Il 10 apc

We assessed the phenotype of Tregs by flow cytometry. CBMCs and PBMCs were cultured with PHA and the indicated concentration of l-arginine at 37°C for 48 h, then washed with fluorescence-activated cell sorting (FACS) buffer prior to evaluation of their Treg subsets. At the end of the stimulation, the cells were stained with anti-CD4-PerCP (BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD25-FITC (Beckman Coulter, Brea, CA, USA) for 30 min, then fixed with paraformaldehyde/phosphate-buffered saline (PBS), and permeabilized using FACS permeabilizing solution (Sigma-Aldrich, St. Louis, MO, USA). For intracellular staining of FoxP3 and IL-10, cells were stained with anti-human FoxP3-PE (eBioscience, San Diego, CA, USA) and IL-10-APC (BD Biosciences). We analyzed the percentages of human CD4⁺CD25⁻, CD4⁻CD25⁺, and CD4⁺CD25⁺ cells, and the intracellular expression of FoxP3 and IL-10 in the subsets, using a FACSCalibur flow cytometer (BD Biosciences).

The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Intracellular flow cytometry was utilized to determine the percentage of IFN-γ, IL-4, IL-17 and IL-10-producing lymphocytes within the CD4⁺subset in 10 patients, in both IC and PB samples. Briefly, heparinized whole blood was diluted 1:1 with RPMI medium (Sigma-Aldrich Co., St Louis, MO, USA), and cells were stimulated for 5 h at 37 °C, in 5 % CO2 atmosphere with 0.5 ng/ml ionomycine (Sigma-Aldrich) and 1 γg/ml phorbol myristate acetate (PMA; Sigma-Aldrich) in the presence of 5 mg/ml Brefeldin A (Sigma-Aldrich) to induce cytokine production (Grille et al. 2010 (link)). Lymphocytes were then stained with anti-human CD4 and anti-human CD3 (BD Pharmingen, San Diego, USA). Red blood cells were lysed with FACS Lysing Solution (BD Biosciences), and lymphocytes were permeabilized with FACS Permeabilizing Solution (BD Biosciences). To detect intracellular cytokines, cells were stained with the following cytokine-specific antibodies: anti-human IL-17-PE, IFN-γ-APC, IL-4-PE and IL-10-APC (BD Pharmingen, San Diego, USA), washed, and fixed with 1 % paraformaldehyde. Data acquisition and analysis was performed as previously described. A gate was set on CD4⁺CD3⁺ lymphocytes and at least 5000 cells were counted and evaluated for the expression of IFN-γ, IL-17, IL-4 and IL-10.

During the last 6 h of culture, brefeldin A (1 µg/mL) (eBioscience) was added to improve cytokine detection. The cells were then washed and stained for surface markers, and fixed using 2% formaldehyde (Sigma-Aldrich). Fixed cells were permeabilized and stained using anticytokine monoclonal antibodies. Immunoglobulin control antibodies and a control of unstimulated PBMC were included in all experiments [45] (link).
Monoclonal antibodies directly conjugated with fluorocromes were: anti-CD4 APC-Cy7 (BD Biosciences), CD25 PE-Cy7 (eBioscience), CD14 PerCP (eBioscience), Foxp3 Alexa fluor 488 (BD Biosciences), TGF- β1 PerCP (R & D Systems), IL-10 APC (BD Biosciences) and IL-17F PE (eBioscience).
Preparations were acquired on BD FACSCanto II (BD Biosciences) and 30,000 events were acquired for the analysis. Data was processed using FlowJo software, version 7.6.5 (Tree Star Inc, Ashland, OR, USA). Specific gating strategies to select Treg CD4⁺CD25^highFoxp3⁺, CD4⁺CD25⁻Foxp3⁻ and monocytes (CD14⁺) subsets are presented through representative density plots which are shown in Figure 1. Treg cells were stained and analyzed for TGF-β1, IL-10 and IL-17, while monocytes were stained for IL-10.

CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.

PBMCs were cultured in RPMI-1640 media (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), with or without LPS (100 ng/mL, STEMCELL Technologies, Vancouver, Canada) and Golgiplug (BD Biosciences, San Diego, CA, USA) for 3 h. The cells were surface-stained with CD45-BV786, CD14-Alexa Fluor 700, CD16-BV711, HLA-DR-APC-H7, TIM-3-BB515 (BD Biosciences, San Diego, CA, USA), and TIGIT-PE-Cy7 (eBioscience, San Diego, CA, USA), and intracellularly stained with antibodies against IL-10-APC, IL-1β-Pacific blue, TNF-α-BV650 (BD Biosciences), IL-6-PE (eBioscience), GM-CSF-PE-CF594 (BioLegend), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Kidneys were collected from sacrificed animals for flow cytometry analysis following the standard manufacturer's protocol. Briefly, kidneys were reperfused, excised, and incubated in collagenase IV for 30 min. Then, monocytes were separated by Percoll (Sigma, St. Louis) gradient. We analyzed the renal cells by multicolor flow cytometry. The monoclonal antibodies used were F4/80 PerCP, CD11b PE, CD206 FITC, MIG PE, IL10 APC, p40 (IL12/IL23) PE‐Cy7, CCR7 PerCP‐Cy5.5, IFNγ FITC, CD45.1 Pacific Blue, CD45.2 APC‐Cy7, GM‐CSF PE, MHC II IAB PerCP, and CD36 FITC (all purchased from BD Biosciences, Franklin Lakes). Samples were acquired on a FACSCanto using FACSDIVA software (BD Biosciences), followed by analysis with FLOWJO software (Tree Star, San Carlo, CA). Fluorescence voltages were determined using matched unstained cells. Compensation was performed using cells (BD Biosciences) single‐stained with CD3 PErCP, CD4 FITC, CD8 APC‐CY7, CD4 PE‐CY7, CD4 PerCP‐Cy5.5, CD3 PE, or CD3 APC. Samples were acquired up to at least 200,000 events in a live mononuclear gate, followed by a doublets exclusion gate, following the standard manufacturer's procedure. The compensation process was performed according to the “fluorescence minus one” method.