD14 mAb-conjugated microbeads (Milteniy Biotec, Bergisch Gladbach, Germany). Purity of the CD14+ cells by flow cytometry analysis was >98%. For generation of monocyte-derived DC (Mo-DC), CD14+ monocytes from healthy donors and leukemic blasts were cultured for 5 days in RPMI 1640 medium containing 10% FCS in the presence of 100 ng/ml GM-CSF (Novartis) and 20 ng/ml IL-4 (Schering-Plough). On day 5, final maturation of DC was induced by adding 75-Gy-irradiated CD40L-transfected cells (2 × 105/well). The medium was replenished with cytokines every 3 days.

Confocal microscopy

Cells were adhered to polylysine-coated glass slides for 30 min at room temperature, fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton in PBS. Cells were then labeled with primary mAb and revealed by species-specific Alexa 488, TRITC (Molecular Probes, Eugene, OR, USA) or Cyanin 5-labeled secondary antibodies (Jackson Immunoresearch, West Baltimore Pike, PA, USA). Slides were then mounted using fluorescent mounting medium (Dako, Trappes, France). Confocal analysis was performed with a TCS NT microscope equipped with argon and krypton ion lasers and a ×100 1.3NA PL fluotar objective (Leica Microsystem, Heidelberg, Germany).

Flow cytometry analysis

The following mAb were used in this study for flow cytometry: CD1a, CD13, CD14, CD54, CD58, CD80, CD83, anti-HLA-DR, anti-HLA-A, B, C, isotypic IgG1, IgG2a, IgG2b and IgM controls from Beckman-Coulter. CD86 and CCR-7 (2H4) were purchased from Pharmingen (San Diego, CA, USA). All mAbs were used as FITC-, PE-, Cy-5-conjugated mAbs or with PE-conjugated F(ab′)2 fragments of goat anti-mouse IgM antibody when using CCR-7 mAb. Cells were analyzed using a FACSCalibur cytometer (BD Biosciences). Data for at least 10 × 103 cells/sample were acquired and analyzed using CellQuest software (BD Biosciences).

FISH analysis

Interphase fluorescence in situ hybridization (FISH) was performed as previously described18 on cytospin preparations with sorted leukemic DCs from two patients selected for detection of their cytogenetic abnormality. For detection of trisomy 8 (UPN9 and UPN16), metaphases were analyzed with a centromeric probe specific for chromosome 8, directly labeled with spectrum green, purchased from Vysis (Voisins le Bretonneux, France). At least 200 nuclei were examined under fluorescence microscopy by two independent observers.

FITC-Dextran capture analysis

To assess endocytosis, FITC-Dextran (Sigma, St Louis, MO, USA) was used according to the method described previously.19 Briefly, the cells were incubated with 0.1 mg/ml FITC-dextran at 37°C for 1 h and analyzed by flow cytometry. The background uptake was obtained at 0°C.

Mixed lymphocyte reaction (MLR)

To evaluate T cell proliferation capacity, various numbers of mature leukemic MDC, normal and leukemic CD14+-derived DC were co-cultured with 1 × 105 allogeneic naive CD4+ T cells in 96-well flat-bottomed plates for 6 days. Proliferation of T cells was monitored by measuring methyl-3H thymidine (Amersham, Little Chalfont, UK) incorporation during the last 16 h of culture on a gas-phase β counter (Matrix 9600, Packard, IL, USA). Naive CD4+ T cells were prepared from adult donor PBMC, negatively depleted of CD8, CD14, CD56 (D Olive, INSERM U119), CD19 (Diaclone, Besancon, France), and CD45RO+ (Beckman-Coulter) cells using goat anti-mouse Ig-coated magnetic beads (Beckman-Coulter). Ninety-eight percent of the resulting cells were CD4+CD45RA+ as controlled by FACS analysis.

Intracellular analysis of cytokine production

Allogeneic naive CD4+/CD45RA+ T cells were cocultured with fresh AML blasts, immature normal Mo-DC, mature normal and leukemic CD14+-derived DC. Cells were harvested after 6 days, and replated in 48-well culture plates at 5 × 105 cells/well for 5 h in the presence of PMA (Sigma; 25 ng/ml), ionomycin (Sigma; 1 μg/ml) and 10 μg/ml of brefeldin A (Sigma). Anti-IL-4-FITC, anti-IL-10-PE, anti-IFN-γ-APC and FITC/PE/APC conjugated isotypic mAbs (Pharmingen) were used according to the manufacturer's instruction. Cells were collected, washed, fixed, and permeabilized using the CytoStain Kit (Pharmingen), and stained with 0.5 μg/test of cytokine-specific mAbs.

