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Originally published online as doi:10.1189/jlb.0705419 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:747-756.)
© 2006 by Society for Leukocyte Biology

Imatinib mesylate minimally affects bcr-abl+ and normal monocyte-derived dendritic cells but strongly inhibits T cell expansion despite reciprocal dendritic cell-T cell activation

Nicolas Boissel*, Philippe Rousselot{dagger},{ddagger}, Emmanuel Raffoux{dagger}, Jean-Michel Cayuela§, Jean Soulier§, Nuala Mooney*, Dominique Charron*, Hervé Dombret{dagger}, Antoine Toubert* and Delphine Rea*,¶,1

* Laboratoire d’Immunologie et Histocompatibilité, INSERM U662, Institut Universitaire d’Hématologie, Paris, France;
{dagger} Service des Maladies du Sang and
§ Laboratoire Central d’Hématologie,
Unité de Thérapie Cellulaire et Clinique Transfusionnelle, AP-HP, Hôpital Saint-Louis, Paris, France; and
{ddagger} Service d’Onco-Hématologie, Centre Hospitalier de Versailles, Le Chesnay, France

1Correspondence: Unité de Thérapie Cellulaire et Clinique Transfusionnelle, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75010 Paris, France. E-mail: delphine.rea{at}sls.ap-hop-paris.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In chronic myeloid leukemia, bcr-abl+ monocytes provide a unique opportunity to generate dendritic cells (DC) expressing a broad spectrum of leukemic antigens, and bcr-abl+ DC vaccines may allow immunological eradication of leukemic cells persisting under treatment with the tyrosine kinase inhibitor imatinib. However, the efficiency of bcr-abl+ DC vaccines will critically depend on the absence of deleterious effects of bcr-abl and of imatinib on DC functions. We show that bcr-abl+ monocytes, devoid of contamination of CD14low granulocytic precursors, differentiate into DC with typical immunophenotypical and functional features, and bcr-abl transcription decreases simultaneously. During differentiation, imatinib induces a slight increase of DC apoptosis and prevents CD1a up-regulation in a dose-dependent manner in bcr-abl+ and normal monocyte-derived DC, but at most, 25% of DC fail to acquire CD1a. When DC maturation is induced in the presence of imatinib, bcr-abl+ and normal monocyte-derived DC up-regulate major histocompatibility complex and costimulatory molecules, CC chemokine receptor 7 and CD83. However, secretion of interleukin-12p70 is decreased in a dose-dependent manner. Imatinib exposure of bcr-abl+ and normal monocyte-derived DC during differentiation and maturation is not detrimental to T cell immunostimulatory functions of DC. In sharp contrast, imatinib, when added to DC-T cell cultures, profoundly suppresses DC-mediated T cell proliferation, despite reciprocal DC-T cell activation attested by up-regulation of CD25 on T cells and of CD86 on DC. Our findings demonstrate that T cells, not normal or bcr-abl+ monocyte-derived DC, are major targets for imatinib immunomodulatory effects. It can be envisioned already that imatinib-free windows will be required to enable vaccination-induced, leukemia-specific T cell expansion.

Key Words: CML • vaccines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recent introduction of the bcr-abl tyrosine kinase inhibitor imatinib mesylate into clinical practice has brought major improvements in the management of chronic-phase chronic myeloid leukemia (CP-CML) with remarkably high rates of hematological remissions and cytogenetic responses [1 ]. However, only a few patients achieve a complete molecular response and the persistence of residual bcr-abl+ hematopoietic progenitors, some of which, with secondary imatinib resistance, represents a major concern and suggests that imatinib as a single agent may not be curative [2 3 4 ]. Thus, novel strategies designed to eradicate residual leukemic cells are needed, among which, combination immunotherapy with imatinib is an attractive option.

The power of cellular immunity to clear CML, demonstrated by the graft-versus-leukemia effect following allogeneic stem cell transplantation (ASCT) and by the emergence of CML-specific T cells in patients responding to interferon-{alpha} (IFN-{alpha}) and ASCT, provides a rationale for developing anti-CML vaccines [5 6 7 ]. Leukemic target antigens include the oncogenic p210bcr-abl fusion protein and overexpressed self-proteins such as proteinase 3, preferentially expressed antigen in melanoma, and Wilms’ tumor gene 1 [8 ]. Dendritic cells (DC), the most potent antigen-presenting cells of the immune system, play a crucial role in the induction of T cell immunity against malignancies. Results from tumor-bearing animals and phase I/II clinical trials in cancer patients show that DC engineered to express tumor antigens represent highly promising cellular vaccines [9 ]. In CML, the presence of the bcr-abl gene in CD34+ progenitors and monocytes [10 ] provides a unique opportunity to generate DC with endogenous expression of leukemic antigens. Two pilot studies have tested bcr-abl+ DC vaccines in late CP-CML patients resistant to IFN. Ossenkoppele et al. [11] used DC obtained from total peripheral blood mononuclear cell (PBMC) cultures, and Takahashi et al. [12] performed sequential injections of blood DC and of immature and mature DC derived from adherent monocytes. Some immune responses were raised but without clinical improvement. The high leukemic burden at the time of vaccination is a potential reason, which could have precluded clinical efficacy in these settings. The addition of DC vaccines in CML patients with low leukemic burden under imatinib may yield better results by favoring further reduction in residual disease and increasing the proportion of patients with a complete molecular response. This approach requires investigations of potential immunoregulatory effects of imatinib on DC. The fact that imatinib impairs the development and maturation of healthy donors’ CD34+ progenitors and monocytes toward DC and macrophages in vitro [13 14 15 ], combined with our recent finding that patients responding to imatinib show reduced blood DC in comparison with healthy individuals [16 ], is indeed raising concerns about the efficacy of DC vaccines in imatinib-treated patients. Until now, the impact of imatinib on monocyte-derived DC harboring the therapeutic target of imatinib in CML, the bcr-abl oncoprotein, has never been explored.

