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Histone deacetylase inhibition improves differentiation of dendritic cells from leukemic blasts of patients with TEL/AML1-positive acute lymphoblastic leukemia Kerstin Schmidt,* Karl Seeger,† Carmen Scheibenbogen,‡ Roderich Bender,* Majd Abdulla,* Sina Su¨ssmilch,* Abdulgabar Salama,* and Anja Moldenhauer*,1 Institutes for *Transfusion Medicine and ‡Immunology and †Department of Pediatric Oncology, Charite´ Campus Virchow-Klinikum and Campus Mitte, Charite´–Universita¨tsmedizin Berlin, Germany

Abstract: Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore, bone marrow samples from 100 children with acute lymphoblastic leukemia (ALL) were cultured in the cytokines TNF-␣, GM-CSF, c-kit ligand, and fetal liver tyrosine kinase 3 ligand, with or without IL-3 and -4 and after administration of HdI valproic acid (VAL), suberoylanilide hydroxamic acid (SAHA), isobutyramid, or trichostatin A. Among the tested samples, 25 were positive for the chromosomal translocation t(12;21), encoding the fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 (TEL/AML1). SAHA increased CD83 expression of TEL/AML1-positive blasts in conditions without ILs, and SAHA and VAL increased the number of CD86(ⴙ)80(–) cells in the presence of ILs. VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive, leukemic DC; VAL and SAHA reduced those of TEL/AML1-negative DC. Cytotoxic T cells sensitized with leukemic DC produced more IFN-␥ and TNF-␣ upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of nondifferentiated blasts, which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. Therefore, the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended. J. Leukoc. Biol. 85: 563–573; 2009. Key Words: valproic acid 䡠 suberoylanilide hydroxamic acid 䡠 cytotoxic T cells 䡠 vaccine 䡠 cytokines

INTRODUCTION The fusion gene translocation ETS-like leukemia/acute myeloid leukemia 1 TEL/AML1, resulting from the translocation t(12; 21), is present in 20 –30% of childhood acute lymphoblastic leukemia (ALL) [1]. By altering cellular self-renewal and survival properties, the fusion protein TEL/AML1 generates a preleukemic state [2], giving rise to leukemia in combination with further genetic abnormalities [3]. Clinical reports about 0741-5400/09/0085-563 © Society for Leukocyte Biology

frontline and relapse ALL trials [4, 5] associate TEL/AML1 positivity with favorable parameters such as confinement to B cell lineage, good response to polychemotherapy, low white blood cell count, and favorable age distribution; TEL/AML1 has therefore been identified as an independent, positive prognostic factor. However, similar frequencies at disease onset and subsequent relapses of ALL cast doubt on the prognostic reliability of this molecular determinant as well as on the efficacy of sole chemotherapy in a substantial proportion of patients with TEL/AML1-positive ALL. Therefore, alternative, therapeutic strategies need to be developed, for example, a vaccine-based immunotherapy. Immunotherapy of acute leukemias includes the use of specific leukemia-associated epitopes with recurring fusion proteins serving as specific target antigens [6, 7]. One approach can depend on leukemia cells to present an array of known and unknown antigens as dendritic cells (DC) [8] by converting leukemic cells directly into DC [9, 10]. The TEL/AML1 fusion represents a recurrent, cytogenetic abnormality leading to the expression of a unique, leukemiaspecific antigen [11]. TEL recruits nuclear corepressor molecules [12] and histone deacetylases [13, 14] to DNA transcriptional regulatory elements. In response to the acetylation of core histone proteins, the accessibility of transcription factors to DNA is disturbed [15]. This leads to transcriptional repression of AML1-responsive genes [16], thereby impairing normal hematopoiesis [17]. Reversion of transcriptional repression, for example, by histone deacetylase inhibition [18], leads to the expression of AML1-regulated genes [19, 20] as well as the up-regulation of adhesion molecules and cell differentiation [21]. Histone deacetylase inhibitors (HdI) can therefore stop tumor growth [22], inducing the apoptosis of cancer [23] and leukemic cells [24 –26]. As HdI promote lymphopoiesis of TEL/AML-positive blasts [27], we hypothesized that histone deacetylase inhibition could also support DC differentiation. In fact, leukemic DC differen-

1 Correspondence: Institute for Transfusion Medicine, Campus VirchowKlinikum, Charite´–Universita¨tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: [email protected] Received August 6, 2008; revised November 19, 2008; accepted December 2, 2008. doi: 10.1189/jlb.0808469

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tiation of three different cell lines positive for AML1 fusion proteins was enhanced after HdI application [28]. Leukemic DC from all of these blasts only induced a blast-specific T cell response if the DC had been differentiated after the application of trichostatin A (TSA), a fungicide antibiotic. TSA reversibly inhibits histone deacetylases of eukaryotic cells, inducing a morphological reversion of transformed cells [29]. However, its toxicity generally precludes its clinical administration. In contrast, methyl-isobutyrate (ISO), a derivative of short lipoid acids, is usually well-tolerated [30]. Other HdI used in clinical practice are suberoylanilide hydroxamic acid (SAHA), also called vorinostat [31], and valproic acid (VAL) [32–34]. Here, we present our first preclinical data about the generation of an antileukemic DC vaccine from patients with ALL.

