Supplemental methods
This study was approved by the local ethics committee of the Provincial Government of
Salzburg, Austria and was conducted according to the Declaration of Helsinki. Peripheral
blood samples from patients were collected during routine examinations after obtaining
informed consent. Immunohistochemistry (IHC) and immunofluorescence (IF) stainings were
performed on formalin-fixed paraffin-embedded (FFPE) tissue samples (lymph node, bone
marrow) obtained for diagnostic purposes. A detailed list of patients participated in this study,
including prognostically relevant parameters and detailed experimental involvement, is
provided as supplemental table S2.
Cell lines
NIH/3T3 murine fibroblasts transfected with human CD40L or empty vectors (Mock) were
kindly provided by Katja Zirlik, Freiburg, Germany. NIH/3T3 cells were periodically
authenticated by morphological inspection. CD40L expression on CD40L-transfected and
control cells was constantly monitored by flow cytometry.
Cell culture
Peripheral blood monocytes (PBMCs) were isolated using Bicoll Separating solution
(Biochrom) from whole blood samples. Cells were cultured in RPMI 1640 medium (PAA
laboratories) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (PAA
laboratories), 2 mmol/L L-glutamine (Sigma-Aldrich), and 10 mg/L Penicilin/Streptomycin
(Biochrom), and incubated at 37°C in a humidified atmosphere containing 5% CO2.
Lymphocyte separation
Purification of CD19+ B cells was performed by immunomagnetic positive selection (Miltenyi
Biotec) following manufacturer’s recommendations. Isolation of CLL cells was performed
using Easy Sep kit for B cell enrichment w/o CD43 depletion (StemCell) and CD19-positive
selection (Miltenyi Biotec), respectively. Purity of isolated cells was more than 95%. CD4+ T
cells were isolated by negative selection using CD4+ T cell isolation kit (Miltenyi Biotec)
yielding purities between 65-98%;
Flow cytometry (FC)
Cells were stained with a mixture of fluorescence-labelled antibodies depending on the
respective experimental setup; for a detailed list see supplemental table S2. Lymphocytes
were pre-gated according to size and granularity parameters. CLL cells were further identified
according to their CD5-CD19 expression profile. All Samples were analysed using a FC500
or Gallios flow cytometer and RXP1.0 software (Beckman Coulter) or Flowjo 7.5.2 software.
Activation assays
PBMCs from CLL patients were cultured at a concentration of 3 x 106/ml. Activation was
performed using phorbol-12-myristat-13-acetat (PMA) and ionomycin (Sigma-Aldrich)
(PMA: [10 nM]; ionomycin: [175 nM] 3).
Proliferation assays
For cell cycle activation, CLL cells were co-cultured in PBMCs at a concentration of 1 x
106/mL enriched with α-CD3/CD28 microbeads-stimulated (invitrogen) autologous T4 cells
in ratios from CLL:T4 = 1:1 to 5:1 (also see Asslaber, 2013, Mimicking the
microenvironment in chronic lymphocytic leukaemia - where does the journey go?). CLL
cells were either stained with carboxyfluorescein succinimidyl ester (CFSE) (invitrogen) and
cultured for five to six days depending on their proliferative capacity, or remained unstained
in culture for three days, each on a cell layer of murine fibroblasts (M2-10B4).
Inhibition of proliferation
CLL PBMCs (containing at least 5% T cells) were pre-stimulated at a concentration of 1 x
106/mL on a confluent murine fibroblast layer (NIH-3T3) transfected with human CD40L for
24 h or control MOCK transfected fibroblasts (also see Asslaber, 2013, Mimicking the
microenvironment in chronic lymphocytic leukaemia - where does the journey go?). Cells
were then incubated with K+ channel blockers: PAP-1 (synthesized by H. Wulff, UC Davis,
CA, USA), Psora-4, TRAM-34, Clotrimazole (Sigma-Aldrich), in given concentrations, 30
min before T cells within PBMCs were activated with α-CD3/CD28 beads (invitrogen). For
toll-like receptor (TLR) stimulation, cells were pre-stimulated at a concentration of 1 x
106/mL with CpG DSP30 oligonucleotide (1 µM) (eurofins mwg operon) (Liang, 1996,
Activation of human B cells by phosphorothioate oligodeoxynucleotides) on a murine
fibroblast layer (M2-10B4) for 24 h, before blockers were added in given concentrations and
further activated by IL-2 (100 ng/mL) (BD biosciences). Ki-67 expression was determined
after an additional 48 h of culturing.
Quantitative real-time PCR (qRT-PCR)
Total RNA isolation was performed with RNeasy® isolation kit (QIAGEN) according to
manufacturer’s instructions. RNA yield was measured by photospectrometry. The amount of
total RNA was used for cDNA synthesis using oligo(dT)15 primers and iScriptTM cDNA
synthesis kit following manufacturer’s recommendations (BIO RAD). RT-PCR was performed
using TaqMan® RT-PCR gene expression kit, see manufacturer’s instructions (Applied
Biosystems). TaqMan® probes for human K+ channels were used: Kv1.3: Hs 00704943_s1;
KCa3.1: Hs 00158470_m1; (herg1: Hs 00918382_m1); All cDNAs were standardized to 18S
RNA.
Electrophysiology
CLL cells were seeded on poly-D-lysine-coated coverslips at 37°C and 5% CO2 for 30 min.
