Characterization of dendritic-like cells derived from t(9;22) acute lymphoblastic leukemia blasts

Jaewoo Lee1, Sheila N. Sait2 and Meir Wetzler1,3

1 Department of Immunology, 2 Clinical Cytogenetics Laboratory and 3 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

Correspondence to: M. Wetzler; E-mail: meir.wetzler{at}roswellpark.org


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We asked herein whether functional dendritic-like cells could be generated from t(9;22) acute lymphoblastic leukemia (ALL) blasts. We first determined that the combination of interleukin (IL)-1ß, IL-3, IL-7, tumor necrosis factor-{alpha}, stem cell factor and CD40 ligand was optimal for generating dendritic-like cells from t(9;22) ALL cell lines. Following 6 days in culture, four of five cell lines demonstrated dendrite-like morphology, upregulation of CCR7, CD54, CD80 and CD86, uptake of extracellular proteins and activation of T cells, and similar results were obtained with blasts from three t(9;22) ALL patients. The dendritic-like cells appeared to be composed of populations resembling both immature and mature dendritic cells (DCs), distinguished by CD80 expression. CD80–CD86+ cells were classified as immature DCs, demonstrating high endocytic capability and inducing minimal allogeneic T cell proliferation, while CD80+CD86+ cells were classified as mature DCs, exhibiting negligible endocytic capability and inducing robust allogeneic T cell proliferation. These mature dendritic-like cells induced autologous cytotoxic T cell responses against the unmodified blasts in a patient who achieved prolonged remission. In summary, CD80+CD86+ cells generated from t(9;22) ALL blasts may be useful in adoptive immunotherapy for t(9;22) ALL.

Keywords: dendritic-like cell, acute lymphoblastic leukemia, maturation, t(9;22), Philadelphia chromosome


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Acute lymphoblastic leukemia (ALL) with translocation (9;22) is a clonal malignancy originating from early hematopoietic progenitor cells. Adult ALL with t(9;22) has a B-lineage immunophenotype in the majority of cases (1). Most t(9;22) ALL patients achieve remission, but these remissions are characteristically short (26) unless patients undergo allogeneic stem cell transplantation (7,8). The availability and benefits of allogeneic transplantation are unfortunately limited by age restrictions, lack of availability of HLA-matched donors, and significant treatment-associated morbidity and mortality (9). Moreover, the introduction of imatinib mesylate (Gleevec®) as a single agent has not significantly improved outcome for patients with relapsed or refractory t(9;22) ALL (10,11). New treatment approaches are needed.

Immunotherapy with monoclonal antibodies specific for B-lineage ALL blasts has been investigated as a new treatment modality, but clinical outcomes have been disappointing (12). Adoptive immunotherapy using dendritic-like cells differentiated from leukemic blasts has been proposed as an alternative immunotherapy approach (1317). Four groups (16,1820) have reported success in generating dendritic-like cells from ALL blasts, while one group (21) reported lack of success. Cignetti et al. (16) and Mohty et al. (19) used interleukin (IL)-4 and CD40 ligand (L) to generate dendritic-like cells from ALL blasts, but a potential problem with this approach is that IL-4 is known to be cytotoxic to t(9;22) ALL blasts (22). Although Blair et al. (18) and Tsuchiya et al. (20) generated dendritic-like cells from ALL blasts using cytokine combinations containing granulocyte–macrophage colony stimulating factor (GM-CSF), B-lineage ALL blasts express GM-CSF receptor (R) in only one third of cases (23). Thus, the cytokine combinations described so far appear not to be optimal. Further, the leukemic origin of the dendritic-like cells was confirmed in only one study (16).

In the study reported here, we developed optimal culture conditions to generate dendritic-like cells from t(9;22) ALL cell lines, and then tested those conditions on primary blasts. We confirmed the leukemic nature of the dendritic-like cells and characterized them with respect to morphology, immunophenotype, endocytosis, T cell stimulation, cytotoxic T lymphocyte (CTL) response and maturation stage.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and culture condition
Five human t(9;22) ALL cell lines (24) were studied, including ALL-1 (kindly provided by Dr G. Rovera, Wistar Institute, Philadelphia, PA), Z33, Z119 and Z181 (kindly provided by Dr Z. Estrov, M. D. Anderson Cancer Center, Houston, TX), and OM9;22 (kindly provided by Dr K. Ohyashiki, Tokyo Medical University, Tokyo, Japan). Cryopreserved bone marrow samples from three newly diagnosed t(9;22) ALL patients (each containing >80% blasts) and peripheral blood mononuclear cells (PBMC) from eight normal volunteers were also studied.

ALL-1, Z33, Z119 cells and bone marrow samples from the three patients were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml) (all from Life Technology, Grand Island, NY). OM9;22 cells were cultured in the same conditions as the other cell lines but in the presence of 15% FBS. Z181 cells were cultured in MEM-{alpha} medium (Life Technology) with 10% FBS, L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 µg/ml). Cells were continuously incubated at 37°C in a humidified atmosphere with 5% CO2.

The study was approved by the Roswell Park Cancer Institute (RPCI) Scientific Review Committee and Institutional Review Board.

