Apch7 anti cd8

PBMC were stimulated for 18 hours with heat-inactivated B. pseudomallei (50μg/well) or media. Brefeldin A (Ebioscience, USA) was added at 10 ug/ml and following 4 hours of further incubation, staining for intracellular APC-IFN-γ and for the immune cell surface markers PCP-anti-CD3, FITC-anti-CD4, APCH7-anti-CD8, PE-anti-CD56 and V450-CD14 all BD Biosciences, USA) was performed. Samples were analysed using a MACSQuant Analyzer 10 (Miltenyi Biotec, Germany) with Flowjo software (Treestar Inc, USA). The gating strategy used to identify the cellular source of IFN-γ after stimulation with heat-killed B. pseudomallei was firstly selection of singlets to exclude aggregates. The lymphocyte populations were then selected for immunophenotyping: double positive CD3+CD4+ for CD4 T cells, double positive CD3+CD8+ for CD8 T cells, double positive CD3+CD56+ for NKT cells and positive CD56 negative CD3 for NK cells. For CD14 positive, cells were gated from singlets. IFN-γ expression levels were then determined by analysis of each cell phenotype. Responses were quantitated for the negative control-subtracted percentage of IFN-γ secreted CD4+ or CD8+ cells from the gated lymphocyte population.

The following fluorochrome-labeled monoclonal antibodies (mAb) against human molecules were used: APC-anti-CD3 (SK7), PerCP/Cy5.5-anti-CD4 (SK3), APC-H7-anti-CD8 (SK1), PE-anti-HLA-DR (TU36), PE-Cy7-anti-CD19 (SJ25C1), FITC-anti-CD45RA (L48), Brilliant Violet 421-anti-CD27 (M-T271), PE-anti-CD25 (2A3), Alexa 488-anti-FOXP3 (259D/C7), Pacific Blue-anti-CD4 (RPA-T4), PE-Cy5-anti-CD21 (B-ly4), FITC-anti-IgD (IA6-2), APC-anti-IgM (G20-127), APC-H7-anti-CD38 (HB7), PE-anti-CD24 (ML5) from BD; APC-anti-CXCR5 (J252D4), PE-Cy7-anti-CD45RA (HI100), Pacific Blue-anti-CXCR3 (G025H7), and Brilliant Violet-anti-CCR6 (G034E3) from BioLegend.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples and used for the quantification of cellular activation by flow cytometry. PBMCs were stained with antibodies: BUV BUV395-anti-CD45 (BD Horizon), BV605-anti-CD3 (Biolegend), ECD-anti-CD4 (Beckman Coulter), APC-H7-anti-CD8 (BD Pharmingen), AF700-anti-CD38 (BD Pharmingen), BV785-anti-HLA-DR (Biolegend), as well as Aqua intracellular dye for dead cells (Invitrogen; L34957). Specimens were analyzed using a LSRFortessa X-20 cytometer (BD Biosciences), and inter-experiment consistency was calibrated using rainbow bead (Spherotech URFP-30-2). Proportional quantification of cellular activation subsets was done using FlowJo (Treestar, Woodburn, Oregon, USA).