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.
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
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.
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.
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).
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.
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.
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.
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.
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).
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.
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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.
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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
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