Cytokine expression in memory CD4+ T cells
Cytokine expression in memory CD4+ T cells were
evaluated ex vivo as previously described [16]. Briefly, peripheral
blood mononuclear cells (PBMCs) were activated with PMA (50 ng/mL) and
ionomycin (1 µM) for 5 hours in presence of GolgiStop and GolgiPlug
(BD). Cells were stained with LIVE/DEAD Fixable Blue (ThermoFisher
Scientific) with the addition of FcR blocking reagent (Miltenyi
Biotech). Cells were then stained with (CD8, CCR7, CD45RA, supplementary
Table 1), fixed, permeabilized with FOXP3/Transcription factor staining
buffer kit (eBioscience), followed by staining with antibodies against
CD3, CD4, IL-17A, IL-4, IL-22, and IFN-γ-(supplementary Table 1). Data
was acquired on a Cytek Aurora 5 laser flow cytometer and data was
analyzed with FlowJo (Tree Star).
Results