MATERIALS AND METHODS Patient samples and DC generation Leukemic blasts were obtained from bone marrow specimens of 136 children with confirmed ALL after written, informed consent was obtained from the parents or guardians. Twenty-six of the 136 ALL samples were positive for the translocation t(12;21) carrying the fusion TEL/AML1. Samples with blast fractions below 75% before Ficoll density centrifugation were not included. Twenty samples dropped out from further testing, three (including one TEL/ AML1-positive specimen) because of bacterial contamination, one because of low cell viability, and 16 as a result of cell counts below 106. A total of 100 different blast specimens were cultured in various basic media, and 25 of the blasts were positive for the TEL/AML1 fusion transcript. The best basic medium turned out to be StemSpan 3000 (Stem Cell Technologies, Vancouver, BC, Canada), supplemented with 10% pooled human serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Other media tested were RPMI, supplemented with 10% or 20% FBS and/or 10% horse serum and QBSF-60 (Quality Biological, Gaithersburg, MD, USA), supplemented with 5% human serum. Leukemia cells (0.5–1⫻106) were cultured in six-well plates with the following cytokines (all PeproTech, St. Katharinen, Germany): 25 ng/mL GM-CSF (G; specific activity, 2.5⫻107 U/mg), 50 ng/mL TNF-␣ (T; specific activity, 1⫻108 U/mg), 50 ng/ml fetal liver kinase 2/fetal liver tyrosine kinase 3 ligand [Flt3L (F); specific activity, 1⫻106 U/mg], 50 ng/ml stem cell factor [c-kit ligand (K); specific activity, 5⫻105 U/mg], with or without 20 ng/ml IL-3 (I3; 1⫻107 U/mg) and 10 ng/mL IL-4 (I4; specific activity, 1⫻107 U/mg). A mix of TNF-␣, GM-CSF, c-kit ligand, and FLT3L (TGKF) was compared with a cytokine combination of these four plus IL-3 and IL-4 (TGKFI3I4). The following HdI (all Sigma-Aldrich, Dreieich, Germany) were added to the cells on the first day of culture: TSA (64 nM), SAHA (500 nM), VAL (250 nM), and ISO (250 nM). Concentrations were chosen according to preliminary, dosedependent toxicity studies (not shown). TSA and ISO were dissolved in 98% ethanol, and SAHA and VAL in calcium- and magnesium-free PBS (pH 7.4).

Cytospin The morphology and maturation features of the cells in the aqueous cultures were documented weekly by phase-contrast microscopy and cytospin preparations as described previously [35]. A Leica microscope, in combination with the Leitz DMRBE digital camera (Visitron Systems Imaging, Puchheim, Germany), was used for digital photography. Pictures were taken at 1000⫻ magnification using the SPOT advanced program, Version 3.5.5 (Diagnostic Instruments, Sterling Heights, MI, USA).

Flow cytometry Cells with detectable, morphological changes were analyzed for DC-specific markers on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) as described previously [35]. The following directly conjugated anti-human mAb were used: CD83-phycoerythrin (Immunotech, Marseille, France), CD1a-PE (Becton Dickinson), CD80-PE (BD PharMingen, San Diego, CA,

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USA), CD86-fluorescence isothiocyanate (BD PharMingen), HLA-DR-FITC (BD PharMingen), and CD14-FITC (Immunotech). Isotype-matched IgG antibodies served as negative controls.

Expression of the TEL/AML1 fusion protein PCR After cytokine treatment, leukemic DC were analyzed by quantitative real-time RT-PCR as described previously [28]. Briefly, 5 ⫻ 106 DC were pelleted and incubated with 1 ml TRIzol reagent for 5 min. Cells were then mixed with 0.2 ml chloroform for 3 min at room temperature and centrifuged at 11,500 rpm for 15 min at 4°C. The upper aqueous phase of the sample was collected into RNase-free Eppendorf tubes and mixed with 0.5 ml isopropanol for 10 min. Samples were recentrifuged, supernatants were aspirated, and pellets were resuspended in 75% ethanol in diethyl pyrocarbonate water by vortexing. Following air-drying, RNA quantities were measured via spectrophotometry. Total RNA was quantified using a spectrophotometer (Biophotometer, Eppendorf, Hercules, Hamburg, Germany), and 2 ␮g per sample was reversetranscribed using the SuperScript II kit (Invitrogen, Karlsruhe, Germany) using the recommended reaction conditions. Equivalents (50 ng) of cDNA were then used for quantitative TaqMan PCR using 5 U/ml Taq polymerase (Platinum DNA polymerase, Life Technologies, Inc., Gaithersburg, MD, USA), custommade TEL (5⬘-TggCTTACATgAACCACATCATgg, exon 5), and AML1 primers (5⬘-ggAAggCggCgTgAAgC, exon 3; TIB Molbiol, Berlin, Germany) [36]. Analysis was performed on the i-Cycler MyiQ (Biorad, Munich, Germany). Relative gene expression was calculated using the ⌬ comparative threshold method with ␤-actin expression as a normalization reference. TEL/AML1(⫹) DC differentiated by TGKFI3I4 with VAL (250 nM) were analyzed after 1 week in culture and compared with blasts differentiated in cytokines only.

Fluorescence in situ hybridization (FISH) TEL/AML1 fusion chromosomes were detected by a Vysis LSI TEL/AML1 ES dual-color translocation probe (Abbott GmbH and Co. KG, Wiesbaden-Delkenheim, Germany), according to the manufacturer’s instructions; the TEL probe was labeled directly with SpectrumGreen, and the AML1 probe was labeled with SpectrumOrange. The TEL probe hybridizes to a 350-kb region telomeric to the TEL breakpoint cluster. The AML1 probe, ⬃500 kb in size, hybridizes to the entire gene and therefore, gives an extra split signal when the TEL/AML1 fusion chromosome is present. A minimum of 100 interphase nuclei was analyzed by an Axiovert 200 (Zeiss, Germany) using the AxionVision Rel.4.5 program. Images were edited with the Adobe Photoshop CS3 software package.

