In vitro T cell proliferation assay

PBMCs were prepared, cultured, and exposed to the biotherapeutics using the same method described for the T cell activation assay. Cell plates were placed in a 5% CO₂ incubator at 37°C for 7 days. After incubation, the cells were washed and stained with viability dye (Thermo Fisher) and CD4 (clone SK3). The cells were fixed and permeabilized with ethanol and stained with anti-Ki67 for 30 minutes at room temperature. The cells were analyzed by FACS Canto II (BD Inc.; Franklin Lakes, NJ), and FACS plots were analyzed using FlowJo FACS analysis software (Tree Star, Inc.; Ashland, OR).