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Cytokine-Driven Differentiation of Blasts from Patients with Acute Myelogenous and Lymphoblastic Leukemia into Dendritic Cells

Medizinische Klinik und Poliklinik I,
^a Institute for Immunology, Universitätsklinikum Carl Gustav Carus, Dresden, Germany

Key Words. Dendritic cells • Acute leukemia • Chromosomal aberrations • Immunotherapy

Martin Bornhäuser, M.D., Medizinische Klinik I, University Hospital Carl Gustav Carus, Fetscherstra ße 74 01307 Dresden, Germany. Telephone: 49-351-458-4186; Fax:-49-351-458-5362; e-mail: bornhaeuser{at}oncocenter.de

	Abstract

Top Abstract Introduction Patients And Methods Results Conclusion References

 
We investigated the ability of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts to differentiate into dendritic cells (DC) in vitro.

Cytokine-supplemented suspension cultures of leukemic blasts in 98 patients with AML and five patients with ALL (normal karyotype, n = 2; BCR/ABL, n = 3) were performed. Mononuclear cells out of peripheral blood or bone marrow containing between 60% and 90% leukemic blasts were cultured for eight days using different growth factor combinations.

The highest yield of CD1a⁺/CD14^– cells could be obtained with stem cell factor, transforming growth factor-ß, tumor necrosis factor-, GM-CSF, and FLT-3-ligand. In the AML samples the median content of CD1a⁺/CD14^– cells after eight days of culture was 3.5% (r = 0%-82%). In five informed patients CD1a⁺/CD14^– cells were sorted by fluorescence-activated cell sorting or immunomagnetic separation. Cytogenetic and polymerase chain reaction analyses showed known primary chromosomal aberrations (monosomy 7 and inversion 16) in the sorted fractions, respectively. Dendritic cells (DC) could be generated out of leukemic blasts in 68% of AML patients. Leukemic DC showed no phagocytosis of latex beads, but stimulated allogeneic naive cord blood-derived T cells more efficiently than did uncultured blasts. In ALL patients the median percentage of CD1a⁺/CD14^– cells was 1.2% (r = 0.7%-3.8%) after culture. The sorted CD1⁺/CD14^– fractions were BCR/ABL-negative when analyzed with fluorescence in situ hybridization, indicating their nonleukemic origin.

Leukemic DC can be generated out of leukemic progenitors in patients with AML. These cells might become relevant for autologous and allogeneic immunotherapy in selected patients. BCR/ABL-positive lymphoblasts could not be transformed into cells with an early dendritic phenotype with the cytokines used in our experiments.

	Introduction

Top Abstract Introduction Patients And Methods Results Conclusion References

 
The malignant progenitor cells in acute myelogenous leukemia (AML) show variable degrees of differentiation, and characteristic cytogenetic aberrations can be found in some cases. Phenotype and cytogenetics have been correlated with clinical outcome in large studies [1]. Unfortunately, long-term remission can only be achieved in a small percentage of patients [2]. Besides allogeneic blood stem cell transplantation, autologous immunotherapy might optimize the treatment outcome, especially in older patients.

Dendritic cells (DC) derived from CD34⁺ progenitors or monocytes are professional antigen-presenting cells (APC) eliciting specific T-cell responses. APC carrying and presenting leukemic antigens are supposed to be helpful in targeting residual leukemic stem cells. Since the development of leukemic progeny resembles normal hematopoiesis, the in vitro generation of leukemic DC seems possible with similar cytokine combinations [3, 4]. Those DC might elicit potent antileukemic T-cell responses with more specificity and might be useful for in vivo vaccination strategies.

Efforts to generate DC with a leukemic genotype in serum-free cultures have been successful in AML and chronic myelogenous leukemia (CML) [5, 6]. DC presenting leukemic antigens seem to be potent effectors for induction of cytotoxic T cell responses in vivo or in vitro [7]. Only recently two groups have suggested that DC with the leukemic genotype or leukemia-specific antigen markers can be grown in vitro out of AML cells [8, 9]. Those DC were able to induce specific cytotoxicity of autologous T cells against leukemic target cells. Although this seems to be a promising approach to immunotherapy of AML, both groups reported cases where no DC could be cultured at all. Some AML cells behave differently in cytokine-driven cultures, and leukemic stem cells cannot be differentiated into DC.

