Facsdiva 2

At the same time as phenotype detection, expression of intracellular IFN-γ, tumor necrosis factor (TNF)-α, CD107a or Granzyme B was determined by ICS. Total leukocytes were obtained from whole blood by lysis of erythrocytes using PBS containing 0.85% NH₄Cl. The cells were adjusted to ~5×10⁶/mL in RPMI 1640 culture medium supplemented with 10% FCS and stimulated with 100 ng/mL phorbol myristate acetate (PMA) plus 1 μg/mL ionomycin for 4 hours, in the presence of the secretion inhibitor monensin (BD Biosciences). Cells were stained with APC-conjugated anti-TCR γδ mAb, followed by washing with PBS, and fixation in 4% paraformaldehyde. Stained cells were permeabilized using 0.1% saponin (Sigma, St. Louis, MO, USA). Cells were incubated with FITC-conjugated anti-IFN-γ (clone 4S.B3; eBioscience, San Diego, CA, USA), PE-conjugated anti-TNF-α (clone Mab11; BD Biosciences), PE-conjugated anti-CD107a (clone H4A3; BD Biosciences), or FITC-conjugated anti-Granzyme B (clone GB11; BD Biosciences) for 30 minutes at 4°C. Finally, cells were washed with PBS, and analyzed using the FACS Canto II flow cytometer (BD Immunocytometry Systems). Data were analyzed using FACSDiva 2.0 software (BD Immunocytometry Systems).

Blood samples from 10 CHB patients were collected for determination of the phenotype and immune function of γδ T cells before and during IFN-α treatment at 4, 8, 12, 24, 36 and 48 weeks. The phenotypes T cell receptor (TCR) γδ, Vδ1, Vδ2, CD45RA or CD27 were detected according to the manufacturers’ instructions (BD Biosciences, La Jolla, CA, USA). The following fluorochrome-conjugated monoclonal antibodies (mAbs) were used: Peridinin chlorophyll (PerCP)-conjugated anti-CD3 mAb (clone SK7), allophycocyanin (APC)-conjugated anti-TCR γδ mAb (clone B1), phycoerythrin (PE)-conjugated anti-Vδ2 mAb (clone B6), PE-conjugated anti-CD45RA mAb, and fluorescein isothiocyanate (FITC)-conjugated anti-CD27 mAb, and were purchased from BD Biosciences(La Jolla, CA, USA). FITC-conjugated anti-Vδ1 mAb was purchased from Thermo Fisher Scientific (clone TS8.2; Rockford, IL, USA). The appropriate volume of antibody was added to 100 μL fresh peripheral anticoagulated blood and incubated for 30 minutes in the dark at 4°C. Erythrocytes were lysed using BD FACS lysing solution, and cells were washed in PBS supplemented with 1% fetal calf serum (FCS). Stained cells were immediately analyzed using the FACS Canto II flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA). Data were analyzed using FACSDiva 2.0 software (BD Immunocytometry Systems).