This study was conducted from January 2009 to January 2011. Patients with clinical evidence of acute severe cerebral infarction were eligible for enrolment in the study (n=30). Other inclusion criteria were age 40 – 80 years, radiographic evidence of acute cerebral infarction, patients hospitalized within 6 hours after the onset of stroke, a score of not less than 16 as per the National Institutes of Health Stroke Scale (NIHSS) on admission. The exclusion criteria were: if the onset was along with infectious and autoimmune diseases, patients who received thrombolytic therapy after the onset, patients receiving immune regulation therapy within 6 months before the onset, patients with history of cerebral stroke within 12 months before the onset, patients who had history of severe cerebral trauma or neurosurgery, seizures during the onset and at the time of admission, renal or hepatic insufficiency at admission, history of blood transfusion within 12 months before the onset, severe trauma or surgery within 2 weeks before the onset, and history of severe psychological disease. A group of healthy adult volunteers (n=30; age: 40-80 years) were also included in the study for comparison. All patients or their family members provided signed informed consent and the protocol was approved by The Committee of Medical Ethics, East Hospital, Tongji University.
Antibodies and reagents
Only mouse anti-human antibodies (purchased from BD Biosciences, USA) were used in the study. This included allophycocyanin (APC) conjugated to CD3 (CD3-APC), peridinin chlorophyll protein (PerCP) conjugated to CD8 (CD8-PerCP), fluorescein isothiocyanate (FITC) conjugated to cluster of differentiation 107a (CD107a-FITC), and phycoerythrin (PE) conjugated to interferon-γ (IFN-γ-PE) and tumor necrosis factor-α (TNF-α-PE). Ficoll-Biocoll, a cell isolation liquid and phosphate-buffered saline (PBS) were used (Biochrom AG, Germany). Perm/Wash™ buffer was used in the analysis to serve as an antibody diluent and cell wash buffer (BD Biosciences, USA). Cytofix/Cytoperm™ solution (BD Biosciences, USA) was used to increase the permeability of cell membrane. BD GolgiStop™ (BD Biosciences, USA), was used as a protein transport inhibitor containing monensin and was required to promote cytokine accumulation in the Golgi complex. RPMI-1640 (Roswell Park Memorial Institute medium, Sigma, Germany) was used as the cell culture medium (CTM). Cells were cultured in RPMI-1640 supplemented with 20 mM HEPES ([4, (2-hydroxyethyl]-1-piperazineethanesulfonic acid), 2 mM glutamine, 1% penicillin/streptomycin (Sigma, Germany), and heat-inactivated 10% human AB serum (3H Biomedical AB, Sweden). CellTrace™ carboxyl fluorescein diacetate succinimidyl ester (CFSE) was used as a cell proliferation reagent (Invitrogen, USA). Trypan blue solution (0.4%) was used to assess cell proliferation and viability by dye exclusion method (GIBCO, UK). Recombinant human interleukin-2 (rhIL-2) was purchased from ProSpec (Rehovot, Israel) and CD28/CD49d costimulatory antibody from BD Biosciences (USA). Mixed virus peptide used in the study was CTL-CEF-Class I peptide pool “Plus” (CEF peptide) (Cell Technology Ltd, USA). CEF peptide was a pool of 32 peptides, with sequences derived from the human cytomegalovirus, Epstein-Barr virus, and influenza (flu) virus.
Isolation of peripheral blood mononuclear cells
PBMCs were isolated from whole blood as the red cells could have interfered with the flow cytometer analysis. In our preliminary experiments, we observed the apoptotic ratio of isolated PBMCs with Ficoll-Biocoll method and with lysate method (data not shown). Annexin V Apoptosis Detection Kit was used to detect apoptosis. The viability of PBMCs obtained was always > 95% with Ficoll-Biocoll method and < 50% with Lysate method. Hence, Ficoll separation method was used in the study.
Peripheral venous blood (20 ml) samples were collected from patients with cerebral infarction 6 hours after the onset of stroke in a heparinized test tube. The entire blood sample was diluted to a final volume of 20 ml with PBS. This suspension was then poured into 7 ml Ficoll-Biocoll separating solution in a conical tube. After density gradient centrifugation (1500 rpm, 20°C, 30 minutes, without brake), peripheral blood mononuclear cells (PBMCs) were isolated. The isolated cells were collected carefully and washed with sterile PBS and re-suspended in RPMI-1640. Cell viability was determined by trypan blue staining and cells were counted; cell viability should always be > 95%. Final density of the cell in CTM was adjusted to 5-6 × 106 cells/ml. PBMCs from healthy volunteers were isolated in the same way. Collection of blood from the patient population and healthy volunteers was carried out at Philipps-University Marburg, Germany.