MLR and stimulation of HLA-matched or autologous T cells When the highest purity of leukemic CD83(⫹)DR(⫹) cells was achieved, an aliquot of the differentiated cells was irradiated at 30 Gy, and an allogeneic MLR was performed using 3H-thymidine incorporation as described previously [35]. Autologous-like stimulation was achieved by culturing HLA-A and -B matched PBMC from healthy volunteers with leukemic DC. PBMC were isolated from heparinized blood by Ficoll density gradient (Biochrom AG, Berlin, Germany). To obtain effector cells for cytotoxicity and IFN release assays, isolated PBMC were pretreated with IL-2 (100 U/ml, PeproTech) plus IL-7 (100 U/ml, PeproTech) in RPMI 1640 supplemented with pooled human serum and 2 mM L-glutamine and 100 U/mL penicillin/streptomycin (all supplied by Biochrom AG) for 1 week. Then, 1 ⫻ 106 PBMC per well were cultured with 3.3 ⫻ 105 irradiated DC [yielding a stimulator/responder (S/R) ratio of 1/3] in 24-well plates for 3 days. Afterwards, IL-2 and IL-7 were readded, and sensitization was continued for another 4 days (for details, see ref. [28]). If ⬎108 leukemic cells were obtained from the patients, autologous stimulations were performed: One aliquot of at least 3 ⫻ 106 cells was cultured immediately as future target cells; one-third of the leukemic blasts was used for DC generation, and one-half was cultured in cytokines alone and one-half cultured in cytokines plus HdI. In cases with ⬎5 ⫻ 108 leukemic cells at baseline, two HdI were administered to cell populations in separate flasks. Two-thirds of the leukemic blasts were cultured in IL-2 and IL-7 as the future effector cells (CTL). Irradiated DC and effector cells were mixed 1:3 after 1 week and cocultured for an additional week.

http://www.jleukbio.org

Cytotoxicity assay Target cells (5⫻103 naı¨ve blasts) were cultured with sensitized effector cells at E/T ratios of 25/1, 12.5/1, and 6.25/1 for 5 h in 96-well round-bottom plates containing phenol red-free RPMI-1640 medium plus 5% human albumin (200 g/L, Baxter, Heidelberg, Germany). Cytotoxicity was measured using a lactate dehydrogenase (LDH) release assay, according to the manufacturer’s instructions (Promega, Madison, WI, USA). Light absorption was determined at 490 nm by a microplate reader (MR 5000, Dynatech Laboratories, Denkendorf, Germany). The percentage of specific lysis was calculated using the following formula: 100⫻ (experimental release–spontaneous release target–spontaneous release effector)/(maximum release–spontaneous release target). Maximum releases were determined by lysing leukemic blasts and TEL/AML1-positive REH cells. Effector cells cultured in ILs alone without DC priming helped to rule out nonspecific reactions.

Germany) was added after 2 h. After 16 h, cell suspensions were washed with PBS, incubated with 1 mM EDTA for 10 min, and washed again, this time, with 2% polyclonal IgG (Endoglobuline, Baxter Hyland-Immuno Division, Unterschleissheim, Germany). For extra- and intracellular IFN-␥ staining, fluorescence-conjugated antibodies (Becton Dickinson, Heidelberg, Germany) were used after the cells had been treated by FACS lysing and FACS permeabilization solution (Becton Dickinson). Flow cytometry data were acquired on a FACSCalibur in a minimum of 250,000 events and analyzed using the CellQuest software (both Becton Dickinson).

Statistical analysis, ethics Student t-test was used on Microsoft Excel 2000 to evaluate the differences between two groups. P ⬍ 0.05 was considered to be statistically significant. The Ethical Commission of the Charite´–Universita¨tsmedizin Berlin (Germany) approved the study.

Intracellular IFN-␥ staining We analyzed T cell responses against the 15-mer peptide NH2-MP IGRIAE CIL GMN PS-COOH, which covers the TEL/AML1 fusion region and was shown to stimulate proliferation of TEL/AML-reactive CD4⫹ T cells [37]. Our assumption that the 15-mer peptide will bind to HLA-A2 is based on the paper by Yotnda and colleagues [11], who identified the nonapeptide RIAECILGM as binding to HLA-A2.1, in particular. In fact, the published sequence corresponds to positions 4 –12 of our 15-mer fusion protein. It does not need antigen processing and can be recognized by CD4(⫹) and CD8(⫹) T cells (unpublished observation). Intracellular IFN-␥ staining was performed as described previously [38]. In brief, 2 ⫻ 106 PBMC or nondifferentiated blasts were incubated at 37°C and 5% CO2 with 10 ␮g/ml TEL/AML1 fusion protein in Iscove’s medium supplemented with 10% FBS, 2% L-glutamine, and 1% penicillin/streptomycin. PBMC incubated with an irrelevant HIV epitope served as a negative control. Peptide-presenting cells and DC-sensitized T cells were coincubated, and 7.5 ␮g/ml brefeldin A (Sigma-Aldrich, Steinheim,

RESULTS Morphology When leukemic TEL/AML1-negative (Fig. 1A) and TEL/ AML1-positive (Fig. 1B) blasts were cultured in four cytokines consisting of TGKF, they developed membrane bulges and increased in size, and the nucleus/plasma ratio shifted toward plasma. Culturing the blasts in six cytokines including IL-3 and IL-4 (TGKFI3I4) induced the formation of hairy dendrites. Histone deacetylase inhibition shifted the plasma/nucleus ratio toward plasma but did not further influence the dendritic

Fig. 1. Morphology. Leukemic blasts cultured in TGKF were larger in size with an increased cytoplasm content. Further addition of I3 and I4 induced the development of prominent dendrites, which were not enhanced after application of HdI. Cytospin preparations were performed after 1 and 2 weeks of culture. Shown are 1-week results of one TEL/AML1-negative (A) and one TEL/AML1-positive (B) example.

Schmidt et al. Blast differentiation after histone deacetylase inhibition

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morphology. Overall, 81% of TEL/AML1-negative and 76% of TEL/AML1-positive samples could be differentiated into DC, as determined by morphology and/or flow cytometry.

