Anti cd8 pacific blue

Frozen PBMCs were thawed, washed then left to settle for 2 h in complete medium (RPMI supplemented with 10% fetal calf serum, penicillin, and streptomycin). For cell surface antibodies, PBMCs were incubated for 30 min on ice with directly labeled antibodies, washed and resuspended in PBS and DAPI as a viability dye to exclude nonviable cells. Intracellular staining of FOXP3 was performed using a kit (eBioscience/Thermo Scientific) as per the manufacturer’s instructions. For the T cell proliferation assay, cells were labeled with CSFE (Invitrogen) for 15 min at 37°C, washed three times in complete medium, then incubated (25,000 cells/well) with CD3/CD28 beads (Invitrogen) for 72 h. Data were acquired using an LSR II Flow Cytometer using the BD FACSDiva operating software. Positive staining was considered based on the negativity of an isotype control and a minimum of 20,000 events were recorded for all samples. Antibodies used were AmCyan-anti-CD3, APC-anti-CD4, PerCP-Cy5.5-anti-CD4, Pacific Blue-anti-CD8, PE-Cy7-anti-CD8 (all BD Biosciences), FITC-anti-CD45RO, PE-anti-CD27 (DAKO), and APC-anti-CD25, PE-Cy7-anti-CD127 (eBioscience).

PBMCs from HLA-B*35:01⁺ individuals infected chronically with HIV-1 were stimulated for 14 days with an epitope peptide (100 nM) to induce peptide-specific bulk T cells. Epitope-specific T-cell clones were generated from bulk T cells using the limiting dilution method in 96-U plates, together with 200 μl of cloning mixture (5 × 10⁵ irradiated PBMCs from healthy donors, 1 × 10⁵ irradiated stimulator cells, and epitope peptides at a concentration of 100 nM in RPMI medium containing FCS, 200 U/ml of recombinant interleukin 2 [rIL-2], and 2.5% phytohemagglutinin [PHA]). In order to establish epitope-specific T-cell lines, PBMCs were stained with both phycoerythrin (PE)-conjugated YF9-tetramer and allophycocyanin (APC)-conjugated FF9-tetramer, followed by staining with fluorescein isothiocyanate (FITC)-anti-CD3 (Dako), Pacific Blue-anti-CD8 (BD Pharmingen, USA), and 7-aminoactinomycin D (7-AAD). Among CD3⁺CD8⁺7-AAD⁻ cells, YF9-specific, cross-reactive, or FF9-specific cells were sorted into a 96-well plate using a fluorescence-activated cell sorter (FACS) Aria I instrument. The sorted cells were stimulated with the corresponding epitope peptide and cultured for 2 to 3 weeks.

PBMCs from patient KI-705 were stained with both PE-conjugated YF9-tetramer and APC-conjugated FF9-tetramer and then stained with FITC-anti-CD3 (Dako), Pacific Blue-anti-CD8 (BD Pharmingen, USA), and 7-AAD. Among CD3⁺CD8⁺7-AAD⁻ cells, WT-specific, cross-reactive, or MT-specific cells were sorted into a 96-well plate by using a FACS Aria I instrument. Unbiased identification of TCR gene usage was assessed as described previously (51 (link)).