Cytokines
Cytokines used in the study included CD40L (500 ng/ml), GM-CSF (80 ng/ml) (both kindly provided by Immunex, Seattle, WA), human recombinant IL-1ß (0.2 ng/ml), IL-3 (400 ng/ml), IL-4 (20 ng/ml), IL-7 (10 ng/ml), tumor necrosis factor (TNF)-{alpha} (1 ng/ml), stem cell factor (SCF) (10 ng/ml) (all obtained from RD, Minneapolis, MN) and IL-2 (10 U/ml) (Sigma, Saint Louis, MO).

Generation of DC
ALL-derived dendritic-like cells
To generate dendritic-like cells, t(9;22) ALL blasts were cultured at 1.5 x 106 cells/ml in one of four cytokine combinations. These combinations were selected based on our review of the literature as well as on cytokine receptor expression on the different cell lines. The first two cytokine combinations have been reported to generate dendritic-like cells from human B-lineage ALL blasts (16) and murine pro-B cells (25). In Combination III, we tested whether CD40L enhanced the efficacy of Combination II based on data showing a role for CD40L in human lymphoid DC maturation (26). Combination IV is the standard combination used for generating myeloid/monocytic-lineage DCs from human bone marrow cells; we tested this combination because B-lineage ALL blasts co-express myeloid antigens in approximately one third of cases (27) and some ALL cells express the GM-CSFR (23). Medium with or without an additional cytokine mixture was replaced every 3 days. On days 2, 4 and 6, aliquots of cells were removed, treated with ethylenediaminetetraacetic acid (EDTA; 5 mM) (Sigma) to prevent clumping (28) and studied for morphology, immunophenotype and function. Cells from the five cell lines did not show any dendritic-like changes during culture without cytokines, and served as controls for cytokine-treated cells. Because thawed primary blasts did not survive in culture without cytokines, primary leukemic blasts were used as controls.

PBMC-DCs
Human myeloid/monocytic DCs were used as normal DC controls; they were generated from PBMCs as previously described (29,30) with modifications. Briefly, PBMCs from normal volunteers were cultured for 9 days with GM-CSF and IL-4 to generate immature DCs and were further cultured for two additional days with TNF-{alpha} to induce maturation.

Morphologic analysis
Cytospins were prepared on days 2, 4 and 6. Following fixation with methanol, cells were stained with Giemsa May–Grünwald (Sigma). Stained cells were analyzed by light microscopy (Nikon Inc., Melville, NY).

Antibodies
Monoclonal antibodies (mAb) to the following antigens directly labeled with phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC), tri-color (TC), peridinin chlorophyll protein (PerCP) or Cychrome (Cy) were used for flow cytometric analysis: CD10 (5-1B4), CD19 (SJ25-C1), CD22 (RFB4) and CD83 (HB15e) (all from Caltag, Burlingame, CA); CD13 (WM15), CD33 (HIM3-4), CD40 (5C3), CD58 (1C3), CD80 (L307.4), CD86 (FUN-1) and CCR7 (2H4) (all from BD Pharmingen, San Diego, CA); CD11b (ICRF44), CD11c (B-ly6), CD20 (2H7), CD34 (581), CCR5 (2D7/CCR5) and HLA-DR (G46-6) (all from BD Biosciences, San Jose, CA); CD54 (84H10) (Immunotech, Marseille, France); and IL-7R (40131.111) (RD). mAbs to the following antigens were conjugated with biotin: GM-CSFR (BVD2-21C11), IL-3R{alpha} (7G3) and TNF-{alpha}R (MABTNFR1-B1) (all from BD Pharmingen); and c-kit (104D2) (Caltag). APC- or PerCP-conjugated streptavidins (BD Bioscience) were used for labeling biotin-conjugated antibodies. mAbs against IL-4R (hIL4R-M57) (BD Pharmingen), HLA class I (TP25.99.8.4), human ß2-microglobulin (ß2m) (SJJ-6) (kindly provided by Dr Soldano Ferrone, RPCI, Buffalo, NY) and IgM heavy chain (G20-127) (BD Pharmingen) were used as primary antibodies. FITC- or PE-conjugated anti-mouse IgG (Caltag) and biotin-conjugated anti-mouse IgM (BD Pharmingen) were used as secondary antibodies. FITC-conjugated mouse IgG1, TC-conjugated mouse IgG2a, PE-conjugated mouse IgG2b and APC-conjugated mouse IgG1 (all from Caltag) were used as isotype controls.

Flow cytometric analysis
Before treatment and on days 4 and 6 following cytokine treatment, cells were stained with antibody panels and fixed with 2% ultrapure formaldehyde (Polyscience Inc., Warington, PA). Data were evaluated on a FACSCalibur (BD Bioscience). To ensure analysis of viable cells, all cells were stained with ethidium monoazide (Sigma) and viable cells were determined by ethidium monoazide labeling. All samples had at least 80% viable cells in the regions gated by forward and side scatters and were thus appropriate for analysis.

Cell sorting
To enrich for mature dendritic-like cells, cells cultured for 6 days were sorted based on CD80 and CD86 expression with a FACSVantageTM (BD, San Jose, CA). CD80–CD86+ cells were defined as immature, and CD80+CD86+ cells as mature, dendritic-like cells.