Cytokine production assay

Supernatants of DC cultures were harvested at day 3 for mature leukemic MDC, and day 7 for normal and leukemic CD14+-derived DC. After thawing, IL-10, IL-12p70 and TNF-α concentrations were measured by ELISA assays using the ELISA set OptEIA purchased from Pharmingen.

Results

Generation of leukemic DC from AML patients

DC were generated in vitro from the bulk blast population of 30 leukemic AML samples (Table 1) cultured in the presence of GM-CSF, IL-4 and CD40L. As previously reported by different authors,6,7,8,9,10,11,12,13,14 various percentages of cells with DC features could be obtained in this large group of patients. When we compared the percentage of cells with DC features in vitro to the proportion of circulating leukemic MDC and CD14+ cells in the initial population before the start of culture, we found a striking correlation between the number of MDC, CD14 expression at diagnosis, and the ability to acquire CD83, a marker of mature DC (Figure 1).20 Moreover, as shown in Table 1, in patients who have a majority of peripheral CD14+ leukemic cells at diagnosis without or with little circulating MDC, there was a correlation between the percentage of CD83+ leukemic DC and the initial proportion of CD14+ cells, further supporting the fact that CD14 expression by leukemic cells can predict differentiation of leukemic DC. In contrast, under the same conditions, culture of the non-MDC and non-CD14+ cells in these patients could never give rise to DC, based on the expression of CD83 and costimulatory molecules (data not shown). This observation suggests that in AML patients, only cells expressing dendritic phenotype (MDC) or CD14+ cells which correspond to cells with intermediate status between CD34+ immature hematopoietic progenitors and fully differentiated monocytes, can be induced to acquire DC features in the presence of GM-CSF and IL-4. Based on this observation, we wanted to establish the functional properties of these two different leukemic subsets that might lead to the generation of leukemic DC in vitro. Therefore, CD14+ cells and leukemic MDC were sorted as described in ‘Patients and methods’ and compared for their functional properties to the classical normal monocyte-derived DC subset.19 Cells were cultured in the presence of GM-CSF and IL-4. We examined their capacity to undergo maturation after either simultaneous in vitro culture with appropriate cytokines and CD40L for 3 days (MDC) or 5 days with cytokines followed by 2 days of stimulation by CD40L (CD14+ cells). Cells displayed dramatic morphological changes with variable size increase, cytoplasmic protrusion and irregular cellular membrane as soon as day 1 for leukemic MDC and day 3 for monocytes and CD14+ cells derived DC (Mo-DC) (Figure 2). Before culture, leukemic cells variably expressed HLA-DR, did not express CD83 marker, and expressed little, if any, CD80 and CD86 markers (Figure 3a). After culture, immunophenotypic analysis indicated that in comparison to normal Mo-DC, both subsets of leukemic DC could acquire the expression of CD83. As expected, CD14 was down-regulated on normal and leukemic Mo-DC. Both leukemic DC subsets, like normal Mo-DC, expressed the costimulatory molecules CD80 and CD86. The expression of HLA class I and HLA-DR was up-regulated (Figure 3). Acquisition of CD83 on leukemic MDC and Mo-DC was further confirmed by confocal microscopy staining (Figure 2). We also examined the expression of the adhesion molecules CD54 and CD58, which were up-regulated (Figure 3). These results indicated strikingly that circulating leukemic MDC and CD14+ cells are the leukemic compartments that can be induced in vitro in the presence of GM-CSF, IL-4 and CD40L to acquire mature DC phenotype.

Figure 1
figure1

Correlation between the % of CD83+ leukemic DC generated in vitro and the % of MDC and CD14+ cells composition of the initial leukemic bulk population.

Figure 2
figure2

Morphological and confocal microscopy analysis of leukemic DC subsets. Leukemic MDC and CD14+-derived DCs were isolated and cultured as described in Patients and methods. (a) They display dendritic morphology as shown by interferential contrast transmission microscopy (×100). Two-color immunofluorescence staining was performed. (b) HLA-DR expression is shown in red and (c) CD83 in blue. Data are from patient UPN24, and representative of three independent experiments.