The objectives of our study were to ask whether imatinib could affect bcr-abl+ monocyte-derived DC differentiation, maturation, and immunostimulatory functions and to compare bcr-abl+ and normal monocyte-derived DC. Toward this goal, we had to deplete granulocytic precursors abnormally present in the peripheral blood from untreated CP-CML patients prior to CD14-based monocyte purification to properly analyze bcr-abl+ monocyte-derived DC. Unlike monocytes, these granulocytic precursors indeed express low levels of CD14 but are unable to differentiate into DC. We found that imatinib had a modest inhibitory impact on bcr-abl+ and normal monocyte-derived DC differentiation and decreased interleukin (IL)-12p70 production by mature DC without altering their T cell immunostimulatory functions. However, when imatinib was present during DC-T cell interactions, the capacity of T cells to proliferate was compromised severely.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and healthy donors
Thirty-one CP-CML patients above 18 years of age, without previous treatment except for hydroxyurea, entered this study. Patients with a history of malignant solid tumors, chronic viral infections, and autoimmune disorders were excluded. Peripheral blood was obtained after informed consent before initiation of imatinib treatment under a protocol approved by the Institutional Ethical Committee (Hôpital Saint-Louis, Paris, France). Peripheral blood from 22 healthy donors was obtained at Hôpital Saint-Louis Transfusion Center.

Differentiation of DC from monocytes
Monocytes were purified from PBMC by positive selection with CD14 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). In CML, CD15+ granulocytic precursors circulating in the peripheral blood expressed low levels of CD14. Thus, depletion with CD15 microbeads (Miltenyi Biotec) was performed prior to monocyte isolation. Monocytes (>95% pure) were cultured for 6 days with medium change every 2 days in RPMI 1640 (Life Technologies, Gaithersburg, MD) with 2 mM glutamine, 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 µg/ml streptomycin, 800 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Leucomax, Novartis Pharma, Basel, Switzerland), and 500 U/ml IL-4 (R&D Systems, Minneapolis, MN). As previously reported [10 ], 85% of CML-DC carried the bcr-abl fusion gene by fluorescence in situ hybridization (FISH) analysis (not shown). For maturation, DC were plated at a density of 106/ml and incubated with 250 ng/ml lipopolysaccharide (LPS) from Escherichia coli strain 055:B5 (Sigma Chemical Co., St. Louis, MO) for 48 h. In some experiments, 1–10 µM imatinib (kindly provided by Novartis Pharma), dissolved in culture medium from 10 mM stock solution in dimethyl sulfoxide (DMSO), was added at initiation of DC differentiation every other day during medium change and during DC maturation. Equal amounts of DMSO were added to control DC to exclude any effect of the solvent. Of note: The doses of imatinib used in this study are in the range of the concentrations pharmacologically achieved in vivo in patients treated with 400 mg daily imatinib [17 ].

Quantification of bcr-abl transcripts in monocytes and DC
After purification, monocytes (2x106) were stored at –80°C in TriReagent (Molecular Research Center, Cincinnati, OH). The remaining monocytes were differentiated into immature DC in the presence of GM-CSF and IL-4 as above and stored at –80°C in TriReagent. RNA was extracted, and measurement of bcr-abl transcripts in monocytes before and after DC differentiation was performed by real-time quantitative-polymerase chain reaction (RQ-PCR), as published [18 ].

Apoptosis and necrosis assay
Apoptosis of monocyte-derived DC from healthy donors and CML patients and of CD4+ T cells cultured in the presence of imatinib or control DMSO was measured by double-staining with fluorescein isothiocyanate (FITC)-conjugated annexin V-FITC and propidium iodide (PI; Bender MedSystems, Vienna, Austria), according to the instruction of the manufacturer, followed by flow cytometry analysis. Apoptotic cells were defined as annexin V-FITC+/PI–.

