Anti cd8 apc h7

One ml of whole blood was collected in a sodium heparin tube from each volunteer at study day 0, day 38, day 98 and day 154 during the period of the study, which coincided with a clinical follow-up visit day. One hundred fifty μl of whole blood was used for staining with a cocktail of antibodies against: anti-CD3 BV650, anti-CD8 APC-H7, anti-γδTCR PE, anti-Vδ2 FITC purchased from e-bioscience. Red blood cells were lysed using the BD FACS Lyse solution and the cells were washed two times with PBS and acquired on a LSRII flow cytometer equipped with a Blue (488 nm), Red (633 nm) and Violet laser (405 nm). γδT cells (Vδ2+ and Vδ2− populations) were enumerated as a percentage of total CD3 T cells.

Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).