CD107a degranulation analysis
For the expression of CD107a, 20 μl of CD107a-FITC antibody was added into 80 μl of PBMCs suspension (2-3 × 106 cells/ml). PBMCs suspension mixed with CD107a was dispensed in a flat-bottom 96-well plate. Into each well, 2 μl of CD28/CD49d costimulatory antibody was also added. Further, 100 μl of CEF peptides with concentrations of 64 μg/ml were added in the CEF-treated-control group, and the same amount of CTM in the negative control group. The culture was incubated for 60 minutes at 37°C in 5% CO2 incubator after which 0.5 μl BD GolgiStop containing monensin was added. The incubation was continued for 120 minutes. After washing, cells were stained with CD3-APC and CD8-PreCP antibodies and incubated for 30 minutes at 4°C in the dark. Cells were centrifuged, supernatant was discarded and resuspended in 130 μl of CTM. Cells were analyzed on the four-color flow cytometer (FACSCalibur®, CellQuest® software, Becton Dickinson). At least 50000 events (events refer to the number of particles recorded by flow cytometry) were collected per cell. In the lymphocyte gate, CD8+ T lymphocyte for CD107a expression was defined as CD3+/CD8+/CD107a.
Intracellular IFN-γ and TNF-α analysis
For analysis of intracellular IFN-γ and TNF-α, 2 μl CD28/CD49d costimulatory antibody was added to 100 μl of PBMCs. This suspension was dispensed into flat-bottom 96-well plate. Further, 100 μl of CEF peptides with concentration of 64 μg/ml was added in the CEF-treated-control group, and the same amount of CTM in the negative control group. The culture was incubated for 60 minutes at 37°C in 5% CO2 incubator and 0.5 μl BD GolgiStop containing monensin was added. This was further added at an interval of 6 hours during the next 24-hour incubation. Cells were centrifuged and supernatant was discarded. After washing, cells were stained with CD3-APC and CD8-PerCP antibodies, and incubated at 4°C for 30 minutes in the dark. Cells were then fixed with the 100 μl of BD Cytofix/Cytoperm solution and incubated for 20 minutes at 4°C in the dark. After washing and centrifugation, cells were suspended in Perm/Wash buffer solution and 20 μl of IFN-γ-PE and 20 μl of TNF-α-PE antibodies were added and incubated for 30 minutes at 4°C in the dark. Cells were analyzed on the four-color cytometer (FACSCalibur®, CellQuest® software, Becton Dickinson). Data from at least 50000 events per cell were acquired. In the lymphocyte gate, IFN-γ positive CD8+ T lymphocyte was defined as CD3+/CD8+/IFN-γ + and TNF-α positive CD8+ T lymphocyte was defined as CD3+/CD8+/TNF-α + .
Cell proliferation analysis
CFSE stock solution was prepared and added into PBMCs in culture media. The working concentration of CFSE used was 0.4 μM. The culture media was incubated in the dark for 10 minutes at 37°C. The freed CFSE was inactivated with ice on CTM. It was again centrifuged and washed and cells were resuspended in CTM. The cell density was set at 2-3 × 106 cells/ml. Cell suspension (stained with CFSE) was then dispensed into a flat-bottom 96-well plate. In cell suspension, 2 μl of CD28/CD49d costimulatory antibody was added. Then 100 μl of CEF peptides with concentration of 64 μg/ml and rhIL-2 with final concentration of 40 IU/ml was added in the CEF-treated-control group, and the same amount of CTM was added in the negative control group. After 5 days of incubation at 37°C in 5% CO2 incubator, cells were centrifuged, supernatant was eliminated and CD3-APC and CD8PerCP antibodies were added and incubated for 30 minutes at 4°C in the dark. Cells were again centrifuged, supernatant was discarded, washing was repeated, and cells were resuspended in 130 μl of CTM. Cells were analyzed on the four-color flow cytometer (FACSCalibur®, CellQuest® software, Becton Dickinson). At least 50000 events were collected per cell. The percentage of proliferating cells was measured by the percentage of low CFSE cells in CD3+/CD8+ gate (in the upper left quadrant of each plot). The definition for low CFSE cells was defined according to the distribution of CFSE dye in baseline, which was measured in unstimulated cells. CFSE decrease was a result of dye dilution in each cell division.
All values were expressed as mean ± standard deviation (SD). Continuous data were analyzed using the paired t-test. For statistical comparisons, a p value less than 0.05 was considered to be significant.