Figure 3
figure3

Maturation capacities of leukemic DC subsets. (a) Expression of phenotypic markers on bulk leukemic cells before culture and (b) after culture. Results are presented as the mean and standard deviation of positive cells in the patients included in this study. (c) Normal Mo-DC and the two leukemic DC subsets obtained after culture of sorted fresh cells, (d) MDC and (e) CD14+-derived DC were analyzed by flow cytometry after 72 h of culture with either GM-CSF, IL-4 and CD40L (MDC) or 5 days with GM-CSF and IL-4 followed by 2 days of CD40L (normal and leukemic Mo-DC). Open histograms represent cells stained with isotype-matched control mAbs. Results indicated are representative of those obtained from at least 10 healthy donors and 15 patients in this series (UPN 2, 9, 10, 11, 14, 16, 17, 18,19, 20, 22, 24, 26, 29, 30).

CCR-7 expression on leukemic CD14+-derived-DC

As a way to understand the regulation of leukemic Mo-DC traffic, we examined the expression of chemokine receptor CCR-7 on their surface in comparison with mature normal Mo-DC. Flow cytometric analysis revealed that mature leukemic CD14+-derived DC express CCR-7 which is usually induced on normal mature Mo-DC (Figure 3).

Leukemic origin of leukemia derived DCs

The leukemic origin of leukemic DCs derived from MDC was established in our previous studies by confirming the presence of cytogenetic abnormalities in fluorescence in situ hybridization (FISH) experiments.15 For the purpose of this study, the leukemic origin of leukemic DCs derived from CD14+ cells was also investigated by FISH. Purified CD83+ DCs derived from CD14+ cells (patients UPN9 and UPN16) were analyzed for the persistence of the initial cytogenetic abnormality. An example from patient UPN9 with trisomy 8 at diagnosis is depicted in Figure 4. A green hybridization signal on all of the analyzed nuclei showed the presence of trisomy 8. These results further confirm the leukemic status of leukemic DCs derived from CD14+ leukemic cells.

Figure 4
figure4

Leukemic status of CD14+-derived DC. Leukemic CD14+-derived DC from patient UPN 9 were analyzed by FISH for the presence of trisomy 8 using a specific probe for chromosome 8.

Th1 polarization capacity of leukemic DCs

The ability to stimulate a T cell primary response is one of the key features of DC function. Thus, leukemic subsets (MDC and CD14+ cells from the same patient) that can give rise to leukemic DC in vitro, were assessed for their ability to stimulate naive CD4+ T cells in an allogeneic MLR. Leukemic DC obtained either from leukemic MDC or CD14+ cells from the same patient could efficiently and similarly stimulate the proliferation of naive CD4+ T cells (Figure 5). In the same experiments, fresh leukemic cells and normal PBMC, induced weak, if any, proliferation of naive allogeneic T cells (Figure 5). We next examined the nature of primary allogeneic T cell responses induced by normal and leukemic CD14+-derived DC. Naive CD4+CD45RA+ T cells isolated from human peripheral blood were cocultured with fresh leukemic cells, normal mature Mo-DC and mature leukemic CD14+-derived DC. The cultured cells were counted and restimulated with PMA and ionomycin for single-cell cytokine analysis by flow cytometry. Only T cells originally cultured with mature normal and leukemic CD14+-derived DC secreted the highest amounts of IFN-γ (Figure 6) but undetectable IL-4 and IL-10 (Figure 6 and data not shown). Thus, leukemic CD14+-derived DC can drive naive T cells towards a potent Th1 response profile.

Figure 5
figure5

T cell stimulatory capacity of leukemic DC subsets. Leukemic DC were cultured as described in Patients and methods. CD4+/CD45RA+ naive T cells were purified by negative selection of adult blood PBMC. Serial dilutions (10 × 103 to 3 × 102 cells/well) of irradiated stimulating cells were cultured in triplicate with 105 allogeneic naive T cells. Proliferation of T cells was monitored by measuring methyl-3H thymidine incorporation during the last 16 h of a 6-day culture. Normal PBMC and fresh leukemic PBMCs were used as control stimulators. The mean results obtained from at least four independent experiments from different patients in this series are indicated.

