Flow cytometry and immune phenotyping.

Fluorophore-conjugated antibodies used for multiparametric flow cytometry analysis of patient PBMCs are listed in Supplemental Table 4. Where indicated, stimulation for the detection of intracellular cytokines was by PMA/ionomycin for 4 hours in the presence of brefeldin A followed by fixation and permeabilization with BD Cytofix/Cytoperm. For intracellular staining of IRF8 by flow cytometry, cells were permeabilized using the FoxP₃ transcription buffer staining kit (eBioscience) followed by incubation with IRF8 eFluor 710 (clone V3GYWCH, eBioscience). Flow cytometry was acquired on a BD Fortessa configured for 14 colors. All analysis was done in FlowJo X (Tree Star). Positive populations were identified as those with fluorescence greater than that of isotype or fluorescence-minus-one controls. Cytokine bead arrays were performed with custom BD Biosciences CBA Flex Sets using IL-12, IFN-γ, TNF-α, and IL-10 probes. PBMCs (2 × 10⁶) were incubated for 4 hours with 10⁵ EBV-transformed B cells previously induced to lytic cycle with 200 ng/ml phorbol 12-myristate 13-acetate. Data were analyzed with FlowJo X (Tree Star), and statistical analysis was performed and graphed using Prism 6.0 software (GraphPad Software).