Analysis of DC and CD4+ T cell phenotype by flow cytometry
Cells were stained on ice for 20 min in phosphate-buffered saline (PBS)-1% FCS with FITC or phycoerythrin-(PE)-conjugated monoclonal antibodies (mAb) and analyzed by flow cytometry. The following mAb and corresponding isotype controls were used: PE-anti-CD1a (HI149), PE-anti-CD86 (FUN-1), FITC-anti-CD40 (5C3), FITC-anti-human leukocyte antigen (HLA)-A,B,C (G46-2.6), FITC-anti-HLA-DR (G46.6), FITC-anti-HLA-DQ (Tü 169), PE-anti-CD54 (HA58), FITC-anti-class II-associated invariant chain peptide (CLIP; CerCLIP), FITC-anti-CD4 (RPA-T4), and PE-anti-CD25 (M-A51; all from BD PharMingen, San Diego, CA), FITC-anti-CD14 (Miltenyi Biotec), FITC-anti-CC chemokine receptor 7 (CCR7; R&D Systems), and PE-anti-CD83 (HB15A; Immunotech, France).

Antigen-uptake experiments
Antigen-uptake experiments were performed as described by Sallusto et al. [19 ]. Cells were resuspended in medium buffered with 25 mM HEPES. FITC-bovine serum albumin (BSA; Sigma Chemical Co.) was added at the final concentration of 1 mg/ml. Cells were incubated at 37°C or 4°C to determine background uptake. After 1 h, cells were washed extensively with ice-cold PBS and analyzed by flow cytometry.

Cytokine secretion by DC
To compare cytokine production during maturation in healthy donors and CML patients, DC (105), exposed or not to imatinib, were stimulated with 250 ng/ml LPS (Sigma Chemical Co.) for 6, 24, and 48 h, followed by restimulation for 24 h with 1 µg/ml CD40 ligand (CD40L; kindly provided by Amgen, Thousand Oaks, CA), as described by Langenkamp et al. [20 ]. Human IL-12p70 was measured in the supernatants of DC cultures by solid-phase sandwich enzyme-linked immunosorbent assay (ELISA; Diaclone Research, Stamford, CT; sensitivity, 3 pg/ml).

DC-induced, naive CD4+ T cell polarization
Naive CD4+ T cells from healthy donors were purified by negative selection with the human CD4+/CD45RO– naive T cell subset column kit (R&D Systems) according to the manufacturer’s instructions. Immature and mature DC, exposed or not to imatinib, were plated at 104/well in 96-well U-bottom plates. Allogeneic, naive CD4+ T cells were added to the DC at a density of 5 x 104/well. After 7 days, T cells were harvested and stimulated for 6 h with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) and 250 ng/ml ionomycin in the presence of 5 µg/ml brefeldin A (Sigma Chemical Co.). CD4+ T cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.5% BSA and 0.1% saponin, and stained for intracellular cytokines. Polarization toward T helper cell type 1 (Th1) and Th2 was measured by flow cytometry. The following mAb were used: FITC-anti-IFN-{gamma} (4SB.3), PE-anti-IL-4 (8D4-8), and PE-anti-IL-10 (JES3-19F1; BD PharMingen).

Phytohemagglutinin (PHA) and DC-driven CD4+ T cell proliferation
Immature and mature DC, exposed or not to imatinib, were plated at 104/well in 96-well U-bottom plates. Allogeneic CD4+ T cells from healthy donors were isolated after monocyte depletion of PBMC using CD14 microbeads (Miltenyi Biotec) and positive selection of CD4+ T cells with CD4 microbeads (Miltenyi Biotec), as indicated by the manufacturer. T cells were washed in serum-free medium and stained for 5 min with the PKH26 dye (Sigma Chemical Co.) at 2 x 10–6M final concentration, according to the manufacturer’s instructions. After three washes, T cells were added to the DC at 5 x 104/well. Proliferation was assessed on Day 7 by flow cytometry. The PKH26 dye is retained stably in the cell membrane of T cells and is equally divided in daughter cells upon each cell division, allowing the identification of T cells that had divided among cultured cells by flow cytometry. In some experiments, imatinib at 1 µM and 5 µM was added during DC-T cell coculture. To analyze CD4+ T cell response to PHA, PBMC from healthy donors or CML patients were stained with the PKH26 dye as above, plated at 105/well in 96-well U-bottom plates, and stimulated with 5 µg/ml PHA (Sigma Chemical Co.). Proliferation was assessed on Day 7 by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
bcr-abl+ and normal monocytes but not CD14low leukemic cells differentiate into DC
We reproducibly obtained highly purified (>95% pure) monocytes from all CML patients by removal of circulating CD15+/CD14low granulocytic precursors followed by positive immunomagnetic selection of monocytes. This former step was crucial, as under GM-CSF and IL-4 treatment, CD15+/CD14low cells survived in culture and did not acquire morphological DC features or CD1a expression (Fig. 1A ), lacked the costimulatory molecules CD86 and CD40 (Fig. 1B) , failed to uptake antigens efficiently (Fig. 1C) , and did not prime naive CD4+ T cells into polarized Th1 (IFN-{gamma}+/IL-4–) or Th2 effectors (IFN-{gamma}–/IL-4+; Fig. 1D ). When cultured in GM-CSF and IL-4, purified monocytes from CML patients and healthy donors developed into homogeneous and typical immature DC as CD14– and CD1a+. The expression of CD1a did not differ statistically between monocyte-derived DC from CML patients (median MFI, 454; range, 59–2363) and from healthy donors (median MFI, 529; range, 339–2845). The median DC yield from monocytes was 33% (16.3–60) in CML patients and 34% (16–52.5) in healthy donors (not significant), which is comparable with what was reported in the literature with normal DC [21 ]. In conclusion, bcr-abl+ and normal monocytes share a comparable DC differentiation potential, and the removal of CD14low granulocytic CML cells is a critical step in the generation of bcr-abl+ monocyte-derived DC.


