4 week induction with 30-60 mg PF-06700841 once daily, followed by 8 week chronic administration of 10-100 mg PF-06700841 once daily

Test articles were prepared as 30 m stocks in DMSO. A 11-point 2.5 dilution series was created in DMSO with a top concentration of 5 mM. Further dilution was done by adding 4 μΙ of the above test article solutions into 96 μΙ of PBS with a top concentration of 200 μΜ. Human whole blood was collected from healthy donors via vein puncture into Vacutainer collection tubes containing sodium heparin (Catalog No. 366480; Becton Dickinson, Franklin Lakes, NJ). Blood was warmed to 37°C prior to use. Human whole blood was aliquoted in 96-well, deep-well, V-bottom plates and treated with compounds at 11 different concentrations (0.2% DMSO final) at 37°C for 60 minutes. This was followed by a challenge with IFNa for 15 minutes. Samples were treated with warm 1X Lyse/Fix buffer to terminate activation and further incubated at 37°C for 20 minutes to lyse red blood cells. Plates were centrifuged at 300 x g for 5 minutes, supernatant was aspirated, and cells were washed with 800 μΙ per well of staining buffer. The washed cell pellets were resuspended with 350 μΙ per well of pre-chilled 90% methanol, and incubated on ice for 30 minutes. After the removal of 90% methanol, cells were washed once with staining buffer. Cell pellets were resuspended in staining buffer containing anti-pSTAT3-AlexaFluor647, and incubated at room temperature in the dark overnight.