Figure 6
figure6

Th1 polarizing capacity of in vitro-differentiated leukemic DCs derived from CD14+ cells. Production of IFN-γ and IL-10 was measured by intracellular staining of CD4+ T cells stimulated with the in vitro differentiated mature leukemic CD14+-derived DC and normal mature Mo-DC. Fresh undifferentiated leukemic PBMCs were used as control stimulators. Results are representative of six experiments with six different patients from this series (UPN 12, 16, 17, 18, 20 and 26).

On the other hand, antigen uptake is a specific property of immature DC, which is not shared by mature CD83+ DC and other antigen-presenting cells.19 Thus, we analyzed endocytosis of FITC-dextran by leukemic DC. When compared to normal immature Mo-DC cultured for 5 days with GM-CSF and IL-4 alone, quantification of FITC-dextran endocytosis in both CD83+ leukemic DC subset, showed the absence of endocytic capacities of these cells (data not shown), thus confirming that leukemia-derived DC subsets display in vitro characteristics similar to normal fully mature Mo-DC.

Cytokine secretion by leukemic DC

It has been previously shown that ligation of CD40 on DC triggers IL-12p70 production.21 IL-12p70 stimulates NK cells, mediates T cell development and fosters cytotoxic T lymphocyte (CTL) differentiation. Thus, we assessed cytokine production by both subsets of leukemic-derived DC after CD40L activation. Although not statistically significant, in comparison to leukemic DC derived from leukemic MDC, leukemic DC derived from CD14+ leukemic cells produced the largest amounts of IL-12p70 within 48 h after CD40L activation (Figure 7a). In contrast, leukemic DC derived from leukemic MDC produced in some cases higher amounts of IL-10 (Figure 7b). Also, both subsets of leukemic DCs secreted significant amounts of TNF-α(Figure 7c).

Figure 7
figure7

Cytokine secretion profile of leukemic DC subsets. Culture supernatants from 106 normal control mature Mo-DC, leukemic Mo-DC, and leukemic MDC were harvested and (a) IL-12p70, (b) IL-10 and (c) TNF-α secretion were analyzed by ELISA. Results are represented as the mean and standard deviation obtained from nine independent experiments from nine patients in this series.

Discussion

The goal of this work was to determine the type of leukemic cells that can be induced in vitro to differentiate into functional cells with antigen presenting and DC features. We have demonstrated that leukemic DC generated in AML patients in the presence of GM-CSF, IL-4 and CD40L, are composed of two major subsets: leukemic DC derived from CD14+ cells and leukemic DC derived from in vivo expanded circulating blood MDC. Although no marker that persists throughout the DC differentiation process is presently available to clearly establish the relative contribution of each precursor, the correlation between MDC and CD14+ leukemic cells and DC yield can help to draw a plausible conclusion about the contribution of both subsets to leukemic DC generation. Since leukemic DC are an attractive tool for the design of vaccine immunotherapy protocols for the adjuvant treatment of AML patients, our results could be considered as a prerequisite before immunotherapeutic applications.

Many reports exist on the optimum growth factor cocktails for the generation of DC. In our study, we used the combination of GM-CSF and IL-4 used for the generation of DC from monocytes. Because normal CD34+ stem cells can be differentiated into DC by the use of GM-CSF and TNF-α,22 some authors added TNF-α in their cytokine combination.7,11,23,24 The majority of these cases concerned patients with chronic myeloid leukemia where the leukemic counterpart of the CD34+ stem cells can be found.25 For many reasons, it is unlikely in our series that CD34+ blasts might have contributed to the generation of a major fraction of the leukemic DC. Combined analyses of stem cell phenotypic antigen expression, genetic lesions and biological features provide evidence that major differences exist between normal and malignant progenitors.26 Furthermore, GM-CSF and IL-4 combination did not prove in vitro to induce DC differentiation from normal stem cells. Finally, differentiation of efficient DC from normal stem cells requires a longer period of culture.22 This was not the case in our culture system, where fully mature leukemic DC could be obtained after a maximum of 7 days, and in much shorter periods in the majority of cases. The latter is more compatible with Mo-DC differentiation profile than CD34+ stem cell profile. Moreover, one should bear in mind that to be applicable to the majority of patients, a DC vaccine protocol should avoid complex and sophisticated culture techniques. Our aim in this work was to identify a blast subset (although it may not be the only one) capable after a short-term culture with a minimal combination of cytokines of giving rise to fully functional leukemic DCs. Such a rapid and predefined culture protocol, avoiding patienttailored techniques, long-term in vitro manipulations (increasing the risk of procedure failure) and complex and expensive cytokine combinations would allow access to this strategy to the highest number of patients. The impact and DC differentiating potentials of other cytokines on AML blasts has been under intensive investigation in our laboratory. Unfortunately, even with the use of a large variety of cytokines including IL-4, IL-6, FLT3-L, SCF or TNF-α, we cannot yet obtain any convincing or definitive data as for the possible use of these cytokines for leukemic DC generation.