Further experiments were conducted at room temperature. Subsequently cells were labeled
with a PE-conjugated CD19-antibody (see suppl. table S2), visualized by an AxioVert.A1
fluorescence microscope (Zeiss), connected to the patch clamp setup. Cells were patchclamped in the whole-cell configuration using an EPC10 amplifier (HEKA) and processed via
Patch Master software (HEKA). Standard external solution contained [mM]: 140 NaCl, 2.8
KCl, 2 MgCl2, 1 CaCl2, 10 Hepes, 11 glucose (pH 7.2; 300 mOsm). Kv-currents were elicited
using a pipette solution containing [mM]: 134 KF, 2 MgCl2, 1 CaCl2, 10 Hepes, 10 EGTA
(pH 7.2; 300 mOsm). To identify Kv1.3 currents, cells were clamped at a holding potential of
-80 mV intermitted by repeated 200 ms voltage steps of from -80 to +40 mV using a 10 mVinterval applied every 30 s. Current maxima at +40 mV were used for the calculation of Kv1.3
conductance. Kv1.3-associated cumulative use-dependent inactivation was identified by
applying 10 consecutive voltage steps from -80 to +40 mV every 1 s. Kv-currents were
inhibited by application of 200 nM PAP-1. For KCa3.1 measurements, pipette solution
contained [mM]: 120 K+ glutamate, 2 MgCl2, 8.7 CaCl2, 10 Hepes and 10 EGTA (pH 7.2; 310
mOsm), resulting in a free intracellular Ca2+ concentration of 1 µM. KCa3.1, were elicited by
a voltage ramp protocol from -120 to +40 mV over 200 ms acquired at 0.5 Hz and a holding
potential of -80 mV. Data were sampled with 5 kHz and digitally filtered with 1.25 kHz.
KCa-currents were completely blocked by 500 nM TRAM-34. Conductance of KCa3.1
channels was assessed by the slope at -80 mV calculated from the mean of 50 consecutive
voltage ramps. Patch pipettes used exhibited a mean resistance of about 3 MΩ. To calculate
the number of channels per cell, the single channel conductance for Kv1.3 of 12 pS
(Grissmer, 1990, Expression and chromosomal localization of a lymphocyte K+ channel gene)
and 11 pS for KCa3.1 (Ghanshani, 2000, Up-regulation of the IKCa1 potassium channel
during T-cell activation. Molecular mechanism and functional consequences.) was used. Total
conductance measured was divided by single channel conductance. For assessment of channel
density, the total capacitance of the cell membrane was monitored over the whole period of
the measurement. A relation of 0.01 pF/µm2 cell surface was assumed. Data was analyzed
using Fit Master (HEKA) and Igor Pro (WaveMetrics) software.
Immunohistochemistry (IHC)
FFPE tissue samples from patients or healthy donors were pretreated for IHC as previously
described (Wulff, 2004, K+ channel expression during B cell differentiation: implications for
immunomodulation and autoimmunity) and labeled with antibodies for CD19, Ki-67 and
KCa3.1, respectively (see supplemental table S2). Primary antibodies were labeled with the
appropriate species secondary HRP-labeled antibodies (see supplemental table S2) and
visualized via DAB. Images were obtained using a Olympus IX81 microscope and 4x, 10x or
20x objective and Olympus xcellence software.
Immunofluorescent (IF) staining and confocal microscopy
Multicolor IF stainings on FFPE tissue samples and PBMC cytoblocks were performed in an
automated manner on Dako Autostainer Plus™. For native staining, cells were fixed with 4%
paraformaldehyde (PFA), permeabilized using 0.2% Triton X-100 and blocked with 3 mg/mL
donkey serum (Jackson ImmResLab). Primary antibodies were conjugated to appropriate
donkey whole IgG affinity purified antibodies (see supplemental table S2) for 30 min at RT.
Negative controls for KCa3.1 antibody (Sigma-Aldrich) were performed in human brain and
heart muscle tissue. All secondary antibodies were used alone to deny unspecific binding.
Samples were embedded using ProLong® Gold mounting medium (life technologies),
containing 4,6 diamidino-2-phenylindole (DAPI). Imaging was performed with a confocal
LSM510 microscope (Zeiss) and objective alpha Plan-Fluar 100x/1.45 oil; within each
experiment all fluorescent images were taken under the same conditions. Data was assessed
using Zen 2009 (Zeiss) software and analysed using ImageJ 1.46. Fluorescence intensity (FI)
from confocal images was measured and calculated using ImageJ 1.46 and Windows Excel
2007. For each patient and sample 30 cells were randomly chosen from 3 independent images
and the mean FI of negative controls (= second antibody only) was subtracted from total FI to
obtain final FI values.
Fluorescence-assisted cell sorting (FACS)
Previously isolated and magnetically separated (CD19+) CLL cells were stained with
fluorescently labeled antibodies against CXCR4 (CD184) and CD5 (see supplemental table
S2) and sorted in two population according to their expression profile (see Fig. 2), using a
Coulter Epics Altra cell sorter (Beckman Coulter). Gated populations constituted between 5
and 10% of the whole populations. Purities achieved were more than 96%.
Statistical Analysis
Statistical analysis and graphical representation was performed using GraphPad Prism®
Version 5.02 and Windows Excel 2007. Following a Gaussian distribution (D’Agostino and
Pearson omnibus normality test) p-values were calculated for a confidence interval of 95% by
paired or unpaired Student’s t-test. When calculating p-values for normalized data, onesample t-test for a hypothetical value of 1.0 was used. In case of non-parametric data either
Mann-Whitney U or Wilcoxon-ranked pairs test was used. Box plots were created following
Tukey approach. FC measurements were always based on the detection of at least 5,000 cells
(for exception see Figure S5C, due to limited T cell numbers). Prognostic risk factors of
patients involved (see supplemental table S3) were considered for statistical relevance for
every experiment. No significant differences were detected.