Patients with BCR/ABL-positive acute lymphoblastic leukemia (ALL) have a poor prognosis even after allogeneic blood stem cell transplantation and do not respond to infusion of donor leukocytes in most cases [10, 11]. This seems to be due either to a resistance against the induction of apoptosis or the insufficient presentation of leukemic antigens and costimulatory signals.

We performed experiments using mononuclear cells of 98 patients with AML, three patients with BCR/ABL-positive ALL and two patients with common ALL. In most cases we compared the leukemic cells originating from peripheral blood (PB) and bone marrow (BM). These cells were cultured in serum-free medium with the addition of different cytokines.

In the present study we show the differentiation of leukemic DC in AML with different cytokine combinations. Furthermore, the clonal origin of sorted DC is shown with cytogenetics and polymerase chain reaction (PCR) for the specific aberration in selected cases. No BCR/ABL-positive DC, tested by fluorescence in situ hybridization (FISH), could be generated in ALL patients.

	Patients And Methods

Top Abstract Introduction Patients And Methods Results Conclusion References

 
Patient Characteristics and Therapy
The characteristics of the AML patients are summarized in Table 1. The median age was 60.5 years. Most leukemias belonged to the M1/2 FAB (French-American-British classification) subtype. AML patients less than 60 years of age included in a multicenter trial received induction therapy with cytosine arabinoside (Ara C) 8 x 100 mg/m², etoposide 5 x 100 mg/m², and mitoxantrone 5 x 10 mg/m². On day 28 Ara C 5 x 2,000 mg/m² and amsacrine 5 x 100 mg/m² were infused. Patients older than 65 years were treated with 2 x 45 mg/m² daunorubicine and 7 x 100 mg/m² Ara C. Complete remission was defined as less than 5% atypical blasts in a BM aspirate when the platelet count was above 100 x 10⁹/l. Partial remission was defined as >5%-30% residual blasts.

Patient Samples
Blood or BM from 98 AML patients and five patients with ALL was collected after informed consent. The clinical study including the drawing of all diagnostic and research samples had been approved by the institutional ethical board. The cells were diluted with phosphate-buffered saline (PBS) Dulbecco's without magnesium and calcium, pH 7.2 (GIBCO; Karlsruhe, Germany). Density gradient centrifugation was performed as follows: blood was centrifuged with Immuflot (Immucor GmbH; Rödermark, Germany) at 800 g for 20 min and mononuclear cells were collected and washed twice with PBS, supplemented with 0.5% human serum albumine ([HSA] 5%, Immuno GmbH; Heidelberg, Germany). Pellets were resuspended in 5 ml of X-VIVO 20 (Bio-Whittaker; Verviers, Belgium). Cells were counted automatically by Technicon H 3 RTC^TM (Bayer Diagnostics; Munich, Germany).

Generation of DC from AML and ALL Cells
Mononuclear cells containing AML blasts were cultured at 1 x 10⁶ cells/ml in 25 cm² flasks containing 10 ml serum-free medium (X-VIVO 20, Bio-Whittaker) at 37°C and 5% CO₂ for eight days. Four growth factor cocktails containing GM-CSF (Novartis; Nuernberg, Germany), tumor necrosis factor- (TNF-), FLT-3 ligand, stem cell factor (SCF), transforming growth factor-ß (TGF-ß) (R&D; Wiesbaden, Germany), interleukin 4 (IL-4), and IL-13 (Promocell, Heidelberg; Germany) as seen in Table 2.

To test for vitality, propidium iodide (PI) staining was performed in all experiments. Annexin (R&D; Wiesbaden, Germany) was used to determine the percentage of apoptotic cells. PI-/Annexin⁺ events defined early apoptosis whereas PI⁺/Annexin⁺ signals were counted as late apoptosis. PI⁺/Annexin^– cells are necrotic cells.

