Anti ccr5

NFkB ELISA was performed using a commercially available kit following manufacturer’s instructions (Active Motif, Belgium). Briefly, 105 sorted cells were treated with 50 ng/ml of CCL3/CCL4 (R & D systems, MN, USA), 5 μg/ml anti-CCR5 (mouse monoclonal, clone 45531, R & D Systems, MN, USA), 5 μg/ml anti-PD-L1 (Clone 29E.2A3, Biolegend, San Diego, CA, USA) or 10 μg/ml recombinant PD-L1 (R & D systems, MN, USA). Cells were stimulated with anti-CD3/anti-CD28. Unstimulated cells were used as controls. Cells were lysed after 180 minutes with lysis buffer provided with the kit to make whole cell lysates, 20 μl of which was added to ELISA wells pre-coated with NFkB consensus DNA binding sequence. Bound NFkB was detected with antibody specific to the NFkB p50 subunit. Raji cell nuclear extract was used as a positive control. NFkB activation was expressed as absorbance at 450nm.

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, NJ, USA). Fresh tissues were washed and cut into small fragments, prior to homogenization by tissue disaggregation vessels (BD Medimachine System). The cells in each sample were adjusted to a concentration of 2 × 10⁶ cells/mL, and 0.5 mL cell suspension stimulated with 2μL Leukocyte Activation Cocktail and BD GolgiplugTM (BD Pharmingen, San Diego, CA, USA) for 4 hours.
T cell subsets were phenotyped in isolated PBMCs or tissue cells by flow cytometry (FACSAria flow cytometer, BD Biosciences, NJ, USA) according to manufacturer’s instructions. The cells were labelled with specific monoclonal antibodies; including anti-CD4-PerCP, anti-CD25-PE, anti- Foxp3-FITC, anti-IL-4-PE and anti-IFN-γ-FITC (all from BD Biosciences, NJ, USA). T cell subsets were selected for detailed phenotypic analysis as follow: (1) Th1 cells: IFN-γ⁺IL-4^- CD4⁺ T cells; (2) Th2 cells: IFN-γ^-IL-4⁺ CD4⁺T cells; (3) Treg cells: CD4⁺CD25⁺Foxp3⁺ T cells. A minimum of 10⁶ cells per staining were assessed, with at least 10⁵ events being measured. To measure CCRs in peripheral Treg cells, anti-CCR3, anti-CCR4, anti-CCR5 and anti-CCR8 (R & D Systems, Mineapolis, MN, USA) were used to evaluate corresponding receptor expression in circulating Treg cells of 3 groups.

BTICs cells co-cultured with hAT-MSCs were collected and lysed in protein lysis buffer (Cell Signaling, Danvers, MA). Equal amounts of sample lysate were separated by NuPAGE 4–12% bis-Tris gel (Invitrogen) and transferred to a nitrocellulose iblot gel transfer stack (Invitrogen) using the iblot system (Invitrogen). The membranes were blocked with 5% skim milk in Tris-buffered saline, Tween-20 (TBST) buffer and incubated overnight at 4°C with anti-SDF-1 (1:500, Abnova, Taipei, Taiwan), anti-RANTES (1:250, Abcam), anti-IL-6 (1:200, Thermo Scientific, IL), anti-IL-8, anti-IGF-1 (1:500, Abcam), anti-CXCR4R (1:1000, Abcam), anti-CCR5 (1:500, R&D system), anti-IGF1R (1:250, Abcam) and β-actin (1:5000, Sigma-Aldrich). The membranes were washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies. The membranes were developed with enhanced chemiluminescence kits (Invitrogen) and exposed to film. For quantification, the band densities were normalized to the levels of β-actin and represented as the relative intensity.