HdI improve immunophenotypic differentiation of TEL/AML1-positive DC but have only marginal effects on TEL/AML1-negative blasts The expression of DC surface markers on treated and nontreated leukemic blasts was assessed by flow cytometry. Nondifferentiated blasts cultured in basic medium were highly positive for HLA-DR (⬎80%) and almost negative for CD83 (⬍2%). They were generally positive for CD80, and a small

fraction of cells was CD86(⫹)80(–). The four cytokines led to an up-regulation of CD83 and HLA-DR, which was almost doubled by the addition of IL-3 and -4 to the cytokine cocktail. Although dendritic differentiation of TEL/AML1-negative blasts was not affected by VAL (Fig. 2A), TEL/AML1-positive blasts showed a higher expression of CD83 and HLA-DR in both cytokine conditions and developed more CD80 and CD86 in conditions without ILs (Fig. 2B) if VAL had been applied. TEL/AML1-negative blasts

In comparison with control samples, the percentages of CD83(⫹)DR(⫹) cells were fourfold higher during the 1st week

Fig. 2. CD80, CD83, CD86, and HLA-DR expression of a TEL/AML1-negative and a TEL/AML1-positive leukemic DC. TGKF induced a significant up-regulation of CD83 within 1 week compared with blasts cultured in basic media. This was propagated further in cytokine cocktails using IL-3 and IL-4. Histone deacetylase inhibition by VAL had no effect on TEL/AML1-negative blasts (A) but further increased CD83 and HLA-DR expression in a TEL/AML1-positive sample (B). Here, VAL also enhanced CD80 and CD86 in cytokine conditions without ILs. Shown are the results of two independent experiments. Shaded histograms, sample glycoprotein; black line, IgG control.

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TABLE 1.

Frequency and Absolute Cell Counts of TEL/AML1-Negative CD83(⫹)DR(⫹) and CD86(⫹)80(–) Cells Week 1

Week 2

CD83(⫹)DR(⫹)

Control TGKF ⫹SAHA ⫹VAL ⫹ISO ⫹TSA TGKFI3I4 ⫹SAHA ⫹VAL ⫹ISO ⫹TSA

CD86(⫹)80(–)

CD83(⫹)DR(⫹)

CD86(⫹)80(–)

%

⫻104

%

⫻104

%

⫻104

%

⫻104

5.4 ⫾ 1 21.4 ⫾ 3a 19 ⫾ 3.1a 21.3 ⫾ 3.4a 21.1 ⫾ 3.4a,b 15.9 ⫾ 4.4a,b 27.5 ⫾ 3.3a 25.3 ⫾ 3.3a 28.5 ⫾ 3.5a 29.7 ⫾ 3.5a 31 ⫾ 6.9a

2.8 ⫾ 0.78 8.2 ⫾ 1.4a 6.6 ⫾ 1.2a 6.6 ⫾ 1.2a,b 7.9 ⫾ 1.6a 5.9 ⫾ 1.9a 12.3 ⫾ 2.5a 8 ⫾ 1.1a 11.6 ⫾ 2.5a 10.9 ⫾ 2a 12.2 ⫾ 3.5a

21.4 ⫾ 3.1 29.1 ⫾ 4b 30.4 ⫾ 4.1b 32.3 ⫾ 4.2a,b 31.4 ⫾ 4.5a,b 28.9 ⫾ 6.8 40.5 ⫾ 3.6a 40.1 ⫾ 3.6a 44.8 ⫾ 3.6a 42.6 ⫾ 4.1a 44.7 ⫾ 7.3a

11.7 ⫾ 2.7 13.3 ⫾ 2.7 12.8 ⫾ 2.5 12.2 ⫾ 2.2b 14 ⫾ 3.1 10.9 ⫾ 3.4 18.4 ⫾ 2.9a 14.4 ⫾ 1.9 18.8 ⫾ 2.6a 17.9 ⫾ 3.1 17.8 ⫾ 4.3

3.9 ⫾ 1.1 23.7 ⫾ 4.4a 22.9 ⫾ 4.9a 22 ⫾ 4.3a 21.2 ⫾ 4.4a 17.4 ⫾ 4.5a 25.1 ⫾ 5a 22.5 ⫾ 4.2a 20.4 ⫾ 4.6a 22.5 ⫾ 4.9a 27.2 ⫾ 5.4a

0.79 ⫾ 0.19 5.6 ⫾ 1.2a 5.1 ⫾ 1.1a 4.8 ⫾ 1.1a 5.1 ⫾ 1.1a 3.8 ⫾ 1a 5.4 ⫾ 1.2a 5.4 ⫾ 1.1a 4.8 ⫾ 0.81a 4.7 ⫾ 0.97a 5.8 ⫾ 1.2a

19.4 ⫾ 4.2 38 ⫾ 5.1a 31.9 ⫾ 4.8a,b 37.2 ⫾ 4.9a,b 40.7 ⫾ 5.5a 31.9 ⫾ 7.3 42.4 ⫾ 4.7a 46.8 ⫾ 4.7a 48.6 ⫾ 4.2a 43.5 ⫾ 4.9a 42 ⫾ 7a

5.3 ⫾ 1.6 10.3 ⫾ 2.2a 7.2 ⫾ 1.3b 9.6 ⫾ 2b 10.5 ⫾ 2.3a 7.5 ⫾ 2.2 10 ⫾ 1.6a 11.4 ⫾ 1.4a 14.9 ⫾ 2a,c 9.7 ⫾ 1.5a 9.1 ⫾ 2.7

TEL/AML1-negative leukemic DC (n⫽55) developed in two different cytokine combinations TGFKF and TGKFI314. aSignificantly different compared with control; bsignificantly different compared with cytokines only; csignificant difference between both cytokine conditions.

and twice as high during the 2nd week of culture (P⬍0.05) in all cytokine conditions (Table 1). This led to two- and threefold increases in the absolute numbers of CD83(⫹)DR(⫹) cells. Supplementation with I3 and I4 predominantly enhanced the expression of CD86. After 1 week, TGKF plus I3I4 induced higher frequencies of CD86(⫹)80(–) cells than culture conditions without IL (P⬍0.021). In the absence of ILs, VAL and

TABLE 2A.