DC Purification after In Vitro Culture
Cell sorting of DC was performed after the culture period. Two selection methods were used: in four cases DC were separated with the help of a FACS VANTAGE (Becton Dickinson). All cells were labeled with a double color staining, using anti-CD14-FITC and anti-CD 1a-PE. Unspecific staining was removed, using the correction with the isotype controls IgG1-FITC/IgG1-PE and the compensation with CD14-FITC/IgG1-PE as well as CD1a-PE/IgG1-FITC. Sorted DC that were CD1a⁺/CD14^– were collected in a tube prefilled with PBS and 1% horse serum albumin.

Alternatively, DC were enriched using the MACS system according to the manufacturer's instructions. Briefly, cells were washed and resuspended in PBS, 0.5% bovine serum albumin and 5 mmol/l EDTA. Cells were first incubated with CD1a antibody (mouse antihuman CD1a, Pharmingen) in the presence of human IgG as a blocking reagent. After one cell wash, another incubation step with 100 µl CD14 microbeads (Miltenyi; Bergisch-Gladbach, Germany) per 10⁸ cells followed. Labeled cells were loaded onto a column installed in a magnetic field. Trapped cells were removed and the CD14^– fraction was used for further staining. These cells were incubated with 100 µl rat antimouse IgG1 microbeads per 10⁸ cells. In some experiments CD1a microbeads (Miltenyi) were used for direct marking. Labeled cells were loaded onto another column installed in a magnetic field. Trapped cells were eluted after removal of the column.

Genetic Analysis of Cells by FISH and PCR
To prove the clonal origin of the sorted DC, FISH for monosomy 7 was performed in three cases. Cells were sorted on slides. A commercial SO CEP 7 ( satellite) DNA probe was used together with a centromer-specific probe for chromosome 11 as a control (Vysis; Stuttgart, Germany). In the samples from ALL patients, interphase FISH for the BCR/ABL rearrangement was performed using an LSI bcr-abl dual color DNA probe (Vysis). The probes were used according to the manufacturer's instructions. At least 300 interphase nuclei were examined in each experiment.

In two patients with AML FAB M4eo, a sensitive nested RT-PCR for the fusion transcript CBFß-MYH1/ inversion 16 was performed in the sorted CD1a⁺/CD14^– cells as described before [12].

Longitudinal Analysis
In one patient whose blasts had shown to provide a significant number of DC in previous culture experiments, sequential phenotypic analysis was performed during the culture period. Therefore, flow cytometric measurements using the antibodies mentioned above were done daily for 10 days. Marker combinations were analyzed to specify subsets of DC. The ability to take up latex beads by enriched CD1a⁺/CD14^– cells was tested twice at days 2, 4 and 6 of the culture period.

Phagocytosis
To test the phagocytic activity, a 96-well-plate (flat bottom) was used (Nunc; Wiesbaden, Germany). 1 x 10⁶ latex beads (Sigma; Deisenhof, Germany, particle diameter 0.760 µ), resuspended in 50 µl X-VIVO 20, were placed in one well. Then, 1 x 10⁵ DC in 150 µl medium were added. The analysis was done in triplicate and compared with a positive control of monocytes from a normal donor which internalizes three or more beads in most cases.

Mixed Leukocyte Reactions
To access the ability of cultured DC derived from AML blasts of three patients to activate allogeneic CD4⁺ cord blood T lymphocytes, mixed leucocyte reactions were performed. In brief, sorted CD1a⁺/CD14^– DC generated from AML blasts and, as a control, freshly thawed AML blasts were isolated and irradiated (30 Gy from a ¹³⁷Cs source). These cells were used as stimulator cells and plated to 96-well flat-bottom microtiter plates (Corning; New York, NY) at 5 x 10⁴ cells/well. As responder cells 2 x 10⁵ allogeneic CD4⁺ cord blood T cells were added to each well. Lymphocyte proliferation was determined after five days of culture by uptake of ³H-thymidine (NEN; Zaventem, Belgium) which was added at a concentration of 1 µCi/well 16 h before harvesting. To generate the CD4⁺ responder cells, density-gradient centrifugation was performed with unmanipulated cord blood cells. The mononuclear fraction was incubated with 100 µl CD4 microbeads/10⁸ cells (Miltenyi). After two runs through the column, the trapped cells were eluted and suspended in PBS.

