Pd 146176

T cells were cultured with plate bound α-CD3 (5 µg/ml) and soluble α-CD28 (2 µg/ml) with IL-2 (20 ng/ml) at 37°C in IMDM (Invitrogen) media supplemented with 10% FCS (Invitrogen) unless stated otherwise. All reagents were purchased from Sigma-Aldrich except otherwise stated. The following small molecule inhibitors were tested in threefold dilution series: β-mercaptoethanol (b-ME, 50 µM; Invitrogen), α-tocopherol (aToc, 100 µM), ascorbic acid (L-Asc, 10 µM), PD146176 (1 µM), z-VAD-FMK (100 µM; R&D Systems), Necrostatin-1 (Nec-1, 100 µM; Merck), Necrostatin-1 inactive control (Nec-1i, 100 µM; Merck), 3-methyladenine (3MA, 10 µM), U0126 (100 µM), deferoxamine (DFO, 400 µM), and Ferrostatin-1 (Fer-1, 10 µM; ChemBridge). Ebselen was purchased from Sigma-Aldrich. Cremophor EL (Sigma-Aldrich) was used to solubilize α-tocopherol in aqueous solution according to the manufacturer’s protocol. Whole splenocytes and peripheral LNs were isolated and treated in vitro with plate-bound α-CD3 (5 µg/ml; homemade), soluble α-CD28 (2 µg/ml; BioLegend), and IL-2 (20 ng/ml; homemade) at 37°C with 5% CO₂.
For T cell viability analysis in vivo, mice were injected with α-CD3 (1 µg; eBioscience) i.v., and splenocytes were isolated for analysis.