The recognized characteristic of DC is the ability to present antigens and to stimulate specific T cell responses. Leukemic DC were shown to express the same cytogenetic abnormality of the leukemic clone.6,15 Thus, these cells might express at least some leukemia-related proteins associated with the cytogenetic abnormality. The crucial role of costimulatory molecules in the generation of an antileukemic response has been shown in murine and other leukemia models.27,28,29,30 In the current study, we have demonstrated that both subsets of leukemic DC could acquire CD80 and CD86. The same held true for HLA-DR, HLA-A,B,C and adhesion molecules CD54 and CD58, that were up-regulated after culture. The increased expression of costimulatory molecules correlated with their capacity to induce naive CD4+ T cell proliferation. This maturation process was associated with the expression of CD83. These markers helped to define the maturation pattern of these cells towards the DC lineage.20

Along with the acquisition of adhesion and costimulatory molecules, the migration of DCs from the tumor site to the secondary lymphoid organs is believed to be one of the critical events.31 CCR-7 is an important player in the mechanism by which T lymphocytes and DC enter secondary lymphoid organs through high endothelial venules (HEV). In this study, leukemic DC derived from CD14+ cells expressed CCR-7. After injection in vivo, it could therefore be hypothesized that a high proportion of these cells would be trapped in T cell areas of lymph nodes.

Thus, we can assume that leukemic DC while directing a potent Th1 response profile, will help in generating antileukemic cytotoxic responses better than fresh tumoral cells. It has already been demonstrated that the immune balance (Th1/Th2 balance) controlled by cytokines produced by Th1 and Th2 cells plays an important role in immune regulation, including antitumor immunity.32 The Th1 cells that produce IFN-γ have been shown to exert a powerful antitumor effect, whereas a Th2 profile may have an opposite effect, that is, down-regulation of innate and acquired antitumor immunity.33

Our results established some differences in the function of CD14+ cell-derived leukemic DC compared to circulating MDC precursor-derived DC. Stimulation via CD40 pathway enhanced secretion of IL-12p70 by both subsets, but there was a trend towards a higher secretion of IL-10 by DC derived from leukemic circulating MDC. The latter may not help in the generation of antileukemic cytotoxic CD8+ T cells. Recently, in a large study by Harrison et al,13 who attempted to generate leukemic DC in 40 patients with AML, only cells from 24 samples exhibited morphological and immunophenotypic features of DC, and of 17 samples, low autologous antileukemic cytotoxicity was induced in less than half of the cases (eight samples). Despite the fact that AML encompasses a biologically heterogeneous group of clonal disorders of myeloid precursors, Harrison et al13 did not provide direct evidence explaining the lack of generation of DC in all cases, and moreover, the absence of antileukemic cytotoxicity in more than 50% of tested cases. The findings from our study, depicting the type of cells involved in the generation of leukemic DC and their functional heterogeneity, might provide some explanation for the lack of DC and antileukemic response generation observed by different authors.6,8,13

In summary, our study shows evidence of two subsets of leukemic cells that can be induced through the myeloid DC pathway. The corollary from functional studies, especially from chemokine receptor expression, polarization capacities and cytokine secretion profile, is that leukemic DC derived from CD14+ cells, should be whenever possible the preferred source of DC for immunotherapeutic approaches.

