Cd8 pe tr

PFMC or PBMC were stained with LIVE/DEAD yellow fixable dead cell stain kit (Life Technology, Eugene, OR) and with the following two monoclonal antibody staining panels: In Panel 1, cells were surface-stained by anti-CD3-PerCP, CD4-PE-Cy7, CD45RO-FITC, CD69-APC, CD38-AF700 (BD Bioscience, San Jose, CA), CD8-PE-TR (Invitrogen, Frederick, MD), CCR7-APC-Cy7 and HLADR-Pacific Blue (BioLegend, San Diego, CA). Cells were then fixed, permeabilized, and washed with Transcription Factor Buffer Set (BD Pharmingen) according to the manufacturer and stained with anti-HIV-1 p24-PE (KC57) (Beckman Coulter, Indianapolis IN). In Panel 2, monoclonal antibodies included: anti CD25-APC (BioLegend), CCR5-V450 and intracellular staining for Ki67-BV711 (both from BD Bioscience) in addition to anti-CD3, CD4, CD8, CCR7, CD45RO, and HIV-1 p24 as in panel 1. Gates were set using Fluorescent-Minus-One controls for each sample. T cells were identified as naïve (CD45RO-CCR7+), central memory (Tcm) (CD45RO+CCR7+), effector memory (Tem) (CD45RO+CCR7-), and terminally differentiated effector memory (TemRA) (CD45RO-CCR7-). Stained samples were analyzed by a LSRII cytometer (BD). Data were analyzed using FlowJo (Tree Star, Ashland, OR).