Cell proliferation and viability
The trypan blue dye (Life Technology) exclusion assay was used to study viability. In addition, apoptosis was analyzed with the annexin-V–Fluos staining kit (Roche, Indianapolis, IN) following the manufacturer's instructions; cells were evaluated on the FACSCalibur.

IL-12 concentration in conditioned media
To measure IL-12 production, untreated, sorted immature and mature cells (1.5 x 106 cells/ml) were cultured for 2 days. Supernatants were collected and stored at –80°C for later analyses. Triplicate samples were tested for the production of IL-12 (p70) using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (RD). Analyses were performed in accordance with the manufacturer's instructions. The assays were read in a microplate reader (MTX lab systems, Vienna, VA). The lowest limit of the ELISA was defined as two standard deviations (SD) above the mean optical density of 10 replicates of zero standard, according to the manufacturer's instructions.

Fluid-phase endocytosis
The fluid-phase endocytosis assay was performed as previously described (31), with modifications. Briefly, 1 mg/ml FITC–dextran or rhodamine–dextran (MW 40 000) (Molecular Probes, Eugene, OR) was added to 1 x 106 cells harvested on days 4 and 6 of culture with cytokines. Cells were incubated for either 30 min, 1 h or 1.5 h at 37°C in a humidified atmosphere with 5% CO2. The best results were obtained at 1 h and this incubation period was therefore used in subsequent experiments. Signals were analyzed on a FACSCalibur after washing four times with cold phosphate-buffered saline (PBS) with 0.1% sodium azide, 1 mM EDTA and 1% FBS. Cells incubated with the endocytic tracer at 0°C were used as background controls.

Confocal microscopy
To verify intracellular localization of the endocytic tracers, cells incubated with rhodamine-conjugated dextrans were washed with PBS with 0.1% sodium azide and 1% FBS, fixed with 2% ultrapure formaldehyde, and then incubated with FITC-conjugated mouse anti-HLA–DR antibodies to outline the cell membrane. Ten thousand cells were cytospun onto glass slides and signals were observed with the Leica TCS SP2 Spectral Confocal Microscope (Leica Microsystems Inc., Exton, PA). The images were analyzed using the Leica Confocal Software Version 2.00 (Leica Microsystems Inc.).

Allogeneic-mixed lymphocyte reaction (allo-MLR)
Irradiated (30 Gy) dendritic-like cells from ALL blasts were used as stimulators and T cells isolated from normal PBMCs using nylon-wool column (Polysciences Inc) (32) were used as responders. Stimulator cells at different concentrations (1:1, 1:10 and 1:100) were added to 1 x 104 or 1 x 105 responder cells/well in 96-well flat bottom microtiter plates in triplicate in a final volume of 200 µl of complete medium and co-cultured for 72 h at 37°C in a humidified atmosphere with 5% CO2. Controls for stimulator cells consisted of untreated ALL blasts and PBMC–DCs generated from normal PBMCs. T cells stimulated with 5 µg/ml concanavalin A (Con A) (Sigma) were used as positive controls for responder cells (33). Cells were pulsed with 1 µCi [3H]thymidine (Amersham Pharmacia, Piscataway, NJ) for the last 18 h of incubation and the incorporated radioactivity was measured with a Wallac Oy 1450 microbeta counter (Wallac Oy, Turku, Finland).

Induction of autologous CTL
Irradiated dendritic-like cells from ALL blasts were used to stimulate autologous T cells obtained in remission. The assay was performed as follows: CD3+ T cells from a t(9;22) ALL patient in complete remission were isolated using the pan T cell isolation kit (obtained from Miltenyi Biotec, Auburn, CA). A total of 4 x 106 isolated CD3+ T cells were cocultured for 5 days with or without the same amount of irradiated dendritic-like cells generated from the diagnostic bone marrow sample of the same patient in the presence of human recombinant IL-2.

CTL assay
The 51Cr release assay was used to monitor the cytotoxic potential of the in vitro-stimulated lymphocytes, used as effector cells. Specifically, on day 5, CD8+ T cells were isolated from the in vitro-stimulated CD3+ lymphocytes, using a CD8+ T cell isolation kit (Miltenyi Biotec). These stimulated CD8+ T cells were used as effector cells in the CTL assays. The unmodified diagnostic bone marrow samples from the same patient were used as target cells. In addition, bone marrow samples from the same patient in complete remission were used as normal target cell controls. A total of 2 x 106 target cells were pre-labeled with 1 mCi/ml 51Cr (Amersham Pharmacia) for 2 h at 37°C. Labeled target cells (2 x 104 cells/well) were co-cultured with effector cells at various effector: target ratios (5:1, 10:1, 20:1) for 6 h at 37°C in 96-well U-bottom plates in triplicates. Pre-labeled target cells cultured without effector cells were used to determine spontaneous 51Cr release. In addition, pre-labeled target cells incubated with 2% Triton X-100 (Bio-Rad, Hercules, CA) in dH2O were prepared to determine maximum 51Cr release. Radioactivity was measured with a Packard Cobra {gamma} counter (GMI, Albertville, MN). Percentage lysis was determined for each triplicate experiment as [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x100.

