Journal of Hematology - Supplements - Haematologica

87
the same buffer was added to all tubes and flow
cytometric analysis was performed immediately. 6
Cell culture: stimulation with PHA
Peripheral blood mononuclear cells (PBMC)
from healthy volunteers and patients were purified
by density gradient centrifugation with
Ficoll-Hypaque , washed in Hank’s balanced salt
solution, and resuspended in RPMI 1640 media
(Seromed SPA, Milan, Italy) supplemented with
10% of fetal calf serum (Seromed), 2 mM Glutamine
and antibiotics. Cell density was adjusted
to 0.5x10 6 cells/mL and then treated as just
described in the Drug Treatment section.
Aliquots of treated or untreated samples were
placed into sterile polystyrene round bottom
tubes with caps, without stimulus or with three
different concentrations of PHA 1, 3, 6 µg/mL. 7
Cells were cultured in a humidified incubator at
37°C in 5% CO2.
Immunofluorescence staining for CD69
expression
Stimulated or unstimulated samples were harvested
at 72h, washed twice in HBSS 1%BSA,
and then 50 µL were put into each test tube and
labeled with CD3 PerCP, CD69 PE and CD4
FITC (Caltag). Labeling was performed for 30
min a 4°C followed by a washing step. Samples
were then resuspended in 500 µL of PBS and
analyzed by flow cytometry.
Apoptosis detection
Apoptosis evaluation of cells treated with MP,
VCR and MP + VCR was carried out using flow
cytometric analysis. 8 Briefly, cells were pelleted
at 200 g for 5 min., washed in PBS and resuspended
in 200 µL of a binding buffer [10 mM
Hepes/NaOH, pH 7.4, 140 mM NaCl and 2.5
mM CaCl2 (Bender MedSystem, Austria)]. Staining
and analysis were carried out according manufacturer’s
instructions. Briefly, 5 µL of FITC-
Annexin V (AnV final concentration: 1 mM) and
10mL of propidiun iodide (PI) (20 mg/mL) were
added to each cell suspension. Cells were incubated
at room temperature for 5-15 min in the
dark and subsequently analyzed by flow cytometry.
Annexin V fluorescence emission was detected
in FL-1 (green fluorescence). PI staining is a
dye-exclusion assay that discriminates between
cells with intact membranes (PI-) and those with
permeabilized membranes (PI+).
Flow cytometry
Samples were analyzed by three-color analysis
using a FACScan flow cytometer (Becton Dickinson
Immunocytometry Systems, San Josè, CA,
USA) equipped with an air-cooled argon ion
laser. Ten thousands events were acquired in list
mode and data analyzed with LYSIS II software.
A gate was defined on the lymphocyte population
on the basis of SSC/FSC properties; in addition
a T-cell gate defined by SSC and Fl3 was
used for CD3 + cells. For all experimental conditions
matched subclass controls were employed
to determine the level of non-specific binding.
Results
The experiments performed on healthy controls
show a marked down-regulation of IFN-γ
and IL-4 intracellular production, indicating
that in vitro drug treatment inhibited both the
Th1 and Th2-like responses. Significantly lower
number of CD3 + T-cells producing IFN-γ and IL-
4 were found in treated samples as compared to
in untreated ones. MP alone caused a more
marked decrease of cytokine production than
VCR alone, but the effect was slightly enhanced
by using the two drugs in combination (Figure
1). We also obtained similar results in patients
submitted to allogeneic transplantation. Data,
expressed as means ± SD for healthy controls
and for a group of patients studied (Table 1),
show that treatment with a combination of MP
and VCR results in a significant decrease of CD3
producing IFN-γ (p< 0.0005) and IL-4 (p