No

Initials

Age

Sex

Disease stage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

KL VM BT KH VS LA ZI WL SL GS MD SF GB KK BL DM KJ KT HM KL BJ ET MM KM LT

8 5 8 4 10 6 5 3 10 5 11 9 3 8 6 8 3 1 7 8 3 10 7 7 7

F M M F M F F M F M M M M M F F M M M F F F M M M

1st relapse diagnosis 1st relapse diagnosis 1st relapse 1st relapse diagnosis diagnosis 1st relapse diagnosis 2nd relapse 1st relapse diagnosis diagnosis diagnosis 1st relapse diagnosis diagnosis 1st relapse 1st relapse diagnosis 1st relapse 1st relapse 1st relapse diagnosis

ISO enhanced CD86 expression as compared with control samples. Addition of VAL also led to significantly higher numbers of CD86(⫹)80(–) cells than basic media (P⬍0.048) or cytokine cultures with ILs (P⫽0.032) after 2 weeks. All other HdI did not support leukemic DC glycoprotein expression of TEL/AML1-negative blasts compared with cytokines alone.

Characteristics of TEL/AML1-Positive Patients

ALL phenotype

Blast fraction

Pretreatment

DC morphology

DC phenotype

common pre-B pre-B pre-B common pre-B pre-B common common pre-B pre-B common common common common common common common common common common common pre-B common common

99% 90% 95% 98% 96% 98% 98% 97% 89% 99% 100% 94% 86% 82% 99% 94%a 94% 95% 97% 75% 95% 98% 95% 99%a 89%

ALL-BFM 95 n.a. CO-ALL n.a. ALL-BFM 95 B-NHL BFM 04 n.a. n.a. ALL-BFM 2000 n.a. O-ALL 89; ALL-REZ BFM 96 ALL-BFM 2000 n.a. n.a. n.a. ALL-BFM 2000 n.a. n.a. CO-ALL CO-ALL n.a. CO-ALL ALL-BFM 2000 ALL-BFM 2000 n.a.

– –/⫹ – ⫹ (⫹) ⫹ ⫹ ⫹ ⫹ ⫹ (⫹) – (⫹) ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

– –/⫹ – –/⫹ – –/⫹ n.t. ⫹ ⫹ ⫹ ⫹ – ⫹ ⫹ ⫹ ⫹ ⫹ n.t. ⫹ n.t. ⫹ ⫹ n.t. ⫹ ⫹

Initials, age, therapeutic regime, and sex, as well as the state of disease and blast fraction of blast donors are summarized. DC differentiability according to morphology and immune phenotype is also mentioned. If the dendritic phenotype were not assessed, cytotoxicity tests or TEL/AML1 RT-PCRs were performed. In two patients, DM and KM, the blast fraction in the bone marrow was 0 and 21%, respectively. aBlast fraction in liquor. Therapeutic regimes: ALL-BerlinFrankfort-Munster 95 (BFM 95) [39]; childhood onset (CO)-ALL [40]; B cell non-Hodgkin lymphoma (B-NHL) BFM 04 [41]; ALL-BFM 2000 [42]; ALL-relapse (REZ) BFM 96 [43]. n.a., not applicable; n.t., not tested.


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TABLE 2B.

Frequency and Absolute Cell Counts of TEL/AMLI-Positive CD83(⫹)DR(⫹) and CD86(⫹)80(–) Cells Week 1

CD83(⫹)DR(⫹)

Control TGKF ⫹SAHA ⫹VAL ⫹ISO ⫹TSA TGKFI3I4 ⫹SAHA ⫹VAL ⫹ISO ⫹TSA b

Week 2 CD86(⫹)80(–)

CD83(⫹)DR(⫹)

CD86(⫹)80(–)

%

⫻104

%

⫻104

%

⫻104

%

⫻104

13.6 ⫾ 5.1 38.4 ⫾ 6.7a 38.6 ⫾ 7.7a 43.7 ⫾ 6.9a 43.2 ⫾ 6.7a 30.7 ⫾ 9.2 47.2 ⫾ 7a 45.4 ⫾ 7.6a 44.6 ⫾ 6.7a 49.5 ⫾ 7.4a 27.2 ⫾ 9.1c

6.5 ⫾ 2.8 14.8 ⫾ 2.4a 13.4 ⫾ 3.6 17.9 ⫾ 5a 18.5 ⫾ 4.5a 6.4 ⫾ 1.5c 16.9 ⫾ 3.4a 15.7 ⫾ 3.2a 18.1 ⫾ 3.4a 16.9 ⫾ 3.5a 8.5 ⫾ 3.1

19.5 ⫾ 5.6 21 ⫾ 5.7 24.4 ⫾ 7b 28.3 ⫾ 7c 21 ⫾ 5.3b 11.5 ⫾ 5.1 34.5 ⫾ 7.1a 43.9 ⫾ 9.4a 41.9 ⫾ 7.8a 38.8 ⫾ 8.9a 17 ⫾ 8.3

8.8 ⫾ 3.5 9.2 ⫾ 2.6 10.6 ⫾ 3.3 10.8 ⫾ 3.1 8.3 ⫾ 2.3 1.8 ⫾ 0.6c 12.6 ⫾ 2.7 16 ⫾ 3.5 21 ⫾ 4.5a 13.1 ⫾ 3 2.7 ⫾ 0.7c

10.9 ⫾ 4.7 26.3 ⫾ 7.1 36 ⫾ 6.5a,c 29.3 ⫾ 7.4a 29.6 ⫾ 6.5a,b 21.3 ⫾ 6.7 35.7 ⫾ 7.3a 37.7 ⫾ 6.8a 33.3 ⫾ 6.8a 39.3 ⫾ 8.4a 32 ⫾ 8.6a

2.5 ⫾ 1.3 6 ⫾ 1.8 7.9 ⫾ 2.1a 6.5 ⫾ 1.2a 6.5 ⫾ 1.5a 5.9 ⫾ 2.2 9.5 ⫾ 2.2a 11.8 ⫾ 3.7a 8.4 ⫾ 1.4a 8.3 ⫾ 1.5a 6.8 ⫾ 1.7a

