Cd8 pe cy7

Antibodies for APC-CD3, PE-CD4, PE/Cy7-CD8, Alexa Fluor647-CD56, PE-CD107a, PE-CCR7, PE/Cy7-CD62L, PE/Cy7-perforin, PE-granzyme B, PE-PD-L1 were purchased from BioLegend. Goat anti-B7-H3 antibody (MAB1027) was purchased from R&D System. Humanized anti-B7-H3 IgG (8H9) was expressed and purified in the laboratory (for details, see Additional file 1). Mouse anti-CD16 antibody (3G8) was purchased from BD Biosciences. PE-conjugated anti-human Fc antibody and HRP anti-human IgG antibody were purchased from Invitrogen. HRP-conjugated rabbit anti-goat IgG antibodies were purchased from Jackson ImmunoResearch. Rabbit antihuman PD-L1 antibody (13,684), HRP-conjugated anti-β-actin and anti-GAPDH antibodies were purchased from Cell Signaling.

OPG levels were measured by ELISA kits (Biomedica Gruppe,Vienna, Austria). The detection limit of assay was 0.12 pmol/l. Lymphocyte surface phenotypes were evaluated by flow cytometry using fresh peripheral blood. For the activation and the senescence analysis of T cells CD4⁺ and CD8⁺, the following fluorochrome-labeled antibodies were used: Pacific Blue-CD3 (BioLegend, 500 uL), FITC-CD28 (BioLegend, 2 mL), PE-CD57 (BioLegend, 2 mL), PerCp/Cy5.5-HLA DR (BioLegend, 500 uL), Pe/Cy7-CD8 (BioLegend, 2 mL), CD38-APC (BioLegend, 2 mL), APC/Cy7-CD4 (BioLegend, 2 mL). The flow cytometer was calibrated using MACSQuantTM Calibration Beads and for the automatic compensation marked beads (BDTM CompBeads Anti -Mouse Ig, k) and unmarked beads (BDTM CompBeads Negative Control, FBS) were used. For the lysis of red blood cells FACS Lysing Solution (BD, Lysing Solution) was used. The flow cytometer used for the analysis was the MACSQuant Analyser with 8 channels and the data were analyzed by using the FlowJo V10 Software. Before each acquisition, both calibration and automatic compensation were performed. Immune activation was defined as HLADR⁺ CD38⁺ whereas immunosenescence was defined as CD28^-CD57⁺ [24 (link)–25 (link)].

The surface markers included FITC‐CD3 (MA1‐7640, eBioscience, San Diego, CA, USA), APC‐Cy7‐CD4 (100413, Biolegend, San Diego, CA, USA), APC‐CD4 (100411, Biolegend), PerCP‐CD8 (100,731, Biolegend), PE‐Cy7‐CD8 (100721, Biolegend), PE‐Foxp3 (12‐5773‐82, eBioscience), APC‐Helios (17‐9883‐42, eBioscience), PerCP‐CD44 (103035, Biolegend), FITC‐CD62L (104405, Biolegend), FITC‐lin (22‐7770‐72, eBioscience), PerCP‐CD34 (50‐0341‐82, eBioscience), APC‐Sca‐1 (17‐5981‐82, eBioscience), PE‐Sca‐1 (12‐5981‐82, eBioscience), APC‐c‐kit (17‐1171‐82, eBioscience), PE‐c‐kit (12‐1171‐82, eBioscience), PE‐B220 (12‐0452‐82, eBioscience), FITC‐IgM (11‐5790‐81, eBioscience), PE‐Cy7‐CD19 (12‐0193‐82, eBioscience), APC‐CD19 (17‐0193‐82, eBioscience), PE‐Cy7‐CD16/32 (25‐0161‐82, eBioscience), PE‐Cy7‐CD45 (25‐0451‐82, eBioscience), PerCP‐5.5‐CD45.2 (45‐0454‐82, eBioscience), and APC‐Cy7‐CD45.1 (17‐0453‐82, eBioscience) obtained from Invitrogen. Intracellular staining with PE‐Foxp3 (72‐5775‐40, eBioscience) was performed with Foxp3 staining kits (Invitrogen, Carlsbad, CA, USA).

For analyzing the expression of IFN-γ and IL-17, the single-cell suspensions were incubated for 3 h at 37°C with PMA (Sigma-Aldrich, p1585; 200 ng/ml, Missouri, USA), brefeldin A (BioLegend, 420601; 5 µg/ml, San Diego, CA, USA), and ionomycin (Abcam, ab120116; 1 µg/ml, Cambridge, UK). And then washed and stained with fixable viability stain 620 (FVS 620; BD-Biosciences, Franklin. Lakes, NJ, USA, 564996) for 10 min. Next, stain cells with following surface antibodies: APC/Cy7-CD3 (100222, BioLegend, San Diego, CA, USA), PerCP/Cy5.5-CD4 (100434, BioLegend, San Diego, CA, USA), PE/Cy7-CD8 (100722, BioLegend, San Diego, CA, USA). After performing surface staining as described above, 4% paraformaldehyde was used to fix cells and added PBS solution (containing 0.1% Triton X-100) to permeabilize the cell surface. Intracellular staining antibodies were included: PE-IL-17A (506903, BioLegend, San Diego, CA, USA), FITC-IFN-γ (505806, BioLegend, San Diego, CA, USA). After staining for 30 min, the cells were washed by PBS and using the NovoCyte flow cytometer and ACEA NovoExpress™ software (ACEA Biosciences, San Diego, CA, USA) for analysis.

Tumors were processed for flow cytometry into single cells using mechanical and enzymatic digestion (enzyme mixture—collagenase, DNAse, and hyaluronidase). Red blood cells were lysed as described above and viable tumor cells counted. The following antibodies were used to stain immune cell populations: Ghost violet 510 Live/Dead, Pacific Blue CD45, PE-594 CD3, PE/Cy7 CD8, APC/Cy7 CD4, BV605 PD-1, APC Tim3, FITC CD62L, BV711 CD44 (BioLegend).
Samples were run on an LSR Fortessa machine. Single color compensation controls were performed using compensation beads (Thermo Fisher Scientific, Waltham, MA, USA) to correct for overlap in signal among antibodies. Spleen samples were used to set a gating strategy for CD3+/CD4+ and CD3+/CD8+ T cells. FlowJo software (version 10) was used to perform all downstream analyses on subpopulations.

PBMCs were prepared from RA patients before treatment and Cs using Ficoll hypaque (Lymphoprep, Axis-Shield, Oslo, Norway) density gradient centrifugation. In RA patients, the peripheral blood was collected at baseline and was from 4 R-RA and 3 NR-RA patients. PBMCs were stimulated or not with anti-CD3/CD28 beads (Gibco, Waltham, MA, USA) at a bead:PBMC ratio of 1:1. PBMCs were incubated for 72 h with or without 10 ng/mL of recombinant human IL-6 (IL-6) (ImmunoTools, Friesoythe, Germany). The PBMCs were stained with the following monoclonal antibodies: anti-human CD3-Viogreen (Miltenyi Biotec GmBh), CD39-Fitc and CD73-PE (BD Biosciences), and CD8-PECy7 (Biolegend). Samples were acquired using the MACSQuant Analyzer 10 flow cytometer, and the data were analyzed using FlowJo software.

Flow cytometry was performed on a Fortessa (BD Biosciences) as previously described methods [2 ]. Anti-mouse CD19-Percp, CD8-PE-Cy7, F4/80-APC, CD3-APC-Cy7, CD11c-PE, CD4-FITC, and CD49b-PE were purchased from Biolegend.