Introduction
Recent therapeutic advances are turning cancer into a more chronic disease. With patients being treated on and off with cytotoxic drugs in order to control metastasis, the effects of such treatment on the immune system in the long run should be considered. Safeguarding the immune competence of cancer patients may be vital to their quality of life as well as overall survival. In addition, successful application of novel immunotherapies also requires intact immune effector functions. Dendritic cells (DC) are the main orchestrators of the immune system and have a key function in linking the innate with the adaptive immune response [
28,
33]. For autologous DC vaccination strategies, most often patient-derived material is used as a source of DC precursor cells. We therefore examined whether a long history of drug exposure could hamper DC differentiation.
The human acute-myeloid leukemia (AML)-derived DC cell line MUTZ3 [
16] can develop either into interstitial DC (MUTZ3-IDC) by culturing the cells with granulocyte and macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), and interleukin-4 (IL-4) or into Langerhans cells (MUTZ3-LC) by culturing the cells with GM-CSF, TNFα, and transforming growth factor beta (TGFβ) [
21,
22]. Of note, MUTZ-3 DC development accurately reflects all stages observed in its physiological CD34
+ precursor-derived counterparts, and as such, this cell line presents a relevant and sustainable human DC/LC differentiation [
23]. We recently found that short-term exposure of CD34
+ cells, including MUTZ-3, to the cytotoxic drugs mitoxantrone or doxorubicin induced accelerated DC and LC differentiation with corresponding functionalities like migration, allogeneic T-cell stimulation, and cytotoxic T lymphocyte (CTL) priming (van de Ven et al. manuscript in preparation). These observations support the use of single-dose cytostatic drugs, as a differentiating agent to facilitate in vitro DC culture for therapeutic purposes. In this study, we examined the effect of long-term drug exposure on the capacity of DC precursor cells to differentiate into functional LC. For these long-term cultures, we made use of MUTZ3 cells that had been stably transduced with the gene encoding the catalytic subunit of the telomerase complex, the human telomerase reverse transcriptase (hTERT), in order to avoid replicative senescence and achieve high passage numbers with maintained cytokine-dependent differentiation ability. Human hematological precursor cells in vivo also express telomerase [
13]. Derivation of this cell line for the first time allowed the in vitro study of the effects of truly long-term exposure of human precursor cells to cytostatic drugs on DC differentiation. Drug selection of these CD34
+ DC precursors revealed that the selective loss of a SCF-R/c-KIT
+ subpopulation resulted in an inability for LC differentiation. After removal of doxorubicin from the cultures, this subpopulation re-emerged as did the LC differentiation capacity. The regained capacity to differentiate after drug removal, as well as the capacity of high-passage hTERT-MUTZ3 cells to differentiate, rules out an hTERT-induced effect on LC differentiation. These data strongly suggest that a drug-free period of adequate length should be allowed for when autologous DC-based therapies are considered consecutive to chemotherapy, in order for the required precursors to recover.
Materials and methods
MUTZ3 and dendritic cell cultures
The AML-derived MUTZ3 cell line was cultured as described before [
21]. In short, MUTZ3 progenitors were cultured in MEM-α (minimum essential medium, Lonza, Vervier, Belgium) containing 20% fetal calf serum (FCS), 100 IU/ml sodium-penicillin, 100 μg/ml streptomycin, 2 mM
l-glutamine, 50 μM β-mercaptoethanol (2ME), and 10% 5637 (renal cell carcinoma) conditioned medium (MUTZ3 routine medium) in 12-well plates (Costar) at a concentration of 0.2 million cells/ml and were passaged twice weekly. Langerhans cells (MUTZ3-LC) were cultured from MUTZ-3 progenitors with 10 ng/ml TGF-β1 (Biovision, Mountain View, CA), 1,000 IU/ml rhGM-CSF (Sagramostim, Berlex), and 120 IU/ml TNFα (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 days to obtain immature MUTZ3-LC as described previously [
30]. Immature MUTZ3-LC were matured by adding 33% monocyte condition medium (MCM) and 2,400 IU/ml TNFα for 48 h.