10.6 ⫾ 2.7 12.5 ⫾ 1.9b 19.1 ⫾ 3.1a,b 17.6 ⫾ 2.5b 12 ⫾ 1.7b 10.1 ⫾ 3.6 25.1 ⫾ 5.1a 32.9 ⫾ 6.2a,b 33.9 ⫾ 4.6a 26.5 ⫾ 7.1a 22.3 ⫾ 9.3

2 ⫾ 0.58 3.2 ⫾ 0.47 3.5 ⫾ 0.45a,b 4.9 ⫾ 1.3a 2.7 ⫾ 0.44b 2.1 ⫾ 0.86c 6.3 ⫾ 1.1a 10.2 ⫾ 1.9a,c 12.5 ⫾ 2.8a,c 6.5 ⫾ 1.6a 4.2 ⫾ 1.7

TEL/AML1-positive leukemic DC developed by two different cytokine combinations; TGFKF and TGKFI1314. aSignificantly different compared with control; significantly different compared with cytokines only; csignificant difference between both cytokine conditions.

TEL/AML-positive blasts

The characteristics of children with TEL/AML1-positive ALL are summarized in Table 2A. Stage of disease, age (mean 6.5⫾2.6; range 1–11 years), sex, blast fraction, or therapeutic regime did not influence DC differentiability. All culture combinations except those using TSA induced higher frequencies of CD83(⫹)DR(⫹) cells than found in blasts cultured in basic media alone (P⬍0.05, Table 2B). SAHA increased CD83 expression in TGKF (P⫽0.025). Higher frequencies of CD83(⫹)DR(⫹) cells were also found in cultures with TGKF plus VAL or ISO, and VAL improved CD86 expression significantly in 1-week values (P⬍0.035). After 2 weeks of culture, SAHA and VAL led to higher absolute numbers of CD86(⫹)80(–) cells than TGKFI3I4 alone (Pⱕ0.03). Interestingly, TEL/AML1-positive blasts developed a 10 –20% higher frequency of CD83(⫹)DR(⫹) cells than TEL/AML1-negative blasts, which was significant in cytokine cocktails including ILs (P⬍0.05). CD1a and CD14 remained negative throughout the culture period in all samples tested, arguing against a monocytic differentiation pathway. The immunophenotypes reflected the allostimulatory capacities of leukemic DC.

HdI improve the allostimulatory capacity of TEL/ AML1-positive DC Histone deacetylase inhibition reduced the allostimulatory capacity of TEL/AML1-negative, leukemic DC. The addition of SAHA and VAL led to a significantly lower 3H-thymidine incorporation than TGKF alone (P⬍0.05; Fig. 3A). VAL, in particular, reduced the allostimulatory capacity compared with cytokines alone at S/R ratios of 1/60 and 1/30. At the latter S/R ratio, ILs further enhanced the SAHA-dependent decrease in immunogenity (26.1⫾3.5⫻103 cpm vs. 38.1⫾6.1⫻103 cpm; P⫽0.046; Fig. 3B). In contrast, TEL/AML1-positive, leukemic DC were more immunopotent when they had been differentiated after histone deacetylase inhibition. DC generated with four cytokines in the absence of HdI (Fig. 3C) showed a significantly higher immunostimulatory activity than nondifferentiated blasts, starting at 568


a S/R ratio of 1/240 (21⫾1.9⫻103 cpm vs. 15.6⫾2.2⫻103 cpm; P⫽0.042). TSA increased the allostimulatory capacity significantly so that a significantly higher value than in control cells was already achieved at 1/480 (19.6⫾2.5⫻103 cpm; P⫽0.035). ISO and VAL further increased the alloresponses at high S/R ratios, although differences were not significant. At these ratios, TSA-derived DC led to lower 3H-thymidine incorporation than DC from TGKF alone. Addition of IL-3 and -4 to TGKF increased the allostimulatory capacity: Leukemic DC then induced a more extensive T cell proliferation than control cells, beginning from the lowest ratio of 1/480 (19⫾2.6⫻103 cpm vs. 14.5⫾1.5⫻103 cpm; P⫽0.041; Fig. 3D). The highest allostimulatory capacity was observed in leukemic DC generated in TGKFI3I4 plus ISO (50.3⫾7.1⫻103 cpm; S/R ratio 1/15). Most HdI except TSA and SAHA enhanced the immunogenicity of DC. Addition of VAL, in particular, increased the allostimulatory potential of TEL/AML1-positive, leukemic DC significantly, compared with cytokines alone at S/R ratios of 1/240, 1/60, and 1/30 (P⬍0.025).

Retention of fusion transcripts and chromosomes despite DC differentiation In five out of six ALL, TEL/AML1 mRNA levels remained the same after DC differentiation and histone deacetylase inhibition (Fig. 4A). Only the blasts from Patient 14 showed a significantly lower transcript mRNA level after administration of VAL than in cytokines alone. In FISH, TEL/AML1 fusion chromosomes were present in more than 65% of nuclei of leukemic DC, independent of HdI application (Fig. 4, B and C). In 10 –15%, two TEL/AML1 fusion chromosomes were detected.

Leukemic DC induce a TEL/AML1-specific CTL response When TEL/AML1 was loaded on autologous blasts, an increase in IFN-␥- and TNF-␣-producing CTL was observed (Fig. 5, A and B). In two cases tested, CTL produced more IFN-␥ than upon HIV peptide presentation, when leukemic DC generated after histone deacetylase inhibition had sensitized them. CTL http://www.jleukbio.org