Fluorescence in situ hybridization (FISH)
To verify the leukemic origin of the dendritic-like cells generated from primary ALL blasts, cells were examined for the presence of the BCR/ABL fusion gene by FISH (34). A total of 1 x 104 CD80+CD86+ dendritic-like cells sorted by FACS were cytospun onto glass slides and fixed in methanol. Fixed cells were hybridized with commercially available dual-color BCR/ABL DNA probes (Vysis Inc., Downers Grove, IL). Images were captured using a monochrome CCD camera (Cohu Inc., San Diego, CA) and analyzed using image analysis software (MacProbe, Perceptive Scientific Instruments, Houston, TX). Signals were visualized with a Nikon Optiphot-II epifluorescence microscope (Nikon Inc.) with appropriate filters. Non-malignant cells were expected to display two red (BCR gene) and two green (ABL gene) hybridization signals, randomly distributed. Dendritic-like cells from leukemic blasts were expected to display a yellow signal (BCR/ABL fusion gene) in addition to one red (normal BCR gene) and one green (normal ABL gene) signal.

Western blot
Bcr/Abl fusion proteins were detected by western blot analysis as previously described (35). Briefly, whole-cell extracts were separated on 8% polyacrylamide sodium dodecyl sulfate gels and proteins were transferred to nitrocellulose membranes. Membranes were incubated with rabbit polyclonal anti-human Bcr (N-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and then with anti-rabbit horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia). Immune complexes were detected using a chemoluminescent detection method (Amersham Pharmacia).

Statistical analysis
Statistical analysis was performed by the Student's paired two-tailed t-test using Microsoft Excel 98.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Optimization of in vitro culture conditions to generate dendritic-like cells from B-lineage t(9;22) ALL cell lines
We tested the efficacy of four cytokine combinations (Table 1) in generating dendritic-like cells from five human t(9;22) ALL cell lines, all expressing p190BCR/ABL. The immunophenotypes and cytokine receptor profiles of the ALL cell lines are summarized in Table 2. On the basis of receptor expression profiles, we tested Combination I (IL-4, CD40L) in all cell lines, Combinations II (IL-1ß, IL-3, IL-7, TNF-{alpha}, SCF) and III (IL-1ß, IL-3, IL-7, TNF-{alpha}, SCF, CD40L) in all except Z33, and Combination IV (GM-CSF, IL-4, TNF-{alpha}) only in Z33 and Z181. The efficacy of the cytokine combinations was tested by morphologic and immunophenotypic analyses.


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Table 1. Cytokine combinations used in this study

 

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Table 2. Immunophenotypes and cytokine receptor profiles of t(9;22) ALL blastsa

 
Cytokine combinations induce morphologic changes in t(9;22) ALL blasts
To determine the ability of the cytokine combinations to generate dendritic-like cells from ALL blasts, we studied cell morphology after treatment with the different cytokine combinations. On day 3 or 4 of cytokine treatment, a large number of cells had ruffle-like membrane structures and a small number of cells developed dendrite-like projections on their surface. On day 6, more cells demonstrated dendrite-like projections (Fig. 1). The OM9;22 cell line responded to Combinations II and III, and the Z181 cell line only responded to Combination III, while the Z119 cell line, like the ALL-1 cell line, responded to Combinations I, II and III. Interestingly, ALL-1, OM9;22 and Z119 exhibited cell clumps of various sizes following cytokine treatments (data not shown). EDTA was used to reduce clumping in subsequent studies. The Z33 cell line did not respond to any cytokine combination. Further, Combination IV (tested in the Z33 and Z181 cell lines) did not induce any morphological changes.



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Fig. 1. Upregulation of CD80 and CD86 on cells treated with Combinations I-III. (A) Untreated cells and cells treated for 6 days with one of four different cytokine combinations were stained with PE–CD86 and APC–CD80 antibodies and the signals were analyzed by flow cytometry. Four of four t(9;22) ALL cell lines treated with Combinations I–III showed upregulation of CD80 and CD86. The Z33 cell line did not respond to any of the cytokine combinations. Combination IV did not induce any significant change in the two cell lines tested, Z33 and Z181. The values are mean percent stained cells from three independent experiments with standard deviation (SD). (B) OM9;22 cell morphology was studied in cytospin preparations and immunophenotype was studied by flow cytometry before treatment and on days 4 and 6 following culture with Combination III.

 
Combination III is most effective in upregulating costimulatory molecules
Upregulation of costimulatory molecules has been used to broadly define mature DCs. Therefore, we used the expression of CD80 and CD86 to test the efficacy of cytokine combinations in generating dendritic-like cells from ALL blasts (Fig. 1A; Table 3). Combination IV (tested in the Z33 and Z181 cell lines) did not alter expression of CD80 and CD86 on any cell line. Combination III upregulated both CD80 and CD86 expression significantly on all responding cell lines except Z181 (P < 0.05; n = 3), and this combination significantly upregulated CD80 (P = 0.01; n = 3), but not CD86, on Z181 cells. Combination I significantly increased both CD80 and CD86 expression on Z119 cells (P < 0.01; n = 3), but only increased CD86 expression on ALL-1 cells (P = 0.02; n = 3). Combination II induced upregulation of both CD80 and CD86 significantly on OM9;22 and Z181 cells (P < 0.05; n = 3), but only increased CD86 expression on ALL-1 and Z119 cells (P < 0.05; n = 3). Interestingly, upregulation of CD80 and CD86 correlated with maturation of the dendritic-like cells. On day 4, when the cells exhibited only initial dendritic-like morphological changes, CD80 expression was minimal, while CD86 was maximally upregulated (Fig. 1B). On day 6, when the cells exhibited dendrite-like morphology, both CD80 and CD86 were maximally upregulated (Fig. 1B). This suggested that CD80 was a marker of maturation of ALL-derived dendritic-like cells.


