2.5. In vitro assay for evaluating cytokine release by human PBMC

To evaluate in vitro cytokine release, PBMC were incubated in 96 well tissue culture plates at 37°C/5% CO₂ for 48 hours in the presence of OKT3, anti‐CD28, or PBS. One day prior to addition of PBMC, plates were coated overnight with OKT3 (0.1 mg/mL) and anti‐CD28 (0.2 mg/mL) in PBS at 4°C. Cryopreserved PBMC were thawed, resuspended in RPMI supplemented with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, 2 mM L‐glutamine, 1% (v/v) nonessential amino acids solution, 50 μM 2‐mercaptoethanol, and 1 mM sodium pyruvate, and washed once. Viability counts were performed, and 1 × 10⁵ viable PBMC were added to each well. Supernatants were harvested 48 hours later, and cytokine levels were measured by a BD CBA Th1/Th2 II kit.