Generation and Characterization of T Cell-Specific SELENOI Knockout Mice

Generation of T cell specific SELENOI KO using lck-Cre (distal promoter) and SELENOI^fl/fl mice was previously described [21 (link)]. Experiments using mice included age/sex matched male and females 8–12 wks of age, and littermate SELENOI^fl/fl mice served as WT controls. Animal protocols were approved by the University of Hawaii Institutional Animal Care and Use Committee. To block Fcγ receptors during flow cytometry, cells were preincubated with anti-CD16/32 (2.4G2; BD Pharmingen) for 15 min followed by antibody stains. BioLegend antibodies used at concentrations recommended by vendor included FITC-anti-CD3 (145–2C11), PE- or APC-anti-CD4 (GK1.5), PE-anti-IL-17 (TC11–18H10.1), PE-anti-IFNγ (XMG1.2), PE-anti-IL-10 (JES5–16E3), PE-anti-TGF-β (TW7–16B4), BV421anti-FoxP3 (MF-14); also used were PE-anti-T-bet (4B10; eBioscience); APC-anti-CD25 (PC61.5; eBioscience), and PE-anti-RORγt (AFKJS-9; Invitrogen). Dead cells were detected with BV421 viable dye or Aqua Dead Cell stain (Invitrogen/ThermoFisher). For immunohistochemistry, antibodies included rabbit polyclonal anti-CD3 (IS503, 1:200; Dako) and SMI-32 Ab (801702, 1:5000; BioLegend). For immunofluorescence, PE-anti-IL-17A was used (17B7, 1:500; Ebioscience). Myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (InSolution AMPK Inhibitor, Compound C) were purchased from Sigma.