Human interferon α

PBMCs were stimulated with cytokines for phosphorylated Signal Transducer and Activator of Transcription (pSTAT) activity [33 (link)–35 (link)]. Cryopreserved human PBMCs were thawed in a water bath at 37°C, washed twice with 10 mL of CTL buffer, according to manufacturer’s instructions, and counted with AO/PI (acridine orange/propidium iodide) staining solution. Viability and number of PBMCs were determined using a cellometer (Nexcelom Bioscience, Lawrence, MA). A total of 1.5 × 10⁶ PBMCs were seeded at 15 × 10⁶ /mL in a deep 96 well round-bottom plate. Cells were stimulated with human Interferon-α (Cell Signaling Technology) at 200 ng/mL for pSTAT1 stimulation, recombinant human IL-2 (Hoffmann-La Roche Inc) at 200 U/mL for pSTAT5, or recombinant IL-10 (PeproTech) 200 ng/mL for pSTAT3 and incubated for 20 min at 37°C. Untreated PBMCs were processed following the same protocol and used as negative control.

The following FCB dyes were used: DyLight 350 NHS ester and Pacific Orange NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies used for surface staining were: mouse anti-human CD3-PerCP-Cy5.5 (clone SK7), mouse anti-human CD4-PE-Cy7 (clone SK3), mouse anti-human CD8-FITC (clone RPA-T8), and mouse anti-human CD20-APC-H7 (clone H1) (BD Biosciences, San Jose, CA, USA); and CD14-PE (clone M5E2) from BioLegend (San Diego, CA). Antibodies used for phosphoproteins were: pSTAT1(pY701)-Alexa Fluor 647 (clone 4a), pSTAT3(pY705)- Alexa Fluor 647 (clone 4/P-STAT3), and pSTAT5(pY694)- Alexa Fluor 647 (clone 47/Stat5 pY694) (BD Biosciences). Phosflow Lyse/Fix Buffer 5X, Phosflow Perm Buffer III, and Phosflow Barcoding Wash Buffer 4X buffers (BD Biosciences) were prepared and used according to manufacturer’s instructions. Phosflow Perm Buffer II (BD Biosciences) was diluted 1:1 with cold PBS and kept on ice before use. For PBMC stimulation, the following cytokines were used: recombinant human IL-10 (PeproTech, Rocky Hill, NJ); human Interferon-α (Cell Signaling Technology, Boston, MA); recombinant human IL-2 (Hoffmann-La Roche Inc, Nutley, NJ). The CTL Anti-Aggregate (CTL) wash buffer was from Cellular Technology Limited (Shaker Heights, OH).