Superoxide Modulation of CD4 T-Cell Responses

Splenic CD4 T cells from BDC-2.5 and BDC-2.5.Ncf1^m1J mice were purified by negative selection according to the manufacturer’s protocol using the EasySep CD4 T-cell enrichment kit (STEMCELL Technologies). CD4 T-cell purity was routinely assessed by flow cytometry and found to be >90% (data not shown). T cells were stimulated with plate-bound anti-CD3ε (0.1 µg/mL) and anti-CD28 (1 µg/mL), and exogenous superoxide was added via 1 mU/mL xanthine oxidase (XO) (17 (link)). BDC-2.5 and BDC-2.5.Ncf1^m1J splenocytes were incubated with 1 µmol/L BDC-2.5 mimotope or 10⁴ dispersed NOD.Rag islet cells in the presence or absence of 1 mU/mL XO (9 (link),15 (link)). Greiss assay and ELISAs were read on a Synergy2 microplate reader with Gen5, version 1.10, software (BioTek) as previously described (17 (link)). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess T-cell proliferation was performed according to the manufacturer’s protocol (Sigma-Aldrich).