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Table 3. Upregulation of CD80 and CD86 on t(9;22) ALL cell lines exposed to Combinations I–IV

 
t(9;22) ALL blast-derived dendritic-like cells exhibit decreased proliferation
Differentiation of ALL blasts into dendritic-like cells was associated with decreased proliferation. These results were concordant with the morphologic and immunophenotypic changes induced in these cell lines by the cytokine combinations tested. For example, treatment with Combinations II and III reduced proliferation of OM9;22 cells, while Combination I, which caused neither morphologic nor immunophenotypic dendritic-like changes, did not affect the proliferation of this cell line (Fig. 2A). Annexin V and propidium iodine (PI) staining demonstrated both apoptosis and necrosis during culture with the cytokine combinations (Fig. 2B). Similarly, proliferation of ALL-1, Z119 and Z181 decreased after treatment with all combinations tested (data not shown). The viability of all responding cell lines decreased after 3 days of culture and was <30% on day 8 (data not shown). Proliferation of Z33 cells did not change after exposure to any combination tested.



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Fig. 2. Cell proliferation and death in the OM9;22 cell line after treatment with cytokine combinations. The ALL cell line OM9;22 was cultured without (control) or with Combinations I–III. Cells were counted following staining with Trypan Blue. (A) Cell numbers were measured on the indicated days. Cells treated with Combinations II and III did not proliferate and cells treated with Combination I showed a similar growth rate to untreated controls. Data shown are representative of three individual experiments. (B) Cells were studied to determine whether reduction in cell proliferation was related to apoptosis or necrosis. OM9;22 cells treated with Combination III were stained with FITC-conjugated annexin V and PI on the indicated days and the signals were evaluated by FACS. Increases in both apoptosis and necrosis were seen.

 
On the basis of morphology and CD80 and CD86 expression, Combination III was the most effective combination in generating dendritic-like cells from t(9;22) ALL cell lines. Four of the five ALL cell lines responded to this cytokine combination. We therefore focused on Combination III in subsequent studies of the t(9;22) ALL cell line (OM9;22) and of patient samples. We chose the OM9;22 cell line for the subsequent studies because it responded best to Combination III. Moreover, based on the initial viability results with cell lines, cells were treated for 6 days and harvested on day 6 in subsequent studies. The viability on this day was >50% (data not shown).

Patient samples demonstrate dendritic-like morphology following exposure to Combination III
Bone marrow samples from three patients with t(9;22) ALL, all expressing p190BCR/ABL, developed dendritic-like morphology, similar to the cell lines, after exposure to Combination III (Fig. 3). FISH analysis showed that the dendritic-like cells from t(9;22) ALL patient samples demonstrated BCR/ABL fusion signals, confirming their leukemic origin (Fig. 3C). To enrich for mature (CD80+CD86+) dendritic-like cells, a bone marrow sample from a patient with t(9;22) ALL was cultured for 6 days with Combination III and then sorted. Ninety-one percent of the sorted cells expressed both CD80 and CD86, and 84% demonstrated BCR/ABL fusion signal by FISH.



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Fig. 3. Dendritic-like cells demonstrating BCR-ABL rearrangement generated from primary ALL blasts exposed to Combination III. Primary blasts from t(9;22) ALL patients demonstrated morphological changes similar to those in the ALL cell lines following incubation without (A) or with (B) Combination III. These dendritic-like cells from ALL patients demonstrated BCR-ABL rearrangement: FISH analysis showed that CD80+CD86+ dendritic-like cells derived from primary blasts had co-localization of the BCR (red) and ABL (green) genes (C), resulting in two yellow signals, reflecting the presence of t(9;22) and +der(22)t(9;22) in patient 1.

 
IL-12 production
We analyzed IL-12 production by untreated, immature and mature dendritic-like cells from the four responding cell lines and from primary blasts from two patients (patients 1 and 2). Samples from the other patients were unavailable. The lowest level of detection was 15.6 pg/ml. Untreated and immature dendritic-like cells did not produce IL-12. IL-12 was detected only in conditioned media from mature dendritic-like cells derived from Z119 (36.8 pg/ml) and from primary blasts from patient 2 (17.8 pg/ml).