Fig. 3. MLRs were performed by mixing irradiated, 1-week-old leukemic DC (ranging from 157 to 10,000 stimulators) and freshly isolated PBMC (150,000 responders), which equals S/R ratios of 1/480 –1/15 on the x-axis. After 5 days of coculture, 1 ␮g/well 3H-thymidine was added for overnight incubation, and incorporation was determined as cpm (y-axis). Leukemic DC generated in four (A, C) and six (B, D) cytokine conditions were analyzed. Histone deacetylase inhibition did not increase the T cell stimulatory capacities of TEL/AML1-negative DC (A, B). In cytokine conditions based on TGKF, VAL and SAHA rather reduced the allostimulatory capacities of DC (A). Addition of IL-3 and IL-4 to TGKF increased the reaction response further, and values in SAHA cultures were significantly lower at a S/R ratio of 1/30 (B). In contrast, TEL/AML1-positive, leukemic DC showed higher allostimulatory capacities if they had been generated with HdI (C, D). VAL even significantly increased alloresponses of DC generated in TGKFI3I4 (D). Mean results ⫹ SE of 11 (TEL/AML1–) and seven (TEL/AML1⫹) independent experiments in triplicate. *, Significant difference to naı¨ve leukemic blasts; #, significant difference to cytokines only; ⫹, significant difference between both cytokine conditions.

sensitized by DC generated in cytokines alone showed a peptide-dependent increase of IFN-␥ in one case only.

Leukemic DC sensitize antileukemic CTL According to the immunphenotypic results, VAL and SAHA seemed to be the most promising HdI for leukemic DC differentiation. Therefore, these were tested further in their capacity to stimulate a cytotoxic response. Unprimed T cells induced a maximum cytolysis of 12.7 ⫾ 4.9% at an E/T ratio of 25/1. Effector cells primed by cytokine-derived DC generated without HdI were significantly more efficient than nonprimed T cells (P⫽0.032). At low E/T ratios, cytolysis of TEL/AML1negative blasts was equivalent, regardless of the use of HdI in DC differentiation (Fig. 6A). At the highest E/T ratio of 25/1, however, leukemic DC generated in the absence of HdI induced a significantly higher CTL response than those generated after application of HdI (29.2⫾13.6% vs. 3.8⫾2.9%; P⫽0.047). On the other hand, TEL/AML1-positive, leukemic DC caused higher reactions after the use of HdI (Fig. 6B). DC

differentiated in cytokines alone induced a cytotoxicity of 18.8 ⫾ 12% of nondifferentiated blasts. CTL responses induced by DC generated after histone deacetylase inhibition through VAL were more than twice the amount (48.4⫾21.1%). SAHA-generated DC caused the highest CTL reaction against nondifferentiated blasts at the lowest E/T ratio of 6.25/1 (33.7⫾20% vs. cytokines alone 20.1⫾12.2% and VAL 23.4⫾14.1%). When both HdI culture conditions were tested versus cytokines alone, cytolysis, using TEL/AML1-positive DC generated after histone deacetylation, was significantly higher than without HdI (P⫽0.037), except for VAL at the lowest E/T ratio.

DISCUSSION In our efforts to optimize the generation of leukemic DC, we found that TGKF were sufficient to induce DC differentiation of leukemic blasts from patients with ALL. This differentiation


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Fig. 4. Retention of the TEL/AML1 fusion mRNA and chromosome. Leukemic TEL/AML1-positive leukemic DC were analyzed for their content of a fusion transcript after culture for 1 week in cytokines (TGKFI3I4) plus VAL (A). Values remained constant in five out of six ALL samples. In one ALL (Number 14), histone deacetylase inhibition by VAL reduced the level of TEL/AML1 mRNA expression by –2.128 to 0.47 ⫾ 0.21 (P⫽0.01) as compared with blasts cultured in cytokines alone. All other DC had the same level as in the nondifferentiated specimen. Mean results of two to three different measurements per sample in triplicate. TEL/AML1-associated chromosome aberrations in interphase FISH analysis of leukemic DC cultured in cytokines (B) and in cytokines plus VAL (C) confirmed the retention of TEL/AML1 fusion chromosomes. SpectrumGreen detected the TEL gene; SpectrumOrange detected the AML1 gene. TEL/AML1 fusion chromosomes (white) are indicated by colocalization of one orange and one green signal (arrows). In cytokine cultures alone, two nuclei had a diploid set, and one overexpressed AML1.

was enhanced further by IL-3 and IL-4. IL-3 serves as a survival and expansion factor in hematopoietic progenitor cells [44]. IL-4 plays an important role in the generation of monocytic DC [45], as it can down-regulate the monocytic marker CD14 [46, 47] and suppresses macrophage development [48]. Therefore, CD1a and CD14 remained negative in TEL/AML1negative and -positive cell populations throughout the study period, excluding the presence of monocytic intermediates and contaminants. Mature DC are characterized by their enormous size, high cytoplasm content, spine-like projections [49], the expression of CD83 [50], and their capacity to stimulate T cell proliferation [51]. All of these characteristics were present in our leukemic DC. Culture conditions based on StemSpan, in particular, led to a frequency of up to 50% CD83(⫹)DR(⫹) leukemic DC, which is more than twofold of those achieved by other groups [52–55]. The higher frequency of CD83(⫹) cells in our study might be explained by the use of pooled human serum instead of animal components, as this also improved DC differentiation from peripheral blood monocytes [35]. CD83 and costimulatory molecules could also indicate the differentiation of activated B cells. Given the culture conditions chosen and considering the morphologic appearance of the cells, however, the generation of B cells is rather unlikely. Differentiation of leukemic cells can be supported by histone deacetylase inhibition [18, 25], opening a new arena for epigenetic anticancer therapies [56 –58]. In several AML cell 570


lines, HdI up-regulate costimulatory molecules such as CD80 and CD86 [59]. This is consistent with our observations, as VAL increased the frequencies and numbers of CD86(⫹)80(–) cells in TEL/AML1-negative blasts. In TEL/AML1-positive blasts, the effect of SAHA was even more prominent, as it increased the frequency of CD83(⫹)DR(⫹) cells significantly in cultures with four cytokines after 2 weeks. At the same time-point, SAHA and VAL generated the highest counts of CD86(⫹)80(–) cells, which were up twofold of cytokine conditions consisting of TGKFI3I4. Histone deacetylase inhibition might also influence monocytic DC differentiation positively, but this remains the objective of future studies. HdI not only affected the immunophentoype but also the leukemic DC functional capacities. VAL, ISO, and SAHA increased the allostimulatory potential of TEL/AML1-positive, leukemic DC, as the proliferation of allogeneic T cells was higher compared with cytokines alone. In contrast, TEL/AML1negative, leukemic DC were less immunopotent if they had been differentiated after histone deacetylase inhibition. This could indicate that HdI activated immunosuppressive, regulatory T cells in TEL/AML1-negative specimens [60, 61]. A HdI-induced, antigen-specific anergy in CD4(⫹) T cells [62] could also be the reason for a reduced immunopotency. In our previous study, the leukemic DC carrying an AML1dependent translocation could only induce a leukemia-specific CTL response when they had been differentiated after inhibition of histone deacetylation. Cytotoxicities ranged at approxhttp://www.jleukbio.org

