Restoring Th1 Responses in Ncf1-Deficient T Cells

Splenic CD4 T cells from OT-II and OT-II.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 greater than 90% (data not shown). T cells were stimulated with plate-bound anti-CD3ε (0.1μg/mL) and anti-CD28 (1μg/mL) (6 (link)). CD4 T cell responses were assayed via antigen recall assays, as described previously, in which splenocytes were stimulated with 1μM OVA_323-339 (3 (link)). To assess the ability of exogenous superoxide to rescue Th1 T cell responses, OT-II and OT-II.Ncf1^m1J splenocytes were treated with potassium superoxide as described (22 (link)). A titration response (0.5, 1, and 5μg/mL) with potassium superoxide was performed in preliminary studies with OT-II and OT-II.Ncf1^m1J splenocyte primary recall assays and 0.5μg/mL was chosen as the ideal concentration to restore Th1 cytokine responses without compromising cell viability. MTT assay for viability assessment and cell counting kit-8 (CCK-8) assay for proliferative capacity were performed according to manufacturer’s protocol (21 (link), 23 (link)).