T-cell proliferation in response to anti-CD3 and anti-CD28 mAbs with fresh and precultured PBMCs. Freshly prepared PBMCs were CFSE-labeled and used in proliferation assays either immediately or after HD preculture for 48 hours. (A-B) PBMCs were incubated with OKT3 and UCHT1 (anti-CD3; 0.1 μg/mL), TGN1412 (5 μg/mL), TGN1412 F(ab′)2 (5 μg/mL; precultured PBMCs only), and CD28.1 superagonist (SA) (1 μg/mL) for 4 days. Cells were then labeled with anti-CD8-APC and anti-CD4-PE mAbs and analyzed by flow cytometry to determine proliferation by CFSE dilution. Results are shown as the percentage of cells having undergone one or more divisions. (A) Representative dot plots and (B) the responses from a panel of donors; n = 5 to 7 for fresh PBMCs and n = 30 for precultured PBMCs. (B, right panel) Responses of T cells isolated after HD preculture of PBMCs to the same mAbs.
