Anti tim 3 pe

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.

Cryopreserved PBMCs were first stained with fixable viability stain 510 (BD Biosciences, San Diego, CA, USA) for 15 min to exclude dead cells, then washed, and surface stained with anti-CD3-BV650 (BD Biosciences), anti-CD4-AF700 (Biolegend, San Diego, CA, USA), anti-CD8-Percp-Cy5.5 (BD Biosciences), anti-CD38-BV786 (BD Biosciences), anti-HLA-DR-Pacific blue (Biolegend), anti-CD160-AF488 (eBioscience San Diego, CA, USA), anti-PD-1-BV605 (Biolegend), anti-TIGIT-APC (eBioscience), and anti-TIM-3-PE (R&D Systems, Inc., USA) at room temperature for 20 min. Cells were permeabilized and fixed with intracellular staining reagents according to the manufacturer’s instructions (eBioscience) before intracellular staining with anti-ki67-PE-Cy7 (Biolegend). Samples were then washed before acquisition and analysis on a BD LSRFortessa flow cytometer instrument with Diva software (BD Biosciences). Relative isotype controls or fluorescence minus one samples were prepared to facilitate gating. Data were analyzed using Flowjo Software version 10 (Tree Star Inc., Ashland, OR, USA).