Retroviral constructs
The retroviral vector LZRS-hTERT-IRES-ΔNGFR has been described previously [
25]. The ΔNGFR is a truncated form of the receptor, with no signaling moiety. Retroviruses were propagated in Phoenix A cells by plasmid DNA transfection using Lipofectamin 2000 (Invitrogen, Breda, The Netherlands) in serum-free medium for 3 h. Medium was refreshed after 24 h, and viral supernatants were harvested 48 h post-transfection and were either directly used for transduction or stored at −80°C in 0.5 ml aliquots.
MUTZ3 retroviral transduction
For the generation of MUTZ3 cells expressing hTERT, MUTZ3 progenitor cells were transduced with the LZRS-hTERT-IRES-ΔNGFR retrovirus as described before [
7,
25]. In short, 0.5 million MUTZ3 progenitor cells were transferred to fibronectin-coated (40 μg/ml RetroNectin, Takara, Japan), non-tissue culture–coated plates (BD Biosciences, Heidelberg, Germany) in 0.5 ml viral supernatant supplemented with 10% 5637 conditioned medium. Plates were centrifuged for 90 min at 2,000 rpm at 25°C. Cells were re-transduced with 0.5 ml fresh viral supernatant after 24 h. The retrovirus infected and transduced the CD34
+ proliferative progenitor population of the MUTZ3 cell line. hTERT-MUTZ3 cells were obtained by selecting the NGFR-positive cells by flow sorting, and cells were maintained in MUTZ3 routine medium as described above. Doxorubicin selection was started by culturing the hTERT-MUTZ3 cells with 5 nM doxorubicin (Amersham Pharmacia, Roosendaal, The Netherlands). Cells were passaged twice weekly as described above. Drug concentrations were gradually increased. Differentiation analysis was performed at different time points during selection with 30 nM (dox30+) and 90 nM (dox90+) cells. Control, non-exposed hTERT-MUTZ3 cells were kept in culture alongside the drug-selected cells and were tested for their differentiating capacity using cells with the same passage numbers.
hTERT PCR
RNA was isolated using RNA-Bee (Bio-connect, Huissen, The Netherlands), following manufacturer’s guidelines. The RNA concentration was determined on a Nanodrop spectrophotometer. cDNA synthesis and PCR reaction were carried out essentially as described before and were performed with 200 ng RNA [
5,
26]. Primers and probes for hTERT and the household control gene U1 small nuclear ribonucleoprotein-specific A protein (SnRNP U1A) were used as described previously [
26]: hTERT forward 5′ → 3′ primer: GAAGGCACTGTTCAGCGTGCTCAAC; hTERT reverse 5′ → 3′ primer: GGTTTGATGATGCTGGCGATGACC; hTERT probe: GGCGCCTGAGCTGTACTTTGTCAAGGTGGA. SnRNP U1A forward 5′ → 3′ primer: CAGTATGCCAAGACCGACTCAGA; snRNP U1A reverse 5′ → 3′ primer: GGCCCGGCATGTGGTGCATAA; snRNP U1A probe: AGAAGAGGAAGCCCAAGAGCCA. The RT-PCR reaction was performed at 35 cycles.
XTT cytotoxicity assay
Twenty thousand cells were seeded per well in 96-well round-bottom plates in a volume of 50 μl per well in culture medium. Doxorubicin was diluted to a maximum concentration of 300 nM in culture medium and 1:3 diluted to 120 pM. Fifty microliters of diluted doxorubicin was added per well in triplicate for each concentration, giving rise to a doxorubicin range from 0.06 to 150 nM. Cells were exposed to the drug for 96 h. Cell viability was measured by adding XTT supplemented with phenazine methosulphate (PMS) for 2–4 h, after which optical density was measured at 450 nm. Cell viability was determined relative to the control cells to which no doxorubicin had been added.
