Cd3 cd28 beads

Buffy coats from healthy blood donors were obtained from the Blood Bank at the Copenhagen University Hospital (Denmark), in agreement with the local ethics committee (Region Hovedstaden). PBMC were purified by density centrifugation and incubated for 1 hour with washed pan-mouse beads (Invitrogen, Cat# 11041), and phagocytic cells removed by magnet. Purified PBL were stimulated with CD3/CD28 beads (Invitrogen, Cat # 111.32D) as described by the manufacturer, and cells supplied with media + IL-2 (20 U/mL) as needed.
After 12 days, CD3/CD28 beads were removed by magnet, and the cells set up in new media with 10⁶ cells/mL. For CD8 depletion, Dynabeads Pan Mouse IgG beads (Invitrogen, Cat# 11041) and the CD8α antibody (Cat# 16-0086-81, eBioscience) were used according to manufacturer's protocol. Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 µg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 µg/mL.

Suppression assays were conducted and analyzed as previously reported (2 (link)). In assays using tritiated-thymidine, T-cells were isolated from spleens of naïve C57BL/6 mice and stimulated with CD3/CD28 beads (Thermo Fisher Scientific, Waltham, MA). Splenic DCs from naïve or BEN+TBI or CY+TBI conditioned BALB/c mice were isolated and co-incubated with pre-stimulated T-cells for 3 days at 37°C in 7.5% CO₂ in a 96-well plate. 0.5 μCi of tritiated thymidine was added to each well on day 3 of culture. After an additional 18 h of culture, plates were harvested using a Brandel wash pump harvester. T-cell proliferation was measured as CPM using a MicroBeta2 counter. For supplemental experiments, T-cells were stained with CellTrace Violet (Invitrogen) immediately following isolation from the spleen and pre-stimulated with CD3/CD28 beads then co-cultured with naïve or BEN+TBI or CY+TBI conditioned DCs. After 4 days of co-incubation, flow cytometry was performed and data was analyzed using Modfit Software (Verity Software House, Topsham, ME) to determine the T-cell proliferation index (PI).

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll separation (Lonza) and then cultured in Panserin 401 medium (Dutcher) with 5% human male AB serum (BioWest), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37 °C in a humidified 5% CO₂ incubator. Expansion of T cell blasts was obtained by incubating PBMCs for 72 h with CD3/CD28 beads (LifeTechnologies) in complete Panserin 401. After 3 days, dead cells were removed by Ficoll-Plaque density gradient, and blasts were expanded in complete Panserin supplemented with IL-2 (100 IU/ml). Epstein–Barr-virus (EBV)–transformed B lymphoblastoid cell lines (EBV-B cells) were generated from PBMCs obtained from patients (A1, B1, and D1) and HCs using standard methods and then cultured in RPMI 1640 (LifeTechnologies) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Human embryonic kidney (HEK) 293T cells were cultured in DMEM (LifeTechnologies) supplemented with 10% FBS and penicillin/streptomycin.