Anti cd3 and anti cd28 coated beads

Isolated CD4+ T cells were labelled with CFSE (10 μM) and stimulated with anti-CD3 and anti-CD28-coated beads (Invitrogen) at a bead-to-cell ratio of 1:1 in the presence of IL-2 (30 IU/ml, Invitrogen) 48h prior to co-culture. Then, CFSE-labelled and activated CD4+ T cells were co-cultured with IDO-transfected Huh7.5.1 cells at a ratio of 1:1. After 72 h of co-culture, CD4+ T cells were collected and their proliferative responses were analyzed by flow cytometry as described above. As a control, some cultures were supplemented with 1-DL-1MT (1000 μM) at the time of co-culture.

Peripheral Blood Mononuclear Cells (PBMC) were isolated using Ficoll-Paque PLUS gradient (GE Healthcare). CD4⁺ T cells were isolated using Dynal CD4 Positive Isolation Kit (Invitrogen). Purified cells were cultured in RPMI media containing 10% FBS and activated with anti-CD3 and anti-CD28 coated beads (Invitrogen) at a bead: cell ratio of 1:4 in media containing human rIL-2. VSV-G pseudotyped control or RIP2-shRNA (Sigma) containing viral particles were produced in HEK293T cells by calcium phosphate transfection according to the manufacturer’s protocol (ProFection, Promega). Lentiviral supernatants were filtered, concentrated by centrifugation and filtered as previously described(Unutmaz et al., 1999 (link)). Cells were infected with lentiviral supernatants (at multiplicity of infection, MOI, of 5) at day 1 of activation. For intracellular cytokine staining, cells were activated with PMA (20 ng/ml for CD4⁺ T cells and 40 ng/ml for PBMC) and Ionomycin (500 ng/ml) (Sigma Aldrich) in the presence of GolgiStop protein transport inhibitor (BD) for 4–6 hours. Cells were then stained for surface antigens CD3, CD4, CCR6 and CD45RO, then fixed and permeabilized (ebioscience) and stained for IL-17A. Immunoblot analysis was performed with anti-RIP2 (Abcam) and anti-RORα (Abcam) antibodies.

Whole blood was obtained from de-identified healthy donors at the DeGowin Blood Center at the University of Iowa Hospitals and Clinics. The recruitment protocol and written informed consent document were approved by the Institutional Review Board for the University of Iowa. PBMCs were isolated from leukocyte reduction system (LRS) cones using Hypaque-Ficoll density-gradient separation as previously described (33 (link)). T cells were then expanded and activated with anti-CD3- and anti-CD28-coated beads (Invitrogen) and human IL-2 for 5 days. Cells were then re-suspended in fresh medium without stimulatory beads or IL-2 for 24 hours. Activated T cells were resuspended in serum-free RPMI 1640 before GML treatment. GML was solubilized at room temperature in 95% ethanol and diluted into the appropriate working concentration. We added 95% ethanol as a comparative vehicle control at final concentrations that did not exceed 0.5%. Activated T cells were treated with ethanol or various doses of GML in serum-free medium for various assays.