Figure 1
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  		Figure 1. bcr-abl+ CD14high monocytes but not CD14low leukemic precursors differentiate along the DC pathway. CD14high monocytes (circle) and circulating CD14low/CD15+ granulocytic precursors (square) from CML patients were cultured for 6 days in the presence of GM-CSF and IL-4. Differentiation into DC was assessed by the measurement of CD1a and CD14 expression by flow cytometry. Specific mean fluorescence intensities (MFI) are indicated (A). The surface expression of HLA-DR, HLA-class I, CD86, and CD40 was also analyzed by flow cytometry. Specific MFI are indicated (B). Antigen capture was determined after incubation of the cells with medium containing 1 mg/ml FITC-BSA. Empty histograms show the background autofluorescence, shaded histograms show the background uptake at 0°C, and black-filled histograms show the specific uptake at 37°C. Specific MFI are indicated (C). Induction of naïve, allogeneic CD4+ T cell polarization toward Th1 (IFN-{gamma}+/IL-4–) and Th2 (IFN-{gamma}–/IL-4+) was determined by intracellular cytokine staining after PMA and calcium ionophore treatment of CD4+ T cells stimulated 6 days before with CD14high monocytes or CD14low/CD15+ granulocytic precursors cultured in the presence of GM-CSF and IL-4 (D). Dot-plots with double IFN-{gamma}-FITC and IL-4-PE staining are shown, and percentages of Th1 and Th2 CD4+ T cells are indicated. In each panel, results from one representative CML patient are shown among three independent experiments.

 
Imatinib minimally affects bcr-abl+ and normal monocyte-derived DC differentiation
When imatinib was added to bcr-abl+ and normal monocytes at 1 µM or 5 µM, with concentrations in the range of those achieved in the plasma of 400 mg daily imatinib-treated patients [17 ], we observed a dose-dependent increase in the rate of apoptotic DC, and a concomitant decrease in viable DC yield to the same extent in bcr-abl+ and normal monocyte-derived DC cultures. With control DMSO, 1 µM imatinib and 5 µM imatinib apoptotic DC, respectively, averaged 4.7% (2.5–8.3), 6.2% (5–7.5; P=0.049; 1 µM vs. DMSO), and 11.6% (8.1–14.31; P=0.011; 5 µM vs. DMSO) of total DC (Fig. 2A ), and the mean viable DC yield was, respectively, 32.8% (20.8–52), 29.5% (25.2–36.3), and 20.2% (15.7–27.5). Moreover, although imatinib did not alter DC morphology, an average of 23.7% [22 23 24 25 26 27 ] of DC from CML patients and of 25.3% [19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ] of DC from healthy donors failed to express CD1a when a 5-µM concentration of imatinib was used (Fig. 2B) . CD14 was down-regulated strongly regardless of the amount of imatinib present, indicating that the CD1a-deficient cells fraction was not held at the monocytic stage (Fig. 2B) . When imatinib was added for 48 h at 5 µM to already-differentiated bcr-abl+ and normal monocyte-derived DC, high levels of CD1a expression were maintained (Fig. 2C) . Of note, 10 µM imatinib, which is far above plasmatic concentrations reached in CP-CML patients, did not lead to an increase in the proportion of CD1a-deficient cells but strongly increased DC apoptosis and was therefore not used in further experiments (not shown). Finally, when monocyte-derived DC from three CML patients were analyzed after 1–6 months of 400 mg daily imatinib treatment, without addition of imatinib in vitro, CD14 was down-regulated, and CD1a was highly expressed on 100% of DC (median MFI, 1671; range, 1610–1723). To conclude, imatinib, during in vitro differentiation of monocytes into DC, induces a dose-dependent increase in apoptosis and a blockade in CD1a up-regulation, but only a small fraction of the cells is affected. bcr-abl+, normal monocyte-derived DC, are equally affected by imatinib, and we hypothesized that this latter finding may relate to a modulation of bcr-abl expression during leukemic monocyte differentiation along the DC lineage. The quantification of bcr-abl transcripts in leukemic monocytes freshly isolated and after 6 days of GM-CSF + IL-4 treatment revealed a median bcr-abl/abl ratio of 5.01 x 10–2 (2.58x10–2–6.47x10–2) in monocytes and of 3.22 x 10–2 (1.22x10–2–4.97x10–2) in DC, corresponding to a median decrease in a bcr-abl/abl ratio of 36% (12.3–52.7; P=0.005; Fig. 2D ). Selection of bcr-abl– monocytes by imatinib was excluded, as FISH analysis revealed the presence of the fusion gene in 85% of DC in imatinib-treated and untreated cultures (not shown).


