Aberrant cell signaling in peripheral blood mononuclear cells upon interferon alpha stimulation in patients with primary Sjögren?s syndrome associates with type I interferon signature

To gain insights into potential dysfunctional intracellular signalling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, and samples during a 4 hour time period following activation by interferon alpha.
Conclusion:
The phosphorylation profiles following stimulation with IFNα2b differed significantly between pSS patients and healthy donors, especially regarding STAT1 Y701. PCA further grouped patients according to clinical characteristics such as presence of autoantibodies and extraglandular manifestations.
Notes:
Stimulation with interferon alpha was conducted using a 4 hour reverse time course. Barcoded samples included a PBMC sample from a single donor (IC) to assess assay variation. Key 9x barcoding stimulus are as follows- POlow + PB low= Unstimulated, POlow + PBmid= IC (unstimulated), POlow + PBhigh= 3 hours, POmid + PBlow= 15 minutes, POmid + PBmid= 60 minutes, POmid + PBhigh = 4 hours, POhigh + PBlow = 30 minutes, POhigh + PBmid = 2 hours, and POhigh + PBhigh= IC (stimulated). An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells