Investigating STAT1 and STAT3 Phosphorylation Defects

To investigate for phosphorylation defects in STAT1 and STAT3, PBMCs from SLT patients (n = 13) were isolated and cells were stimulated for 15 min with IL-6 (100 ng/ml), IL-21 (100 ng/ml), and IFNα (11,500 U/ml; R&D Systems, cat#11101-1) according to previously described diagnostic protocols^{71 (link),72 (link)}. Cells were fixed immediately (Cytofix Fixation Buffer; BD Biosciences, cat#554655), permeabilised (Phosflow Perm Buffer III; BD Biosciences, cat#558050), and stained for CD4 (CD4-FITC), pSTAT1 (STAT1 pY701—Alexa647; BD Biosciences, cat#612597) and pSTAT3 (STAT3 pY705 – PE; BD Biosciences, cat#612569). For each subject, the increase in phosphorylation of STAT1 and STAT3 in stimulated compared to unstimulated CD4⁺ and CD4⁻ cells were determined, and this increase was expressed as a ratio with the increase seen in a healthy control subject in each experiment.