Anti il 10 pe

Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

The following mAbs were used for cell surface staining: anti-CD45-PerCpCy5.5, anti-Ly6G(1A8)-PE or FITC, anti-Ly6C-APC or FITC (all from BD Bioscience), anti-CD11b-Pacific blue, anti-CD11c-PECy7, anti-CD4-AF700, anti-IL-4-FITC, and anti-IFN-γ-PECy7 (all from eBioscience); anti-CD3-PE or APC and anti-IL-10-PE (Biolegend); and anti-CD8-APC (Invitrogen). For the exclusion of dead cells, we used the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen). L. major mCherry was detected using Texas red as a positive control, L. mexicana DsRed and L. major Sd-RFP were detected using PE as a positive control. Fluorescent parasites and stained murine cells were acquired using a flow cytometry analyzer, either BD LSRII or BD LSR-Fortessa Series (Beckton Dickinson) machines, the data was acquired with the DIVA software and the results were analyzed with FlowJo 10 software.

Isolated PBMCs were stained first with Aqua live/dead (Life Technologies) and incubated at room temperature in the dark for 30 min. After washing, they were suspended in 50 μl of filtered Flow Cytometry Buffer FCB (1X PBS (Gibco), 0.5% BSA (Sigma-Aldrich) 2 mM EDTA (Thermo Fischer Scientific)) and 50 μl of Staining Buffer (FCB 2% Beriglobin (CSL Behring)) and stained with anti CD3-PercP Cy5.5 (eBioscience), anti CD4-FITC, (BD Biosciences) followed by an incubation at 4 °C in the dark for 30 min. Anti-IL-10-PE, anti-IL-13-BV711, anti-IL-2-BV785, anti-IFNγ-BV421, anti-TNFα-BV605 and anti-IL4-PE CF594 (all Biolegend) intracellular staining was done according to manufacturer’s recommendations (BD Biosciences). Cell acquisition was performed using a Spectral Cell Analyzer cytometer SP6800 (Sony Biotechnology) 15-color cytometer, with 100,000 cells per tube as the total number of cells acquired. Polyfunctional CD4⁺ T cells were defined as CD4⁺ T cells expressing any combination of IFN-γ, IL-2 or TNF. IL-10, IL-4 or IL-13 producing CD4+ T cells are defined as CD3+CD4+IL-10+, CD3+CD4+IL-10−IL-4+ and CD3+CD4+IL-10−IL-13+, respectively.

The flow cytometry analysis was made using thawed single-cell tumour suspension (patient no. 11, Table 1) obtained after collagenase digestion of fresh tumour material. Bregs were identified using anti-CD19 BV510, anti-CD5 APC, anti-CD38 PERCPCy5.5, anti-CD45 APCH7, anti-GZMB FITC (all from BD Biosciences, Franklin Lakes, New Jersey, USA), and anti-IL10 PE (BioLegend, San Diego, CA, USA). The cells were stained for cell surface markers after blocking nonspecific antibody binding to the Fc receptors using the FcR Blocking Reagent (Miltenyi, Bergisch Gladbach, Germany), fixed and permeabilised with Cytofix/Cytoperm buffer (BD Biosciences), and stained with the intracellular markers (IL10 and GZMB). Production of IL10 and GZMB was measured after overnight stimulation in the presence or absence of LPS (10 μg/mL), PMA (50 ng/mL), and ionomycin (1 μg/mL). GolgiStop (0.7 μL/mL) was added after one-and-a-half hours' stimulation. Dead cells were identified using the LIVE-DEAD® Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific) according to the manufacturer's instructions and excluded from the analysis. Data were acquired by flow cytometry (Gallios™, Beckman Coulter, Brea, California, USA), and analysis was done by Kaluza® Software (Beckman Coulter).

Before detection of intracellular cytokine production, human PBMC or isolated mice splenocytes were restimulated with PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 4h in the presence of Brefeldin A (GolgiStop; BD Biosciences). Staining with LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes) was performed before fixation to allow gating on viable cells. Cells were then blocked for 15 mins and stained with antibodies targeting specific surface markers for different lymphocytes (APC anti-CD3, PerCP/Cy5.5 anti-CD4/ PerCP/Cy5.5 anti-CD19/ FITC anti-CD16, APC anti-CD56/ FITC anti-HLA-DR, PerCP/Cy5.5 anti-CD11c/ FITC anti-CD14) at 4°C for 30 mins. After fixation and permeabilization with Fixation and Permeabilization solution (BD Biosciences) for 20 mins, cells were stained with PE anti-IFN-γ, PE anti-IL-4, PE anti-IL-17, PE anti-IL-10 or PE Rat IgG1, κ (BioLegend) 4°C for 30 mins respectively and then submitted to flow cytometry analysis (FACS Calibur; BD Biosciences).