Figure 2
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  		Figure 2. Effect of imatinib on apoptosis and CD1a expression during normal and bcr-abl+ DC differentiation. Normal and bcr-abl+ monocytes were exposed to control DMSO and imatinib at 1 µM and 5 µM, and DC differentiation was initiated by addition of GM-CSF and IL-4. DC were analyzed after 6 days of culture. Apoptosis was assessed by annexin V-FITC/PI staining, and a representative experiment among four CML patients and four healthy donors is shown. The percentages of apoptotic cells (Annexin V-FITC+/PI–, lower right quadrants) are indicated. The 5-µM imatinib forward-scatter/side-scatter dot-plot is also shown (A). The expression of CD14 and CD1a was determined by flow cytometry, and a representative experiment among four CML patients and four healthy donors is shown. The percentage of CD1ahigh cells is indicated (B). CD1a and CD14 expression was also determined on DC differentiated for 6 days in the presence of GM-CSF and IL-4 and exposed to imatinib at 5 µM for an additional period of 48 h. A representative experiment among two CML patients and two healthy donors is shown (C). To analyze the modulation of bcr-abl during leukemic DC differentiation, bcr-abl transcripts were quantified by RQ-PCR in bcr-abl+ monocytes and monocyte-derived DC. Experiments performed in five individual CML patients are shown (D).

 
bcr-abl+ and normal monocyte-derived DC share comparable immunophenotypic features without alteration by imatinib
The decrease in the number of CD1ahigh DC upon 5 µM imatinib exposure made us search for additional imatinib-induced modifications of monocyte-derived DC. The levels of expression of major histocompatibility complex (MHC) class I and class II molecules, of CD86 and CD40 costimulatory molecules, and of the CD54 adhesion marker in immature bcr-abl+monocyte-derived DC did not significantly differ from those of immature, healthy donor, monocyte-derived DC (Fig. 3A ). The expression of the MHC class II-associated invariant chain self-peptide (CLIP), an antagonist of Th1 polarization found to be high in acute myeloid leukemia [22 , 23 ], was similarly expressed by immature bcr-abl+ and normal monocyte-derived-DC (Fig. 3A) . Internalization of FITC-BSA by immature bcr-abl+ and normal monocyte-derived DC was found to be similarly efficient (Fig. 3A) . Upon LPS exposure, bcr-abl+ monocyte-derived DC retained their capacity to mature, as evidenced by a strong up-regulation of MHC class I and II molecules and CLIP, of costimulatory and adhesion molecules, by the acquisition of CD83 and of the lymphoid chemokine receptor CCR7, and by a down-regulation of FITC-BSA uptake (Fig. 3A) . The presence of imatinib at 1 µM or 5 µM did not affect these LPS-induced modifications substantially, neither in bcr-abl+ monocyte-derived DC nor in normal monocyte-derived DC cultures (Fig. 3B) . Consistent with these results, the phenotype of monocyte-derived DC generated from CML patients under 400 mg imatinib daily did not differ from that of monocyte-derived DC obtained prior to initiation of imatinib treatment (not shown).


Figure 3
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  		Figure 3. Normal and bcr-abl+ monocyte-derived DC immunophenotype is not affected by imatinib. Immature and mature, normal and bcr-abl+ DC were compared for their cell-surface immunophenotype and for their capacity to internalize FITC-BSA by flow cytometry. Results from 20 CML patients and 10 healthy donors are shown (A). Normal and bcr-abl+ monocyte-derived DC were incubated in the presence of control DMSO or imatinib at 1 µM and 5 µM during DC development and maturation. The expression of MHC molecules, of costimulatory molecules, of the lymphoid chemokine receptor CCR7, and of the maturation marker CD83 was analyzed by flow cytometry. Results from a representative CML patient are shown among experiments performed in three CML patients and three healthy donors (B).

