Anti pd 1 phycoerythrin pe

The antibodies used in this study are follows: anti-PD-1-phycoerythrin (PE) (Biolegend, San Diego, USA), anti-PD-1-peridinin-chlorophyll-protein complex (PerCP) (Biolegend), anti-CD45-PE-Cyanin 5 (PE-Cy5) (BD PharMingen, San Jose, USA), anti-CD11b-fluorescein isothiocyanate (FITC) (BD PharMingen), anti-CD11b-Allophecocyanin (APC) (BD PharMingen), anti-CD11c-APC (BD PharMingen), and anti-CD206-APC (BD PharMingen). The BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA, USA) was used for the analysis.

For subjects with carriage of the relevant HLA-DRB1 alleles and available PBMCs, ICS with peptide pools was used to examine the phenotype of HCV-specific T cells and cytokine production. These peptide pools contained specific HLA-DRB1 allele-restricted epitopes with a maximum of six peptides per pool at a final concentration of 10μg/ml. The peptide pools tested were dependent on the HLA-DRB1 genotype of the subject. Briefly, approximately 1 × 10x⁶ PBMCs cells were stimulated with peptide pools in the presence of co-stimulatory molecules CD28 and CD49d (BD Biosciences) for two hours before the addition of brefeldin A (GolgiPlug; BD Biosciences) and incubated for a further 12 hours. Cell surface markers and intracellular cytokine production were analyzed using the following monoclonal antibodies: anti-CD3-A700 (BD Biosciences); anti-CD4-PcPCy5.5; anti-CD8-PECF594; anti-CD14-V500; anti-CD19-V500; anti-IFNγ-fluorescein isothiocyanate (FITC); anti-TNFα PE-Cy7; anti-IL2-BV451 pacific blue and anti-PD-1-phycoerythrin (PE) (BioLegend). AquaViD (Life Technologies) was used for the exclusion of dead cells. All samples were acquired using a LSR II flow cytometer (BD Biosciences) and analyzed with BD FACSDiva software (BD Bioscience) or FlowJo v10.2 (FLOWJO). A response was deemed positive if at least three times the background.

To determine the effect of SnMP (PeproTech, Hamburg, Germany) treatment regarding functionality and phenotype on T-cells in an antigen-independent setting, PBMCs from healthy donors (n = 7) were co-cultured with 10 µM SnMP in the absence of WT1-specific antigens. Isolated CD3⁺ T cells (human Pan T cell Isolation Kit (Miltenyi Biotec) from same donors were co-cultured with anti-CD3/CD28 beads (Dynabeads^® Human T-Activator CD3/28, ThermoFisher Scientific, Schwerte, Germany) according to the manufacturer’s instructions in T-Cell Medium 1 (TCM1)-composed of RPMI 1640 medium (Lonza, Verviers, Belgium) with 10% human AB serum (C.C. pro, Oberdorla, Germany) and 50 U/mL human IL-2 (PeproTech, Hamburg, Germany) for six days in the presence and absence of SnMP. Aliquots of cells were collected on days 1, 2, 3, and 6 for T-cell phenotype and PD-1 expression analysis (anti-PD-1-phycoerythrin (PE), BioLegend, London, Great Britain) by multicolor flow cytometry, and for IFN-γ and miRNA-155 gene expression analysis by quantitative real-time PCR.