Ifn γ pe b27

Cell phenotype was analyzed by flow cytometry. 1 ml heparinized blood samples were collected and stimulated with 20 ng/ml phorbol-12-myristate-13-acetate (PMA) and 1000 ng/ml ionomycin in the presence of Golgi-Stop (BD Biosciences) for 5 h, then incubated with anti-human mouse antibodies against CD3-FITC-A, CD4-BV510-A (BD Pharmingen), CD8-APC-H7 (BD Pharmingen), IFN-γ-PE-B27(BD Pharmingen). In all experiments, a control antibody of the respective IgG isotype was included. Flow assay was detected using FACS Calibur flow cytometer (BD, USA). Data was analyzed with FlowJo v.7.6.4 software (Tree Star).

Ex vivo isolated PBMCs from 11 HLA-A*02+ and 1 HLA-A*02- HIV donors were rested overnight in RPMI 1640 supplemented with 5% human serum (Institut Jacques Boy), 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 mM Hepes, 1% nonessential amino acids, 1 mM sodium pyruvate. Cells were pulsed with individual peptides (1 μM) for 2 hours at 37°C and brefeldin A (5 μg/mL; Molecular Probes) was added for 6 hours at 37 °C. Cells were fixed, permeabilized, and stained using standard procedures with CD8-APC (RPA-T8), TNFα-PE (MAb11), IL-2-PE (MQ1-17H12), IFN-γ-PE (B27) (BD Pharmingen), and MIP-1β-FITC (24006, R&D Systems). Cells were analyzed with a FACS-Calibur™ cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo software (Tree Star).