 
IL-12 secretion by maturing bcr-abl+ and normal monocyte-derived DC is decreased by imatinib, but polarization of naive CD4+ T cells is not affected
Innate microbial signals and T cell factors contribute to maximize IL-12p70 production by DC [24 ], and IL-12p70 synthesis is tightly controlled to regulate Th cell priming [20 ]. We thus primed DC with LPS prior to stimulation through CD40, and IL-12p70 secretion was analyzed at different time-points. The capacity of bcr-abl+ and normal monocyte-derived DC to produce IL-12p70 upon CD40 triggering was found to be maximal 6 h after LPS stimulation and was strongly decreased at 24 h and 48 h (Fig. 4A ). Imatinib treatment of DC did not alter the kinetics of cytokine production during maturation but decreased IL-12p70 production in a dose-dependent manner. The maximal amount of IL-12p70 produced by normal DC averaged 8850 pg/ml with control DMSO, 7600 pg/ml with 1 µM imatinib, and 7200 pg/ml with 5 µM imatinib (P=0.06; 5 µM vs. DMSO; Fig. 4A ). The maximal amount of IL-12p70 produced by bcr-abl+ DC was, on average, 7650 pg/ml with control DMSO, 5460 pg/ml with 1 µM imatinib, and 2100 pg/ml with 5 µM imatinib (P=0.038; 5 µM vs. DMSO; Fig. 4A ). As IL-12p70 production by maturing DC is a key feature for the induction of Th1 and Th1-dependent CTL responses, we analyzed the ability of bcr-abl+ and normal monocyte-derived DC to prime CD4+ T cells toward polarized effectors, and we compared the potential of imatinib-treated and imatinib-untreated DC to trigger Th1-type responses. Immature monocyte-derived DC from CML patients and healthy donors induced low levels of Th1 (IFN-{gamma}+/IL-4–) and Th2 (IFN-{gamma}–/IL-4+) polarization of naive CD4+ T cells (Fig. 4B) . Upon maturation, both DC types were found to be potent inducers of Th1 polarization with Th1 cells, respectively, representing, on average, 50% (30–63) and 60% (39–83) of T cells when bcr-abl+ and normal mature DC were used as stimulators (Fig. 4B) . It is important that bcr-abl+ and normal monocyte-derived DC were not found to prime significant numbers of IL-10-producing T cells (Fig. 4B) . The presence of imatinib at 1 µM or 5 µM during DC development and maturation did not have a deleterious effect on the induction of Th1-type responses, whether bcr-abl+ or normal DC were used (Fig. 4C) .


Figure 4
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  		Figure 4. Imatinib induces a dose-dependent decrease in IL-12p70 secretion by normal and bcr-abl+ monocyte-derived DC but does not impair CD4+ T cell polarization. Normal and bcr-abl+ monocyte-derived DC were incubated in the presence of control DMSO or imatinib at 1 µM and 5 µM during DC during differentiation. Maturation was induced by addition LPS for 6 h, 24 h, and 48 h, followed by restimulation for 24 h with CD40L. IL-12p70 production was measured in the culture supernatants by ELISA. Results from four CML patients and three healthy donors are shown (A). bcr-abl+ and normal monocyte-derived DC were compared for their capacity to polarize naive CD4+ T cells. Allogeneic CD4+/CD45RA+ T cells (5x104) were cultured for 7 days with immature and mature bcr-abl+ and normal monocyte-derived DC (1x104). T cells were restimulated for 6 h with calcium ionophore and PMA in the presence of brefeldin A, and expression of intracellular IFN-{gamma}, IL-4, and IL-10 was determined by flow cytometry. The proportion of CD4+ T cells polarized toward Th1 (IFN-{gamma}+/IL-4–), Th2 (IFN-{gamma}–/IL-4+), and producing IL-10 from 11 CML patients and six healthy donors is shown. Pretreatment of immature and mature bcr-abl+ and normal monocyte-derived DC with imatinib at 1 µM and 5 µM did not affect polarization of CD4+ T cells toward Th1 (IFN-{gamma}+/IL-4–). Results from three CML patients and three healthy donors are shown (C).

 
bcr-abl+ and normal monocyte-derived DC are unable to induce T cell proliferation provided that imatinib is present during DC-T cell cocultures
Imatinib was shown recently to suppress normal T cell proliferation induced by the mitogenic lectin PHA and by antibodies specific for the T cell receptor (TCR)-CD3 complex and the CD28 costimulatory molecule [25 26 27 ]. Consistent with these in vitro findings, we found that PHA-driven proliferation of T cells from CML patients on 400 mg daily imatinib therapy was compromised significantly, compared with that of normal donor PHA-stimulated CD4+ T cells (P=0.05, normal vs. imatinib-treated CML; Fig. 5A ). We asked whether imatinib was also able to impair DC-dependent T cell proliferation. The treatment of DC with imatinib at 1 µM or 5 µM during development and maturation prior to culture with allogeneic CD4+ T cells did not have any impact on T cell proliferation, whether bcr-abl+ or normal DC were used (Fig. 5B) . In contrast, the addition of 5 µM imatinib to DC-T cell cultures decreased immature and mature DC-mediated T cell proliferation, on average, by more than 50% (immature DC: P=0.025, 5 µM vs. DMSO; mature DC: P=0.0009, 5 µM vs. DMSO; Fig. 5B ). We attempted to determine whether this inhibitory effect of imatinib was solely a result of impact on T cells or also an indirect impact on DC. It is important that apoptosis of CD4+ T cells measured by annexin V and PI staining did not account for the reduced proliferation in imatinib-treated cultures (not shown). Moreover, the ability of immature and mature DC to activate CD4+ T cells was not compromised by imatinib. The up-regulation of the T cell activation marker CD25 was indeed analyzed 24 h following DC stimulation, and the proportion of CD4+CD25+ T cells was comparable in imatinib-treated and imatinib-untreated cultures (Fig. 6A ). In turn, allogeneic CD4+ T cells from imatinib-treated and imatinib-untreated cultures induced CD86 up-regulation on DC when immature cells were used as stimulators (Fig. 6B) . Altogether, these results favor T cells and not monocyte-derived DC as the main targets for imatinib-induced antiproliferative effects.