References

  1. 1

    Antin JH . Graft-versus-leukemia: no longer an epiphenomenon Blood 1993 82: 2273–2277

    CAS  PubMed  Google Scholar 

  2. 2

    Powles RL, Russell J, Lister TA, Oliver T, Whitehouse JM, Malpas J, Chapuis B, Crowther D, Alexander P . Immunotherapy for acute myelogenous leukaemia: a controlled clinical study 2 1/2 years after entry of the last patient Br J Cancer 1977 35: 265–272

    CAS  Article  Google Scholar 

  3. 3

    Kolb HJ, Schattenberg A, Goldman JM, Hertenstein B, Jacobsen N, Arcese W, Ljungman P, Ferrant A, Verdonck L, Niederwieser D . Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia Blood 1995 86: 2041–2050

    CAS  PubMed  Google Scholar 

  4. 4

    Falkenburg JH, Smit WM, Willemze R . Cytotoxic T-lymphocyte (CTL) responses against acute or chronic myeloid leukemia Immunol Rev 1997 157: 223–230

    CAS  Article  Google Scholar 

  5. 5

    Banchereau J, Steinman RM . Dendritic cells and the control of immunity Nature 1998 392: 245–252

    CAS  Article  Google Scholar 

  6. 6

    Charbonnier A, Gaugler B, Sainty D, Lafage-Pochitaloff M, Olive D . Human acute myeloblastic leukemia cells differentiate in vitro into mature dendritic cells and induce the differentiation of cytotoxic T cells against autologous leukemias Eur J Immunol 1999 29: 2567–2578

    CAS  Article  Google Scholar 

  7. 7

    Choudhury A, Gajewski JL, Liang JC, Popat U, Claxton DF, Kliche KO, Andreeff M, Champlin RE . Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia Blood 1997 89: 1133–1142

    CAS  PubMed  Google Scholar 

  8. 8

    Choudhury BA, Liang JC, Thomas EK, Flores-Romo L, Xie QS, Agusala K, Sutaria S, Sinha I, Champlin RE, Claxton DF . Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses Blood 1999 93: 780–786

    CAS  PubMed  Google Scholar 

  9. 9

    Cignetti A, Bryant E, Allione B, Vitale A, Foa R, Cheever MA . CD34(+) acute myeloid and lymphoid leukemic blasts can be induced to differentiate into dendritic cells Blood 1999 94: 2048–2055

    CAS  PubMed  Google Scholar 

  10. 10

    Robinson SP, English N, Jaju R, Kearney L, Knight SC, Reid CD . The in-vitro generation of dendritic cells from blast cells in acute leukaemia Br J Haematol 1998 103: 763–771

    CAS  PubMed  Google Scholar 

  11. 11

    Smit WM, Rijnbeek M, van Bergen CA, de Paus RA, Vervenne HA, van de Keur M, Willemze R, Falkenburg JH . Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells Hum Immunol 1997 53: 216–223

    CAS  Article  Google Scholar 

  12. 12

    Brouwer RE, van der Hoorn M, Kluin-Nelemans HC, van Zelderen-Bhola S, Willemze R, Falkenburg JH . The generation of dendritic-like cells with increased allostimulatory function from acute myeloid leukemia cells of various FAB subclasses Hum Immunol 2000 61: 565–574

    CAS  Article  Google Scholar 

  13. 13

    Harrison BD, Adams JA, Briggs M, Brereton ML, Yin JA . Stimulation of autologous proliferative and cytotoxic T-cell responses by leukemic dendritic cells derived from blast cells in acute myeloid leukemia Blood 2001 97: 2764–2771

    CAS  Article  Google Scholar 

  14. 14

    Woiciechowsky A, Regn S, Kolb HJ, Roskrow M . Leukemic dendritic cells generated in the presence of FLT3 ligand have the capacity to stimulate an autologous leukemia-specific cytotoxic T cell response from patients with acute myeloid leukemia Leukemia 2001 15: 246–255

    CAS  Article  Google Scholar 

  15. 15

    Mohty M, Jarrossay D, Lafage-Pochitaloff M, Zandotti C, Briere F, de Lamballeri XN, Isnardon D, Sainty D, Olive D, Gaugler B . Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment Blood 2001 98: 3750–3756

    CAS  Article  Google Scholar 

  16. 16

    Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C . Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French–American–British Cooperative Group Ann Intern Med 1985 103: 620–625

    CAS  Article  Google Scholar 

  17. 17

    Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ . The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand J Exp Med 1997 185: 1101–1111

    CAS  Article  Google Scholar 

  18. 18

    Renard N, Lafage-Pochitaloff M, Durand I, Duvert V, Coignet L, Banchereau J, Saeland S . Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand Blood 1996 87: 5162–5170

    CAS  PubMed  Google Scholar 

  19. 19

    Sallusto F, Lanzavecchia A . Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha J Exp Med 1994 179: 1109–1118

