Comprehensive Evaluation of Th17-Related Cytokines

A comprehensive set of Th17-related cytokines was evaluated by ELISA: (i) cytokines that promote Th17 differentiation in naive T cells (IL-1β, IL-6, IL-21, IL-23, and TGFβ1), and (ii) cytokines produced by Th17 cells (IL-17A, IL-21, IL-22) [69 (link),70 (link),71 (link),72 (link)] (Supplementary Figure S1). Additionally, we evaluated the Th1 hallmark cytokines IFNγ and TNFα and the T regulatory (Treg) hallmark cytokine IL-10. All cytokines except IL-22 were evaluated using ELISA Ready-Set-Go! kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. IL-22 was evaluated in a separate ELISA kit purchased from Antigenix America, Huntington Station, NY. All serum samples remained undiluted during the evaluation of cytokine levels except for the evaluation of TGFβ1 levels in which serum was diluted 1:5. In short, the ELISA plates were initially incubated with cytokine-specific capture antibody, followed by the addition of patient serum, and finally the addition of cytokine-specific detection antibody. The binding of the detection antibody was visualized by adding avidin-conjugated horseradish peroxidase and an enzyme-specific substrate. Specific cytokine quantities were measured using a Multiskan FC Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and compared to manufacturer-supplied cytokine standards (presented in pg/mL).