FL cells induce anergy only if CTLA4-Ig is present during primary stimulation. Allogeneic T cells were stimulated in a primary MLR with unstimulated FL cells (A) in the absence or (B) in the presence of CTLA4-Ig. Primary coculture was performed for 5 to 6 days. Cells were then obtained, enriched for viable cells by density-centrifugation, and rested for 48 hours before restimulation. Cells were then restimulated with FL cells, FL cells plus control IgG (2 μg/mL), FL cells plus CD28 MoAb (1 μg/mL), or CD40-FL cells. T-cell proliferation was assessed after 3 (shown here) or 6 days (data not shown) by thymidine incorporation for the last 16 hours of coculture. Approriate controls (T cells, FL cells, CD40-FL cells) were less than 2,000 cpm. Control IgG or CD28 MoAb were added to the T cells 30 minutes before coculture and were present throughout the culture period.