Anti helios pe

For flow cytometry analysis cells were washed with PBS containing 0.5% BSA, either surface stained with anti-CD4-eFluor-780, CD4-FITC (eBioscience), anti-CD25-PE, anti-CTLA4-PE, anti-CD39-PE, anti-Helios-PE, anti-TIGIT-PE or fixed, permeabilized, and stained with anti-, Anti-Ki67-PE, anti-Foxp3-APC and isotype controls antibodies (eBioscience) (1:100) in dark. Samples were analyzed using CyAn ADP Analyzer (Beckman and Coulter) and data analysis was done using Summit v4.3 software.

PBMCs were isolated from heparinized blood samples by Ficoll-Paque plus (Amersham, Uppsala, Sweden) density gradient. After isolation, cells were washed two times with RPMI-1640 (Gibco, Auckland, N.Z.) and prepared for further study. The surface and intracellular stainings were performed using the following fluorochrome-conjugated antibodies: anti-CD4-PerCP, anti-CD45RA-FITC, anti-CCR4-PE, anti-CCR5-PE, anti-CCR7-PE, anti-Ki67-PE (BD Pharmingen, San Diego, CA), anti-Tim3-PE, anti-TNFR2-PE (R&D Systems, Minneapolis, MN), anti-Helios-PE, anti-FoxP3-APC (eBioscience, San Diego, CA). For intracellular staining, cells were fixed and permeabilized using the Human FoxP3 Buffer Set (eBiosciences, San Diego, CA) according to the manufacturer’s instructions. Isotype-matched control antibodies were used to define the positive staining populations. Stained cells were acquired and analyzed using a FACSAria cytometer (Becton Dickinson, San Jose, CA, USA) with FACSDiVa software (BD Biosciences).
Liver biopsy specimen was obtained from six chronic HBV infected patients and was chopped and incubated with collagenase-D (0.1%) (Gibco, Waltham, USA) in RPMI-1640 with 10% fetal bovine serum (Gibco, Grand Island, NY). After incubate and filtered through nylon mesh, cells were suspended in RPMI-1640 and then stained with anti-CD4-PerCP, anti-CD45RA-FITC, anti-Tim3-PE and anti-FoxP3-APC.