The SCNP assay was performed as described previously [7 (link)]. Modulators and concentrations were as follows: 1000 IU/ml IFN-α (PBL); 50 ng/ml IL-4, 5 μg/ml anti-IgD (BD Biosciences); 50 ng/ml IL-2, 50 ng/ml IL-6, 50 ng/ml IL-27, 5 μg/ml R848 (Invivogen); 40 nM PMA (Sigma Aldrich), 3 μg/ml anti-CD3 (eBioscience), 10 μg/ml anti-mouse (Santa Cruz Biotechnology), 10 μg/ml anti-IgM (Southern Biotech). For TCR stimulation, cells were exposed to anti-CD3 for 12 min, with anti-mouse added for the last 2 min; the anti-IgM modulation time was 10 min; all other modulation times were 15 min. Staining was performed using Ab cocktails with each cocktail consisting of 5 Abs to detect phenotypic markers and 2 Abs to detect intracellular protein readouts. Abs used include anti-CD3, -CD4, -CD45RA, -CD20, -p-NF-κB, -c-poly(ADP-ribose) polymerase, -p-Stat1, -p-Stat3, -p-Stat5, -p-Stat6, -p-Erk, -p-ZAP70/Syk (BD Biosciences); -p-Akt, -p-S6 (CST); and -CD14 (Beckman Coulter). Flow cytometry data was acquired on FACS Canto II Flow Cytometers (BD Biosciences). All flow cytometry data were analyzed with WinList (Verity House Software). PBMC subpopulations were delineated according to the immunophenotypic gating scheme described previously [7 (link)].
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