Htlv 1 tax peptide

Analysis of CD107a cell surface mobilization to determine cell-mediated cytotoxicity of CD8+ T cells was quantified using the flow cytometer. PBMCs from ATLL and AC subjects known to display considerable HTLV-1 specific clusters to HLA-A*0201/Tax 11–19 and HLA-A*2402/Tax 301–309 were suspended in medium, RPMI 1640 (supplemented with 10% heat-inactivated FCS, 100 Uml penicillin, 0.1 mg streptomycin) and cultured in a 96 well plate in a 5% CO2 incubator at 37°C for 4 hours with or without 0.02 µM HTLV-1 Tax peptide (Sigma-Aldrich, Japan) in combination with CD107a/IgG1 isotype MAbs-FITC, secretion inhibitor monensin (Immunocyto CD107a detection kit, MBL, Japan). Anti-CD48 antibodies (5 ug/ml) were also added to the culture. Harvested cells were washed and further stained with HLA- tetramer CD8PerCP (BD Bioscience, San Jose, CA). About 1×10⁵ CD8+T cells were acquired by FACScan or FACS Calibur flow cytometer and analyzed with Flowjo Software.

PBMCs were cultured in 96 well plate in 5% CO2 incubator at 37°C for 6 hours in RPMI 1640 medium with or without 0.02 µm HTLV-1 tax peptide (Sigma-Aldrich, Japan) and brefeldin A (BD San Diego, CA) at a final concentration of 10 µg/ml. CD48-blocking antibodies were added at a final well concentration of 5 µg/ml [16] (link). Harvested cells were washed twice with phosphate buffered saline (PBS) and stained with anti-CD8 PerCP (BD Biosciences, San Jose, CA USA) or CD8-PC5 (Cytostat-Coulter, Fullerton USA), PE-conjugated HLA-A*0201 and HLA-A*2402 for 15 minutes at room temperature. Cells were washed and then permeabilized using FACS permeabilizing solution 2 (BD San Diego, CA) for 10 minutes at room temperature. Cells were again washed, suspended in saponin-containing PBS for 10 minutes at room temperature and then incubated with FITC-labeled perforin and isotype control IgG2b κ-FITC (BD) for 20 minutes at room temperature. Finally, cells were washed and analyzed with FACS Calibur flow cytometer and FlowJo Treestar software based on forward and side scatter in the lymphocyte gate as described [31] (link).