Figure 5
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  		Figure 5. Imatinib inhibits PHA- and DC-driven T cell proliferation. PBMC from healthy donors and from imatinib-treated CP-CML patients were stained with PKH26 and stimulated by the mitogenic lectin PHA without addition of exogenous imatinib. PKH26 is divided in daughter cells upon cell division and allows the identification of T cells, which had divided by flow cytometry. Proliferation was assessed on Day 7. Results from three healthy donors and four imatinib-treated CP-CML patients are shown. Typical PKH26 staining of T cells from a healthy donor and a CML patient are also shown, and the proportion of T cells, which have undergone proliferation, is indicated (A). Allogeneic CD4+ T cells were stained with PKH26 and cultured for 7 days with bcr-abl+ or normal, immature and mature monocyte-derived DC, which had been generated previously in the presence of control DMSO or imatinib at 1 µM and 5 µM. Proliferation of CD4+ T cells was measured at Day 7 by flow cytometry. Results performed with DC, three CML patients and two healthy donors, are shown (B, left panel). Allogeneic CD4+ T cells were stained with PKH26 and cultured for 7 days with bcr-abl+ or normal, immature and mature monocyte-derived DC in the presence of control DMSO or imatinib at 1 µM and 5 µM. Proliferation of CD4+ T cells was measured at Day 7 by flow cytometry. Results performed with DC, three CML patients and two healthy donors, are shown (B, middle panel) as well as a typical PKH26 staining of T cells after 7 days culture with bcr-abl+ immature DC in the presence of control DMSO or 5 µM imatinib (B, right panel).

 

Figure 6
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  		Figure 6. Imatinib does not impede DC-T cell reciprocal activation. Allogeneic CD4+ T cells were cultured with immature or mature bcr-abl+ or normal monocyte-derived DC in the presence of control DMSO or imatinib at 5 µM. Expression of the T cell activation marker CD25 was analyzed on CD4+ T cells by flow cytometry after 24 h. Results from three independent experiments are shown as well as a typical CD4/CD25 staining from one experiment (A). Expression of the costimulatory molecule CD86 was analyzed on DC by flow cytometry after 72 h. Results from three independent experiments are shown as well as a typical CD86 staining from one experiment (B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although imatinib displays a remarkable activity in the control of CML disease, resistant, residual leukemic cells persist, and relapses occur when imatinib is discontinued [28 ]. As CML is sensitive to immune-mediated therapies, coupling imatinib with bcr-abl+ DC vaccines, expressing a wide array of leukemic antigens, may offer a clinical benefit, providing that such DC are equally efficient as their normal counterparts and that imatinib do not suppress DC functions.

In this study, we demonstrate that bcr-abl+ and normal monocytes differentiating along the DC pathway share typical DC features with similar surface immunophenotype and functional properties. It is important that we show that depletion of CD15+/CD14low cells prior to isolation of leukemic monocytes is mandatory to obtain pure bcr-abl+ monocyte-derived DC for therapeutic purposes. We indeed found that circulating CD15+ leukemic granulocytic precursors expressed low levels of CD14 but failed to develop into DC when cultured with GM-CSF and IL-4, based on morphology, on the absence of CD1a and CD86 expression, on inefficient antigen internalization, and on the lack of capacity to polarize naïve T cells. In apparent contrast with our findings, Oehler et al. [29 ] reported previously that granulocytic precursors from CML patients, patients with bacterial infections, and patients treated with G-CSF could differentiate into DC-like cells expressing CD1a and CD86. Our culture conditions for the generation of DC differ from those described by Oelher et al. [29] in one important aspect: We used GM-CSF and IL-4 rather than GM-CSF, IL-4, and tumor necrosis factor {alpha} (TNF-{alpha}), and the capacity of this latter cytokine to increase surface expression of CD1a and costimulatory molecules during DC development is well-known [30 ]. Even with TNF-{alpha}, only 40% of the granulocytic precursors up-regulated CD1a; thus, a large proportion of the cells remained unable to acquire critical features of DC in the Oehler et al. [29] study.