Statistics
Whenever applicable, the median or mean with standard deviation was calculated for descriptive purposes.

	Results

Top Abstract Introduction Patients And Methods Results Conclusion References

 
Cytokine Combinations
The cytokine combinations tested are summarized in Table 2. Each patient sample was split and supplemented with the different growth factor combinations. Figure 1 represents the means and standard deviations of 80 experiments with all four combinations. Although not statistically significant, the percentage of CD1a⁺/CD14^– cells and the coexpression of the costimulatory molecules CD80 and CD86 were highest with cocktail 4 containing SCF, TGF-ß, TNF-, GM-CSF, and FLT-3-ligand. The same is true for the expression of HLA-DR on CD1a⁺ cells. The variability between the different samples was significant as shown by the high values for standard deviations. There were no differences between the different cocktails concerning the percentage of apoptotic or necrotic cells after culture. No correlation with FAB classification was found.

View larger version (24K): [in this window] [in a new window]   Figure 1. Cytokine cocktails. The upper bar graph (A) shows the mean percentages of vital, apoptotic and necrotic cells after the culture period with different cytokine combinations obtained in 80 patient samples (Table 2). The other two graphs (B and C) show the mean percentages with standard deviation after eight days in culture for different phenotype markers. Cultures supplemented with DC cocktail 4 (GM-CSF, TNF-, FLT-3 ligand, TGF-ß, and SCF) contained the highest concentration of CD1a+/CD14– and CD1a+/CD80+ cells after culture.

The histograms of one representative experiment before and after the culture period with cocktail DC4 are shown in Figure 2. The CD34 expression decreases during the culture period. The antigens CD1a, CD86, CD80, and CDw123, which could not be found on the initial blast samples, are detected to some extent after culture. In this patient HLA-DR and CD83 expression increased only slightly. The median subset percentages with ranges of pre-and postculture analyses for all patients are provided in Table 3.

View larger version (30K): [in this window] [in a new window]   Figure 2. Phenotype. This graph shows the expression pattern pre-and post-culture of the antigens provided for one representative patient. Cytokine combination DC 4 was used for this culture experiment. The first two graphs across in the upper left represent the isotype controls for FITC and PE, respectively. The black transparent histogram shows the antigen expression before and the grey solid histogram after eight days of culture.

Functional and Morphological Analysis
Figure 3 shows the changes of antigen expression and cell number during the culture with cytokine-cocktail DC 4 in a patient whose blasts had been known to differentiate into DC. A significant decrease of CD34 and CD34⁺/CD38^– early leukemic progenitors was observed after an initial increase, whereas CD1a⁺ cells coexpressing CD80, 86 and 83 peaked between days 4 and 6. The cell count did increase almost fivefold.

View larger version (17K): [in this window] [in a new window]   Figure 3. Longitudinal analysis. The upper two graphs (A and B) provide the percentage of different DC subsets throughout the culture period in one representative culture experiment. The third graph (C) shows the cell count throughout the culture period. Whereas the percentage of CD34+ and CD34+/38– early leukemic progenitors declined after an initial increase, the percentage of CD1a+/CD80+ and CD1a+/CD86+ cells peaked between days 4 and 6 of the culture.

View larger version (8K): [in this window] [in a new window]   Figure 4. Induction of T cell responses. Induction of allogeneic CD4+ cord blood T cells by DC derived from AML blasts of three patients. To determine the proliferation, purified cord blood CD4+ T cells (2 x 105) were cultured with sorted CD1a+/CD14– DC as well as freshly thawed AML blasts (5 x 104). After five days, proliferation of T cells was assayed by thymidine uptake. Columns represent means of triplicate samples; bars indicate SE.

	Conclusion

Top Abstract Introduction Patients And Methods Results Conclusion References

Recent reports have shown that the same might become possible for patients with AML [8, 9, 15, 16]. Although the success of DC cultures varied from patient to patient, leukemic DC could be generated in two-thirds of the patients in our study. Using different flow cytometric analyses, other groups have found a higher percentage of so-called "DC." This might be due either to different culture conditions, different methods of flow cytometry or the heterogeneous patient population investigated.