Concomitant upregulation of CCR-7, CD54, CD80 and CD86 on dendritic-like cells
The cells did not express CCR5, which is known to be expressed on immature myeloid/monocytic DCs (36). Further, CD83 expression was low or absent (37). HLA–DR, CD11c, CD40, CD58 and IL-3R{alpha} were expressed on pretreatment ALL blasts and their expression did not change significantly (data not shown). Significant upregulation of both CCR7 and CD54 along with CD80 and CD86 was seen following 6 days of incubation with Combination III (Fig. 4). Interestingly, CD19, which was highly expressed on pretreatment ALL blasts, was not significantly downregulated following incubation with Combination III. Even though these data might suggest that Combination III induced differentiation into mature B cells, surface and cytosolic IgM heavy chain (µ) were not detected on OM9;22 cells before or after cytokine treatment (data not shown). These data suggest that the differentiation process did not induce B cell maturation. To evaluate differences in immunophenotype between immature (CD80–CD86+) and mature (CD80+CD86+) dendritic-like cells, cells were sorted according to CD80/CD86 expression. Immature (CD80–CD86+) dendritic-like cells expressed CD54 but not CCR7, while mature (CD80+CD86+) dendritic-like cells expressed both CD54 and CCR7 (Fig. 5).



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Fig. 4. Upregulation of CCR7, CD54 as well as co-stimulatory molecules on dendritic-like cells derived from ALL blasts. (A) The ALL cell line, OM9;22, cultured with Combination III demonstrated upregulation of the secondary chemokine receptor CCR7, the adhesion molecule CD54 and the co-stimulatory molecules CD80 and CD86. In addition, these dendritic-like cells upregulated HLA-DR, but not CD83 or CCR5. (B) Similar immunophenotypic changes appeared in primary ALL blasts from both patients with t(9;22) ALL. Data shown are from patient 1. The light gray lines represent the isotype controls and the bold black lines represent the antibodies tested.

 


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Fig. 5. Differences in immunophenotype between immature (CD80–CD86+) and mature (CD80+CD86+) dendritic-like cells generated from the OM9;22 cell line. OM9;22 cells were cultured with Combination III for 6 days. Based on CD80/CD86 expression, two populations, CD80–CD86+ (R2) and CD80+CD86+ (R3), were apparent on day 6 (A). These populations were sorted by FACS and were further analyzed after staining with the PE–CCR7, APC–CD54, FITC–CD80 and Cy–CD86 antibodies. Cells in region R2 expressed CD54 and CD86 (B) while cells in region R3 expressed CCR7 and CD80 as well as CD54 and CD86 (C). The light gray lines represent the isotype controls and the bold black lines represent the antibodies tested.

 
Fluid-phase endocytosis
The functions of immature DCs include endocytosis and antigen processing; both decrease during maturation (36,38). Untreated OM9;22 cells did not demonstrate endocytic function, but significant dextran internalization, indicative of endocytosis, was detected following 1 and 1.5 h of incubation with Combination III (data not shown). Therefore, all subsequent experiments were conducted with 1 h incubations (Fig. 6A). No endocytosis was detected following incubation with Combination I, which did not induce morphologic or immunophenotypic dendritic-like changes in this cell line (Fig. 6A). Primary blasts treated with Combination III also demonstrated increased dextran uptake (Fig. 6B). To evaluate the ability of immature versus mature dendritic-like cells to internalize antigens, cells were sorted according to CD80/CD86 expression. Immature (CD80–CD86+) dendritic-like cells showed intracellular dextran uptake (Fig. 6D–F), but mature (CD80+CD86+) dendritic-like cells did not internalize dextran (Fig. 6H–J), supporting the concept that endocytosis decreases during dendritic-like maturation. In addition, CD80–CD86+ cells did not have dendrite-like morphology (Fig. 6C), whereas this morphology was highly evident in CD80+CD86+ cells (Fig. 6G).



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Fig. 6. Morphology and fluid-phase endocytosis by immature (CD80–CD86+) and mature (CD80+CD86+) dendritic-like cells derived from OM9;22. The OM9;22 cell line (A) and primary ALL blasts (B) untreated (gray line) or cultured for 6 days with Combinations I (black line) and III (blue line) were incubated with 1 mg/ml FITC–dextran in RPMI 1640 supplemented with 10% FBS and penicillin–streptomycin for 1 h at 37°C in a humidified atmosphere of 5% CO2. FITC–dextran (MW 40 000) uptake was measured by flow cytometry. Cells treated with Combination III, but not with Combination I, demonstrated increased endocytosis compared to untreated control. To study antigen uptake by immature and mature dendritic-like cells, OM9;22 cells were cultured for 6 days with Combination III. On day 6, dendritic-like cells were sorted by FACS according to CD80/CD86 expression (C–J). For morphological analysis, sorted CD80–CD86+ (C) and CD80+CD86+ (G) cells were stained with Giemsa–May Grünwald. For the endocytosis assay, CD80–CD86+ (D–F) and CD80+CD86+ (H–J) cells were incubated with rhodamine–dextran (MW 40 000) (red) and stained with FITC-conjugated anti-HLA–DR antibodies (green) to outline the cell membrane. Signals were analyzed by confocal microscopy.