DC vaccines in CML patients may be a promising strategy after an initial phase of imatinib treatment to achieve an immune-mediated control of the residual disease. Whether normal and bcr-abl+ DC vaccines can combine effectively with imatinib is an important issue, as imatinib not only inhibits constitutively activated kinases such as bcr-abl but also affects kinases regulating key functions of immune cells such as mitogen-associated protein kinases (MAPK), Lck, and Abl/Arg. We found a limited impact of imatinib during normal and bcr-abl+ monocyte-derived DC differentiation. At most, 25% of DC did not acquire CD1a, and the expression of costimulatory and MHC molecules was preserved. It is not clear why imatinib affected bcr-abl+ and normal monocyte-derived DC development in a comparable manner, but it is interesting to speculate that the decrease in bcr-abl transcription, which we observed during DC generation, may be involved. Whether this modulation of bcr-abl transcripts lowers leukemic antigen presentation by bcr-abl+ monocyte-derived DC cannot be excluded. The differentiation of monocytes into DC under GM-CSF and IL-4 is accompanied by the activation of multiple transduction pathways including Raf/MAPK kinase/extracellular-signal regulated kinase, phosphatidylinositol-3 kinase (PI-3K)/AKT, and nuclear factor-{kappa}B, and PI-3K/AKT appears especially to play a key role [31 ]. The fact that the PI-3K/AKT pathway is not a direct target of imatinib may explain the poor impact of this drug on normal and bcr-abl+ monocyte-derived DC development. Our results differ strongly from those recently obtained by Appel et al. [15 ]. These authors indeed found a dramatic blockade in CD1a and costimulatory molecule up-regulation when normal, monocyte-derived DC were generated in the presence imatinib at the same concentrations as those used in our study. The reasons for these discrepancies remain unclear, although we purified monocytes through CD14-positive selection, and Appel et al. [15] obtained monocytes through plastic adherence of monocytes in serum-free medium. However, it should be noted that the level of CD1a expression in control cultures was much lower than that we obtained. We focused here on the comparison between bcr-abl+ and normal monocyte-derived DC. It is notable that we found that monocytes from imatinib-treated patients retained their capacity to differentiate into CD14–CD1ahigh DC.

Upon maturation, we showed that bcr-abl+ monocyte-derived DC, like their normal counterparts, lost their ability to capture antigens, increased costimulatory and MHC molecules at high levels, and acquired the maturation marker CD83 and the lymphoid chemokine receptor CCR7. Furthermore, the cognate maturation signal CD40L enhanced IL-12p70 production triggered by the inflammatory stimulus LPS, and the dynamic regulation of IL-12 secretion by bcr-abl+ monocyte-derived DC was comparable with that of normal DC. Although the amount of IL-12 produced by leukemic DC was reduced slightly, mature bcr-abl+ monocyte-derived DC exhibited potent immunostimulatory functions and promoted a strong polarization of naïve CD4+ T cells toward Th1. Therefore, our results are in line with those from Gabriele et al. [32 ], who found that bcr-abl+ monocyte-derived DC were fully competent to stimulate CD8+ T cell responses. When DC maturation was initiated in the presence of imatinib, immunophenotypic changes and T cell immunostimulatory capacity of bcr-abl+ and normal monocyte-derived DC were preserved, but IL-12p70 secretion was decreased in a dose-dependent manner, as also observed by Appel et al. [15] in normal monocyte-derived DC. Nevertheless, we found that the capacity of imatinib-treated DC to drive naive CD4+ T cell responses toward a Th1 profile was not affected in the experimental settings used in our study.

Imatinib was reported recently to inhibit PHA and anti-CD3-driven human T cell proliferation in vitro [25 26 27 ] by targeting TCR signal transduction through the Abl and Lck tyrosine kinases [27 , 33 ]. Our data suggest that imatinib may also be detrimental to T cell expansion in vivo, as the proliferation of CD4+ T cells from imatinib-treated CML patients upon PHA stimulation was decreased. Another striking finding in our study is that normal and bcr-abl+ monocyte-derived DC were unable to overcome the antiproliferative effect of imatinib in vitro. Indeed, exposure to imatinib of CD4+ T cells cocultured with DC dramatically hampered T cell proliferation induced by normal and bcr-abl+ monocyte-derived DC. It is interesting that imatinib did not inhibit DC-mediated T cell activation, as CD4+ T cells from imatinib-treated and untreated cultures demonstrated a similar increase in the T cell activation marker CD25. In turn, up-regulation of the DC costimulatory molecule CD86 upon interaction with responding CD4+ T cells was not suppressed by imatinib. Altogether, our results show that imatinib does not prevent cognate interactions between DC and T cells and suggest that T cells and not monocyte-derived DC are the main targets of imatinib-suppressive action. However, we cannot exclude imatinib-mediated modifications of the immunological DC-T cell synapse. It is indeed important to keep in mind that imatinib was shown recently to alter the cross-talk between DC and other immune effectors such as natural killer cells [34 ]. Moreover, the tyrosine kinase Abl, a target of imatinib, is critical for optimal neuronal and neuromuscular synapse formation [35 ], and neurological and immunological synapses share many similarities [35 36 37 ].

Further research is needed to understand how imatinib may affect immunity more generally and to determine how immunological control of CML in imatinib-treated patients can be achieved and how imatinib should be integrated in the treatment of residual disease in the post-transplantation setting. Our findings readily demonstrate progress toward the clinical applicability of DC vaccines in imatinib-treated patients. As a result of imatinib-induced suppression of T cell proliferation, imatinib-free therapeutic windows during monocyte-derived DC vaccination may be required to enable expansion of leukemia-specific T cell responses.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant by Fondation de France (2002-2386), Délégation Régionale à la Recherche Clinique de l’AP-HP, Allostem (FP6 #503319), and Cancéropôle Ile de France. We thank staff members from the Clinical Investigation Center from Hôpital Saint-Louis for help with collecting patients’ samples.

Received July 28, 2005; revised November 16, 2005; accepted December 8, 2005.


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 DISCUSSION
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