Leukemic DC positive for the t(12;21) fusion generally retained expression of the fusion transcript despite DC differentiation. Blasts from only one patient showed significantly lower mRNA levels of TEL/AML1 after HdI application. We do not have a good explanation for this exception, however it could possibly justify why we did not observe a clear benefit in the peptide-specific response. Leukemic DC are thought to be impaired in comparison with “normal” DC [64], although we could not find any morphologic or immunophenotypic differences [65]. Interestingly, TEL/ AML1-positive blasts developed a higher frequency of CD83(⫹)DR(⫹) cells than TEL/AML1-negative blasts. This could reflect the better prognosis of patients with TEL/AML1positive ALL, as the TEL/AML1 fusion seems to be formed during immunoreceptor gene rearrangement [66]. In agreement

Fig. 5. Analysis of TEL/AML1 peptide-specific T cell responses. When naı¨ve leukemic blasts were used as the peptide-presenting cells (A and B), higher frequencies of IFN-␥- and TNF-␣-specific CTL responses to TEL/AML1loaded than to HIV-loaded blasts were observed if the DC had been generated after histone deacetylase inhibition. In the absence of HdI, only one patient showed a TEL/AML1-induced increase of CD8(⫹)3(⫹) IFN-␥-producing cells. Results of two independent experiments— one with blasts and DC from Patient 19 (A) and one with blasts and DC from Patient 25 (B). y-Axis: Percentage of IFN-␥- or TNF-␣-positive CD8(⫹)CD3(⫹) cells; x-axis: presentation of HIV or TEL/AML1 peptide.

imately 10% in the absence of HdI, and we showed previously that cytolysis is based mainly on CD8(⫹) rather than on CD4(⫹) T cells [28]. In the present study, leukemic DC derived from TEL/AML1-negative blasts sensitized HLAmatched T cells better if they had been grown in the absence of HdI. In contrast, TEL/AML1-positive, leukemic DC induced a blast cytolysis of ⬃50% after histone deacetylase inhibition. Cytolysis was twofold of that induced by DC generated in cytokines alone, which reached 20% standing in accordance with other studies [54, 63]. Furthermore, TEL/AML1-dependent, intracellular IFN-␥ secretions of cytotoxic T cells increased compared with HIV presentation in two cases if the T cells had been primed by DC generated after HdI application. In one case, CTLs, sensitized by DC generated in cytokines without HdI, showed a higher IFN-␥ release as well. Therefore, a HdI-dependent, improved peptide-specific response could be possible but cannot be concluded at this stage.

Fig. 6. Cytotoxic T cell response of leukemic DC. CTL primed by TEL/AML1negative, leukemic DC (n⫽12) differentiated in TGKFI3I4 alone were superior to those generated after application of HdI at the highest E/T ratio (A). Effector cells were obtained from partially HLA-matched volunteer donors or taken from the initial blast specimen and prestimulated with IL-2 and IL-7 before they were primed with leukemic DC. Naive, nondifferentiated leukemic blasts were used as target cells. Percent cytotoxicity (y-axis) was determined by LDH releases. In contrast, TEL/AML1-positive, leukemic DC differentiated in cytokines only induced lower cytotoxic T cell responses than leukemic DC differentiated after administration of VAL or SAHA (B). Here, differences of SAHA and VAL conditions compared with cytokines alone were significant except for VAL at the lowest E/T ratio. Shown are mean values ⫾ SEM of seven (cytokines and VAL) and four (SAHA) independent experiments of TEL/AML1-positive samples in triplicate or quadruplicate. #, Significant difference between CTL sensitized by DC differentiated in cytokines only and CTL sensitized by DC differentiated after addition of HdI.


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with our previous report [65], leukemic DC and cultured blasts showed no migratory activity in response to the chemoattractants used (not shown). This stands in line with reports from clinical vaccination trials, in which the majority of ex vivomanipulated DC remained at the site of injection [67]. Therefore, leukemic DC need to be administered intralymphatically, as recommended by others [68]. In conclusion, our results pay tribute to the role of HdI in the ex vivo generation of leukemic DC. Among the four compounds tested, VAL and SAHA enhanced the “dendriticity” of leukemic DC, most prominently from patients with TEL/AML1positive ALL. Their application led to higher frequencies of CD83(⫹) and CD86(⫹) cells, higher numbers of CD86(⫹)80(–) cells, and higher allostimulatory and cytotoxic T cell reactions than cytokines alone. VAL even increased the number of CD86(⫹)80(–) cells in TEL/AML1-negative blasts. Clinical studies comparing leukemic DC, manufactured with and without HdI, could prove the superiority of either DC type.

ACKNOWLEDGMENTS This study was supported by the German Cancer Aid (Deutsche Krebshilfe e.V., grant 107418). A. M. is currently supported by a Feodor-Lynen scholarship (Alexander von Humboldt-Foundation) and a Rahel-Hirsch scholarship (Charite´). We are indebted to Manuela Ko¨ppen and Willhelmine Keune for excellent technical assistance. We also acknowledge Sandra Bauer for the intracellular IFN-␥ staining and Gabriele Ko¨rner and Thomas Heiden for interphase FISH analysis.

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