 
Allo-MLR
Dendritic-like cells from the OM9;22 cell line and from primary blasts from three t(9;22) ALL patient samples treated with Combination III induced a 4- to 10-fold increase in allogeneic T cell proliferation, compared to untreated cells (P < 0.005; n = 3) (Fig. 7A). The activity of dendritic-like cells generated from the OM9;22 cell line was similar to that of myeloid/monocytic DC controls (P = 0.13; n = 3), but the activity of dendritic-like cells generated from primary blasts was ~30% greater than that of myeloid/monocytic DC controls (P < 0.05; n = 3) (Fig. 7A). Immature (CD80–CD86+) dendritic-like cells did not induce allogeneic T cell proliferation in comparison to untreated cells (P = 0.29; n = 3), but mature (CD80+CD86+) dendritic-like cells induced a greater than 4-fold increase in allogeneic T cell proliferation (P = 0.02; n = 3) (Fig. 7B).



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Fig. 7. Allo-MLR response of dendritic-like cells derived from t(9;22) ALL blasts. (A) OM9;22 cells and primary blasts from patient 1 cultured for 6 days with Combination III. These cells (stimulators) were {gamma}-irradiated (30 Gy) and co-cultured for 72 h with 1 x 105 T cells from normal volunteers (responder cells) in the indicated ratios. Cells were pulsed with 1 µCi [3H]thymidine for the last 18 h of incubation and the incorporated radioactivity was measured. PBMC–DCs were used as a normal DC control. Medium and responder cells alone were used as background controls and responder cells treated with Con A were used as positive controls. The results are presented as the mean of triplicate wells with SD from one representative experiment. (B) Sorted immature (CD80–CD86+) and mature (CD80+CD86+) cells used as stimulator cells were co-cultured with 1 x 104 responder cells. Only CD80+CD86+ cells induced significant allogeneic T cell proliferation, compared with untreated cells (P = 0.02; n = 3).

 
Autologous anti-leukemic CTL activity by dendritic-like cells derived from t(9;22) ALL blasts
Only one patient achieved long-term (>6 months) disease-free survival in the absence of allogeneic transplantation. Autologous CD8+ T cells from this patient, stimulated with dendritic-like cells, were cytotoxic against the diagnostic unmodified leukemic blasts (Fig. 8A), while the same CD8+ T cells were not significantly cytotoxic against autologous bone marrow cells obtained in compete remission (Fig. 8B). This difference was consistent at different effector:target ratios. These data suggest that dendritic-like cells generated from t(9;22) ALL blasts using Combination III can induce an anti-leukemia cellular immune response.



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Fig. 8. CTL response. Effector cells were autologous CD3+ T cells (5 x 106) cultured for 5 days with either 5 x 106 irradiated dendritic-like cells or unmodified blasts from patient 3. On day 5, CD8+ T cells were isolated from the stimulated CD3+ T cells to be used as effector cells. Autologous unmodified leukemic blasts were used as target cells. (A) Cytotoxicity of triplicate cultures obtained at different effector–target ratios. (B) Control experiment with bone marrow cells obtained in complete remission as target cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We tested the efficacy of four different cytokine combinations in generating dendritic-like cells from t(9;22) ALL blasts. The combination of IL-1ß, IL-3, IL-7, SCF, TNF-{alpha} and CD40L (Combination III) was the most effective combination based on induction of changes in morphology and expression of the costimulatory molecules CD80 and CD86. Dendritic-like cells derived from t(9;22) ALL blasts upregulated CCR7 and CD54 along with the costimulatory molecules and internalized extracellular molecules. In addition, these cells induced allogeneic T cell stimulation and anti-leukemia CTL response. Finally, our work strongly suggests that functional changes, including decreased capacity for endocytosis and increased stimulation of T cell proliferation, correlate with maturation of ALL-derived dendritic-like cells, as reflected, at least in part, by CD80 upregulation.

Four of five t(9;22) ALL cell lines and primary blasts from three patients with t(9;22) ALL differentiated into dendritic-like cells in culture with Combination III. However, one cell line, Z33, did not respond to any cytokine combination. This cell line was the only one that did not express the stem cell antigen, CD34, suggesting a more differentiated stage. Further, GM-CSF did not significantly affect the differentiation of t(9;22) ALL cells into dendritic-like cells despite expression of its receptor. Even though GM-CSF is known to be a critical growth factor for generation of myeloid/monocytic DCs, growth and differentiation of non-leukemic (39) and leukemic (current work) lymphoid DCs are independent of GM-CSF. Generation of lymphoid DCs requires different culture conditions than generation of myeloid/monocytic DCs.

Previous work on ALL-derived dendritic-like cells included only bone marrow samples, not cell lines (16,1821), and the malignant origin of the dendritic-like cells was demonstrated in only one study (16). This raised concern that the dendritic-like cells might have originated from non-malignant populations. In the current work, the initial studies were performed on t(9;22) ALL cell lines, excluding the possibility of contamination with non-malignant cells. Further, analysis of bone marrow samples from a t(9;22) ALL patient demonstrated that 84% of the dendritic-like cells were from the malignant clone. Interestingly, the dendritic-like cells derived from t(9;22) ALL blasts did not downregulate the B-lineage marker, CD19, suggesting that these dendritic-like cells were differentiating into mature B cells. However, cytosolic and surface IgM, which are markers that are known to be upregulated upon B cell differentiation (40), were not expressed in any of the t(9;22) ALL blasts or in the derived dendritic-like cells. Therefore, we conclude that the ALL blasts tested in this study did not differentiate into mature B cells. B cell plasticity, i.e. the ability to switch lineages to either DCs (1321,25,41) or macrophages (42), has been described before. Known stimuli for this switch including cytokines (1321,25; this work), expression of oncogenes (42) or other stimuli remain to be characterized (41). The immunophenotypic and functional differences between immature and mature non-malignant myeloid/monocytic DCs have been extensively studied (43,44), but little is known about lymphoid DCs (39). In the current study, the ALL-derived dendritic-like cells obtained following 4 days of culture had ruffle-like membrane structures, while only a few cells had dendrite-like projections on their membranes. Membrane ruffles are specific for fluid-phase endocytosis and therefore characterize immature DCs (43). The cells obtained following 6 days of culture had morphology similar to that of mature myeloid/monocytic DCs. These data suggest that ALL blasts differentiate into dendritic-like cells in at least two developmental stages, as is the case with myeloid/monocytic DCs.

