Multiparametric Immune Profiling Protocol

Fluorescein isothiocyanate (FITC)-conjugated anti-CD38, Alexa 488-anti-CXCR5, Alexa F-647-anti-CXCR5, phycoerythrin (PE)-conjugated anti-IL-21, PE-Cy7-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-IL-6 and FITC-conjugated anti-IFN-gamma were purchased from BD Bioscience and BD Pharmingen™ (San Diego, CA, USA). FITC-conjugated anti-CD3, PE-conjugated anti-ICOS, PE-conjugated anti-IL-17a, peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 and anti-CD8 and APC-conjugated anti-CD279 (PD-1) were purchased from eBioscience (San Diego, CA, USA); and APC-Cy7-conjugated anti-CD3 and anti-CD27, PE-conjugated anti-CD24, PE-Cy7-conjugated anti-CD19 and PerCP-Cy5.5-conjugated anti-IgD were purchased from BioLegend (San Diego, CA, USA). Peripheral blood mononuclear cells (PBMCs) were isolated and phenotypic analysis performed using optimal concentrations of mAbs according to standard protocols (25 (link), 26 (link)). Aliquots of cells were also utilized for flow cytometry and FlowJo software analysis (Tristar Inc, San Carlos, CA, USA). At least 50,000 events per sample were analyzed.