Patients and healthy donors
The study design was approved by the local Bioethical Committee at the Medical University of Wroclaw, Poland, and is in accordance with the Helsinki Declaration of 1975. All participants gave written informed consent after the purpose of the study was explained to them. Thirty-eight previously untreated CLL patients of the Clinic of Haematology, Blood Neoplasms, and Bone Marrow Transplantation, Wroclaw Medical University, Poland, were enrolled in this study. In each of them, the diagnosis was established according to generally accepted criteria including the absolute peripheral blood lymphocytosis ≥5 × 109/L and the co-expression of CD5, CD19 and CD23 antigens on malignant cells. The disease stages were determined according to the Rai classification. Clinical and laboratory features are presented in Table 1.
Table 1 Clinical characteristics of CLL patients
Leukocyte-enriched fractions of peripheral blood donated by 15 healthy volunteers matched for age and sex with the CLL patients were purchased from the Regional Centre of Blood Donation and Treatment in Wroclaw, Poland.
Cell isolation and separation procedures
Peripheral blood mononuclear cells (PBMCs) were separated from heparinised freshly drawn peripheral venous blood of CLL patients and healthy controls by buoyant density gradient centrifugation on Lymphoflot (Bio-Rad Medical Diagnostics GmbH, Dreieich, Germany) and washed three times in phosphate-buffered saline (PBS) (without Ca2+ and Mg2+). The PBMCs were suspended in 95 % foetal calf serum (CytoGen GmbH, Sinn, Germany) containing 5 % DMSO (Sigma-Aldrich, St. Gallen, Switzerland) and stored in liquid nitrogen until used.
CLL cells were isolated from PBMCs by negative selection using EasySep Human B Cell Enrichment Kit without CD43 Depletion (STEMCELL Technologies Inc, Vancouver, Canada) according to the manufacturer’s instructions. Following this separation procedure, more than 98 % of the resulting cell population was CD19+CD5+ as assessed by flow cytometry using anti-CD19 and anti-CD5 monoclonal antibodies (mAbs) (Becton Dickinson, BD Biosciences, San Diego, USA). Normal B cells from healthy individuals were isolated from PBMCs by negative selection using the EasySep Human B Cell Enrichment Kit (STEMCELL Technologies Inc, Vancouver, Canada) according to the manufacturer’s instructions, achieving above 98 % purity as assessed by flow cytometry using anti-CD19 mAbs.
Culture conditions
Purified normal CD19+ lymphocytes or CLL cells were suspended at 1 × 106 cells/ml in RPMI-1640 medium (Gibco, Paisley, UK), supplemented with 10 % foetal calf serum (CytoGen GmbH, Sinn, Germany), 2 mmol/l l-glutamine and 50 μg/ml gentamycin (KRKA-Poland, Warsaw, Poland), and cultured using 24-well U-bottom culture plates (Nunc GmbH & Co. KG, Langenselbold, Germany) at 37 °C in a 5 % CO2 humidified atmosphere for 24 and 72 h either in medium alone or together with 1 μM DSP30 (5′-TCGTCGCTGTCTCCGCTTCTTCTTGCC-3′) (TIB MOLBIOL, Berlin, Germany) [24] and 100 U/ml rIL-2 (Eurocetus, Amsterdam, The Netherlands) [25]. For the blocking experiment, purified CLL cells and normal CD19+ lymphocytes were cultured with 1 μM DSP30 and 100 U/ml IL-2 with the blocking anti-CTLA-4 mAbs (50 μg/ml) (BD Pharmingen, BD Biosciences, San Diego, USA) [26] or control IgG2 (50 μg/ml) (BD Pharmingen, BD Biosciences, San Diego, USA).
Immunostaining of CTLA-4, Ki67 protein and flow cytometric analysis
The expression of these molecules was studied in purified CLL cells and normal CD19+ lymphocytes before and after 24- and 72-h culture by a single immunostaining method.
Briefly, for detection of surface expression of the CTLA-4 molecule, the cells were washed twice in PBS (without Ca2+ and Mg2+), divided into tubes at a concentration of 5 × 105 cells per tube and incubated with anti-CTLA-4 (CD152)/retinal pigment epithelium (RPE) mAbs (BD Pharmingen, BD Biosciences, San Diego, USA) for 30 min at 4 °C in the dark. Excess unbound antibodies were removed by two washes with PBS. Following these washes, the cells were resuspended in PBS and analysed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, BD Biosciences, San Diego, USA). For determination of intracellular CTLA-4 expression, the cells were first fixed for 10 min at room temperature in 2 % paraformaldehyde (Fluka, Sigma-Aldrich, Buchs, Germany), washed in PBS and incubated for 10 min at room temperature in BD Permeabilizing Solution 2 (Becton Dickinson, BD Biosciences, San Diego, USA) according to the manufacturer’s instructions. Then, the cells were incubated with anti-CTLA-4 (CD152)/R-phycoerythrin (R-PE) mAbs for 30 min at 37 °C in the dark.
Ki67 protein was detected by staining of the cells with anti-Ki67/fluorescein isothiocyanate (FITC) mAbs (BD Pharmingen, BD Biosciences, San Diego, USA) after fixation and permeabilisation as described in the case of intracellular detection of the CTLA-4 molecule.
Negative controls were always done by omitting the mAbs and by incubating the cells with mouse Ig of the same isotype as the mAbs conjugated with RPE or FITC. At least 10,000 events per sample were analysed. The results were expressed as the proportion of CTLA-4- or Ki67-positive cells. The CellQuest program was used for statistical analysis of the acquired data.
Apoptosis
Before and after cell culture, the apoptosis was determined by flow cytometry using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany) based on a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay. At least 10,000 events per sample were analysed. The CellQuest program was used for statistical analysis of the acquired data.
Statistical analysis
Statistical analyses of the clinical data and laboratory findings were conducted using Statistica 10.0 or PQStat software. For clinical parameters, the mean values and standard deviation were calculated. For all other analysed variables, the median values and 25th and 75th interquartile range were calculated.
All collected data were examined for normal distribution and for homogeneity of variances using the Shapiro-Wilk test and Levene’s test, respectively. If data were normally distributed and had homogeneous variances, the comparisons between both studied groups of CLL patients and healthy individuals were performed using the one-factor analysis of variance (ANOVA) followed by a post hoc test (Scheffe F test). To test the effects of culture and CTLA-4 blockade on analysed variables, the repeated measures ANOVA and the Student’s t test for dependent samples were used. If data were not normally distributed and/or had heterogeneous variances, the non-parametric Kruskal-Wallis one-way ANOVA by rank, the Friedman ANOVA test followed by a post hoc test (Dunn test) and the non-parametric Wilcoxon signed-rank test were applied. In all analyses, differences were considered significant when P ≤ 0.05.