    CAS  Article  Google Scholar 

  20. 20

    Zhou LJ, Tedder TF . Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily J Immunol 1995 154: 3821–3835

    CAS  PubMed  Google Scholar 

  21. 21

    Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, Liu YJ . Reciprocal control of T helper cell and dendritic cell differentiation Science 1999 283: 1183–1186

    CAS  Article  Google Scholar 

  22. 22

    Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J . GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells Nature 1992 360: 258–261

    CAS  Article  Google Scholar 

  23. 23

    Eibl B, Ebner S, Duba C, Bock G, Romani N, Erdel M, Gachter A, Niederwieser D, Schuler G . Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response Genes Chromosomes Cancer 1997 20: 215–223

    CAS  Article  Google Scholar 

  24. 24

    Coleman S, Throp D, Fisher J, Bailey-Wood R, Lim SH . Cytokine enhancement of immunogenicity in chronic myeloid leukaemia Leukemia 1997 11: 2055–2059

    CAS  Article  Google Scholar 

  25. 25

    Verfaillie CM, Miller WJ, Boylan K, McGlave PB . Selection of benign primitive hematopoietic progenitors in chronic myelogenous leukemia on the basis of HLA-DR antigen expression Blood 1992 79: 1003–1010

    CAS  PubMed  Google Scholar 

  26. 26

    Brendel C, Neubauer A . Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions Leukemia 2000 14: 1711–1717

    CAS  Article  Google Scholar 

  27. 27

    Matulonis U, Dosiou C, Freeman G, Lamont C, Mauch P, Nadler LM, Griffin JD . B7-1 is superior to B7-2 costimulation in the induction and maintenance of T cell-mediated antileukemia immunity. Further evidence that B7-1 and B7-2 are functionally distinct J Immunol 1996 156: 1126–1131

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Mutis T, Schrama E, Melief CJ, Goulmy E . CD80-Transfected acute myeloid leukemia cells induce primary allogeneic T-cell responses directed at patient specific minor histocompatibility antigens and leukemia-associated antigens Blood 1998 92: 1677–1684

    CAS  Google Scholar 

  29. 29

    Boyer MW, Vallera DA, Taylor PA, Gray GS, Katsanis E, Gorden K, Orchard PJ, Blazar BR . The role of B7 costimulation by murine acute myeloid leukemia in the generation and function of a CD8+ T-cell line with potent in vivo graft-versus-leukemia properties Blood 1997 89: 3477–3485

    CAS  PubMed  Google Scholar 

  30. 30

    Costello RT, Mallet F, Sainty D, Maraninchi D, Gastaut JA, Olive D . Regulation of CD80/B7-1 and CD86/B7-2 molecule expression in human primary acute myeloid leukemia and their role in allogenic immune recognition Eur J Immunol 1998 28: 90–103

    CAS  Article  Google Scholar 

  31. 31

    Sallusto F, Lanzavecchia A . Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression Immunol Rev 2000 177: 134–140

    CAS  Article  Google Scholar 

  32. 32

    Mosmann TR, Sad S . The expanding universe of T-cell subsets: Th1, Th2 and more Immunol Today 1996 17: 138–146

    CAS  Article  Google Scholar 

  33. 33

    Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT . Th1/Th2 balance in cancer, transplantation and pregnancy Springer Semin Immunopathol 1999 21: 339–359

    CAS  Article  Google Scholar 

Download references

Acknowledgements

MM was supported by a grant from the ‘Fondation de France’, Paris, France and from the SFGM-TC, Lyon, France. We thank C Mawas (INSERM U119) and D Maraninchi (Institut Paoli-Calmettes) for helpful discussions. We thank D Jarrossay, J Benfares and J Wolfers (Immunotech, Beckman-Coulter, Marseille) for kindly providing the ILT3 mAb. We also thank R Galindeau for assistance in cell sorting; S Just-Landi and N Baratier for excellent technical assistance.

Author information

Affiliations

Authors

Corresponding author

Correspondence to B Gaugler.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mohty, M., Isnardon, D., Blaise, D. et al. Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia. Leukemia 16, 2267–2274 (2002). https://doi.org/10.1038/sj.leu.2402706

Download citation

Keywords

  • dendritic cells
  • AML
  • immunotherapy

Further reading

Search