Flow cytometry
LC were immunophenotyped using FITC- and/or PE-conjugated Mabs directed against CD1a (1:25), CD54 (1:25), CD80 (1:25), CD86 (1:25), CD40 (1:10) (PharMingen, San Diego, CA), CD14 (1:25), HLA-DR (1:25), DC-SIGN (1:10) (BD Biosciences, San Jose, CA), CD83 (1:10), CD34 (1:10), Langerin (1:10) (Immunotech, Marseille, France). hTERT MUTZ3 cells, dox90+ and dox90− cells were phenotyped for cytokine receptors using the following antibodies: CD34-APC, CD116-FITC, CD117-PE (Pharmingen, San Diego, CA), CD14-PerCP_Cy5, and CD123-PE (BD Biosciences, San Jose, CA). In short, 2.5–5 × 104 cells were washed in PBS supplemented with 0.1% BSA and 0.02% NaN3 and incubated with specific or corresponding isotype-matched control Mabs for 30 min at 4°C. Cells were washed and analyzed with a FACS-Calibur flow cytometer (Becton and Dickinson, San Jose, CA) equipped with CellQuest analysis software, and the results were expressed as mean or median fluorescence intensities, the percentage of positive cells, or the relative ratios of positive cells compared with the control cultures.
Mixed leukocyte reaction (MLR)
MLR was performed as described [
30]. In short, 1 × 10
2–3 × 10
4 LC were co-cultured with 1 × 10
5 allogeneic peripheral blood lymphocytes for 4 days in 96-well plates in IMDM containing 10% human pooled serum (HPS) (Sanquin, Amsterdam, The Netherlands), 100 IU/ml sodium penicillin, 100 μg/ml streptomycin, 2 mM
l-glutamine, and 50 μM β-mercaptoethanol. At day 4, 2.5 μCi/ml [
3H]-thymidine (6.7 Ci/mmol, MP Biomedicals, Irvine, CA) was added per well for 16 h. Plates were harvested onto glass fiber filtermats (Packard Instruments, Groningen, The Netherlands) using a Skatron cell harvester (Skatron Instruments, Norway), and [
3H]-thymidine incorporation was quantified using a Topcount NXT Microbetacounter (Packard, Meriden, CT).
Statistical analysis
Statistical analysis of the data was performed using the paired or unpaired two-tailed Student’s t test. Differences were considered statistically significant when P < 0.05.
Discussion
This study describes a possible consequence of long-term exposure of DC precursors to cytostatic drugs, interfering with their capacity to develop into DC. By making use of hTERT-transduced MUTZ3 cells, we have demonstrated that prolonged doxorubicin exposure renders CD34+ precursor cells irresponsive to LC-differentiating cytokines. Fortunately, this detrimental effect of doxorubicin on the differentiation capacity of DC precursors proved to be a reversible phenomenon. The cells regained the capacity to differentiate and consequently their capacity to stimulate T-cell proliferation, after a 3- to 4-month drug-free period. Although this report focused on LC differentiation, we have made similar observations for differentiation of interstitial DC from long-term doxorubicin-exposed hTERT-MUTZ3 cells (data not shown). Analysis of cytokine receptor expression patterns on unexposed hTERT-MUTZ3 cells, doxorubicin-exposed dox90+ hTERT-MUTZ3 cells, and the dox90− hTERT-MUTZ3 cells that had been drug-free for 4 months revealed altered expression of the SCF-R (CD117/c-kit). Whereas a subpopulation of CD34+ hTERT-MUTZ3 cells expressed this receptor, a SCF-R-expressing population was absent in the doxorubicin-selected cells. Interestingly, when deprived of doxorubicin, in conjunction with the regained ability to differentiate, the SCF-R+ CD34+ precursor population re-emerged. Sensitivity analysis showed that SCF-R+ CD34+ precursors were more susceptible to doxorubicin-induced cell death compared to SCF-R− CD34+ precursors.