The immunophenotypes of immature and mature myeloid/monocytic non-malignant DCs are well characterized (36), but, to the best of our knowledge, the immunophenotype of immature lymphoid DCs has not been previously described. Mature non-malignant lymphoid DCs express CD80, CD86, MHC class II antigens and IL-3R, but lack CD83 (36,37). Our work demonstrates that morphologically immature ALL-derived dendritic-like cells do not express CCR7 or CD80, but express CD54 and CD86, while morphologically mature ALL-derived dendritic-like cells express all of these markers. This is the first demonstration of immunophenotypic differences between immature and mature ALL-derived dendritic-like cells.

Immature leukemic myeloid/monocytic DCs demonstrate a high endocytic capability that decreases steadily during their maturation (45). In the current study, immature (CD80–CD86+) ALL-derived dendritic-like cells demonstrated increased uptake of dextran molecules, while mature (CD80+CD86+) ALL-derived dendritic-like cells exhibited only minimal internalization of these molecules. These data clearly confirm that CD80–CD86+ ALL-derived dendritic-like cells are immature DCs, while CD80+CD86+ cells are more mature cells.

The most important function of DCs in the immune system is stimulation of primary and secondary T cells by mature DCs (36). Depending on their maturity, lineage and maturation agents, DCs can polarize different types of T cells, i.e. TH1/TH2/regulatory T (TR) cells, which are related to cellular, humoral and immune regulatory responses, respectively (4648). These functional differences can categorize DCs into three subtypes: type 1 (DC1), type 2 (DC2) and type 3 (tolerogenic) (DC3). DC1 secretes IL-12, expresses costimulatory molecules and induces a TH1-polarized response, while DC2 secretes no or little IL-12, expresses costimulatory molecules and induces a TH2-polarized response (46). DC3 does not secrete IL-12, has no or minimal expression of costimulatory molecules and probably activates TR cells or induces antigen-specific tolerance (46,48). Mature human myeloid/monocytic DCs were previously categorized as DC1 and mature lymphoid DCs as DC2 or DC3 (49). Interestingly, t(9;22) ALL-derived dendritic-like cells may not represent normal lymphoid DCs because the ALL-derived dendritic-like cells induced a leukemia-specific CTL response, in spite of low IL-12 production, suggesting that ALL-derived dendritic-like cells could be categorized as DC1.

CTL responses could occur only if the T cells recognize an antigen on the leukemia-derived dendritic-like cells. The Bcr/Abl fusion protein is present only in leukemic cells and therefore presents a unique target to the T cells. Indeed, several groups have generated Bcr/Abl-specific CTL lines against fusion-region antigens (5052). We do not know if ALL-derived dendritic-like cells indeed present fusion-region antigens or novel antigens not yet recognized. Generating CTL clones against ALL-derived dendritic-like cells may help to identify the antigenic epitopes.

In summary, in the current study we developed a cytokine combination to generate dendritic-like cells from t(9;22) ALL blasts and showed that CD80–CD86+ and CD80+CD86+ dendritic-like cells had the properties of immature and mature DCs, respectively. These t(9;22) ALL-derived dendritic-like cells induced a leukemia-specific CTL response and therefore may be useful in the development of autologous immunotherapy approaches in this disease, likely in conjunction with imatinib-containing regimens (53).


    Acknowledgements
 
We would like to thank Edward Hurley for technical assistance with confocal microscopy, Douglas Nixon for image processing, Dr Carleton Stewart and Sigrid Stewart for flow cytometric analysis, Dr Maria Baer for critical reading and comments and Sanghee Han for preparation of tables and figures. Supported partially by Immunex, Seattle, WA, the Heidi Leukemia Research Fund, Buffalo, NY and National Cancer Institute Grant CA 16056.


    Abbreviations
 
ALL   acute lymphoblastic leukemia
APC   allophycoerythrin
Cy   cychrome
DC   dendritic cell
FBS   fetal bovine serum
FISH   fluorescence in situ hybridization
GM-CSF   granulocyte-macrophage colony stimulating factor
PerCP   peridinin chlorophyll protein
SCF   stem cell factor
TC   tri-color
TNF   tumor necrosis factor

    Notes
 
Transmitting editor: P. W. Kincade

Received 24 January 2004, accepted 6 July 2004.


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 Introduction
 Methods
 Results
 Discussion
 References
 

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