Many reports exist on the optimum growth factor cocktails for the generation of DC out of CD34⁺-enriched progenitor cells [17, 18]. The same growth factors which have been found to provide the highest yield of DC have been included in our experiments, although the mean number of cells with coexpression of costimulatory molecules and typical DC markers seems somewhat lower [3]. These findings underline once more the common phenotypes and origin of normal and leukemic stem cells [19]. Nevertheless, a significant variability was observed with some blast populations behaving like normal progenitors and others with no potential for in vitro differentiation.

Although the CD1a⁺/CD14^– cells in the patient had differentiated out of CD34⁺ cells, they differed somewhat from their nonleukemic counterparts; hence they did not internalize latex beads. On the other hand, it has been shown that the phagocytic activity of DC is impaired by in vitro culture compared to freshly isolated cells [20, 21]. Since the antigens which have to be processed and presented to autologous or allogeneic effector cells should be integrated in the leukemic DC themselves, we hope they are potent stimulators of leukemia-specific responses. The stimulation of a primary naive T-cell response stresses the functional capacity of those cells. Induction of specific antileukemic T-cell activity by priming with leukemic DC was reported by other investigators [9]. We tried to reduce the influence of contaminating B cells and monocytes by performing fluorescence-activated or immunomagnetic enrichment of CD1a⁺/CD14^– DC to a purity of more than 80%. Thereby, we focused on the more immature compartment of the DC. On the other hand, CD1a^– DC might be of interest in terms of functional capacity as well.

The reason we could not induce differentiation of Ph⁺ ALL blasts into DC is not clear. One might argue that lymphoblastic B cells need different cytokines for dendritic differentiation or that different antigens might be expressed on those cells. Nevertheless, DC have been derived from CD10⁺ B cell precursors in healthy individuals [22].

The sorted fractions of five AML patients have provided evidence for the differentiation of leukemic stem cells into DC with different maturation grades. FISH analysis of unselected cultures performed by other groups might be clouded by the mixture of different cells in the samples [9].

In our series, T cells from patients at diagnosis or in remission were difficult to obtain because of low concentrations of CD3⁺ cells in the PB. On the other hand, the T cells obtained at diagnosis did not proliferate adequately in cultures supplemented with IL-2 or anti-CD3 antibodies. Like other groups, we therefore used naive CD4 cord blood cells to test for the potential of the leukemic DC to stimulate CD4 effector cells [16]. To assess the specificity of this activation, cytotoxicity assays using activated autologous T cells together with leukemic targets, as well as animal experiments, have to be performed.

As mentioned above, the clinical use of immunotherapy in AML patients is difficult because they suffer from profound lymphocytopenia, and autologous T cells are difficult to harvest during initial presentation or when in remission. Therefore, the priming of allogeneic T cells might be a potential alternative [23]. Besides leukemic DC, peptides derived and eluted from blasts by high performance liquid chromatography can be used together with allogeneic DC to prime specific responses [24]. These experiments are somehow complicated, and large amounts of leukemic blasts have to be available to elute enough peptides. Lysates of leukemic blasts have to be explored in this context for the induction of specific responses and feasibility in vivo.

Another drawback of immunotherapy in AML is the known downregulation of costimulatory signals also seen in our experiments. This might be overcome to some extent by differential cell cultures in vitro or by transfection of blasts with the genes for CD80 or CD86. First encouraging results have been documented with this approach [25]. Furthermore, the combination of this strategy with the use of allogeneic T cells specific for minor-histocompatibilty antigens appears promising [26, 27].

Our data give further insights into the biology of the AML hierarchy which resembles normal hematopoiesis. Whether the lack of leukemic DC after culture in some patients can be seen as an independent prognostic factor has to be studied with a longer follow-up. Ph⁺ ALL blasts behave differently and showed no significant growth of DC in our hands. Clinical studies using leukemic DC as adjuvant immunotherapy are warranted.

	Acknowledgments

 
The authors thank Uta Oelschlägel and Cornelia Brendel for the cell sorting. We are also grateful to Helga Ochmann for the preparation of the AML samples.

	References

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