SCF-R
+ CD34
+ cells have been reported to be present both in bone marrow and in peripheral blood of healthy donors and AML patients [
4,
6,
8,
12,
15,
17,
27]. In healthy donors, around 50% of the CD34
+ cells have been reported to express SCF-R (range 19–85%), whereas in AML patients SCF-R+ cells were found in the majority of the patients and expression ranged between 3 and 96% of CD34
+ cells co-expressing SCF-R [
4,
6,
8,
12,
15,
17,
27]. It has been reported that SCF, the ligand for the SCF-R, in combination with FLT3-ligand, is crucial for the maintenance of a long-term proliferative pool of CD34
+ DC precursor cells [
10]. While blockade of the SCF-R did not directly inhibit DC differentiation from CD34
+ precursors, it did interfere with continued proliferation of a subset of CD34
+ precursors with the ability to differentiate into DC. Hence, the observation that the dox90+ cells lack this SCF-R
+ subpopulation and simultaneously have lost their ability to differentiate suggests that due to chronic doxorubicin exposure, this DC precursor subset with long-term proliferative potential is selectively depleted from the general precursor population. A similar effect could be expected on the DC precursors among the circulating hematopoietic stem and precursor cells (HSPC) in patients treated with this drug and perhaps with related drugs. Research in patient groups treated with doxorubicin, or related drugs, is required to gain more insight into the effects of repetitive chemotherapy treatment on the presence and quality of DC precursor cells in vivo, in order to determine whether longer drug-free intervals should be considered when applying chemotherapy regimens prior to DC-based immunotherapies. In this regard, the use of allogeneic rather than autologous DC vaccines should be explored for patients with a long history of chemotherapy treatment [
11].
One could argue that the altered differentiation capacity could be caused by the presence of hTERT in the MUTZ3 cells, which could lead to chromosomal instability and hence an altered differentiation potential [
24]. However, it has been shown that hTERT-transduced T cells can be kept in culture for many months, without loosing their antigen specificity or their cytotoxic abilities [
1]. Furthermore, in our studies, non-drug-exposed hTERT-MUTZ3 cells were kept in culture alongside the drug-selected cells and were used as passage-matched controls in the differentiation studies. Despite the continuous expression of hTERT, these cells maintained their cytokine dependence for growth and were still capable of LC differentiation at high passage numbers. Also, the observation that the drug-induced differentiation defect, along with SCF-R/c-
kit expression on CD34
+ cells, could be reversed upon withdrawal of the drug is additional proof that the observed effects are hTERT independent.
The negative effects of long-term exposure of DC precursor cells to cytostatic drugs are in sharp contrast with our own observation that a single low dose of the anthracyclin doxorubicin, or the related anthracenedione mitoxantrone, at the start of in vitro DC differentiation from CD34
+ precursor cells strongly promotes and even accelerates differentiation (van de Ven et al. manuscript in preparation). Also, work by Zitvogel et al. has shown that systemic doxorubicin treatment can promote anti-tumor responses due to the release of danger signals like the high mobility group box 1 protein (HMGB1) released from dying tumor cells. Release of HMGB1 acts as a maturation signal for resident DC and promotes tumor antigen presentation to CD8
+ T cells [
3,
9,
29]. This notwithstanding, our data imply that in the long run, repetitive systemic treatment with cytostatic drugs could be disadvantageous due to its negative effect on the expansion of CD34
+c-
kit
+ DC precursor cells.
It is known that cytostatic drugs can induce the expression of ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1), multidrug resistance protein 1 (MRP1; ABCC1), or the breast cancer resistance protein (BCRP; ABCG2), thereby inducing multidrug resistance in tumor cells [
2,
18]. As the protein expression levels of these ABC transporters were unaltered in doxorubicin-selected cells (data not shown), the effect of progenitor cell selection with doxorubicin seems ABC transporter independent, and to be caused mainly by the selective loss of SCF-R/c-
kit expressing, CD34
+ DC precursor cells with a high proliferative capacity and hence with an increased susceptibility to the DNA damaging effects of cytostatic drugs such as doxorubicin. Altogether our data suggest that, if feasible, sufficiently long drug-free intervals should be included in chemotherapy regimens in order to allow the immune system to recuperate. This is of particular relevance in view of accumulating evidence for a role of immune functions in clinical responsiveness to chemotherapy [
33]. In addition, when DC vaccination is being considered, patients with a long history of chemotherapy treatment should be tested for their suitability for autologous DC vaccination strategies (assessment of the presence SCF-R
+ CD34
+ cells), as their DC precursor cells might give rise to DC of poor quality. These patients might actually benefit more from allogeneic DC vaccination approaches as recently reviewed by us [
11].
Acknowledgments
The authors would like to thank Dr. J. de Wilde (VU University medical center, Amsterdam) for her help with the hTERT RT-PCR. This work was supported by a grant from the Dutch Cancer Society (KWF) to R.J.S., G.L.S. and T.D.G. (KWF2003-2830).