Induction of tumor-specific T-cell memory by BiFP-mediated tumor rejection. (A) BALB/c mice were subcutaneously inoculated with A20-CD20 cells and then treated with CD20-Flex BiFP or control Ig (left). After the first inoculation, the BiFP-treated mice were secondarily inoculated with A20-CD20 cells. Some mice were treated with anti-CD4 mAb, anti-CD8 mAb, anti-CD4, and anti-CD8 mAb or isotype Ig (middle). Naive mice were inoculated with A20-CD20 cells as the control. Irrelevant 4T1 tumor cells were inoculated into A20-CD20–rejecting mice or naive mice (right). (B) BALB/c and SCID mice were inoculated with A20-CD20 cells and then treated with CD20-Flex BiFP mAb (200 μg) (n = 25; 800 μg) or control Ig (n = 25) (left). Five weeks after the first inoculation, the BiFP-treated BALB/c and SCID mice that had rejected A20-CD20 were secondarily inoculated with A20-CD20 cells (middle). Naive BALB/c and SCID mice were inoculated with A20-CD20 cells as the control. Two days after inoculation of A20-CD20 cells, some SCID mice were intravenously transferred with splenic T cells from the BiFP-treated BALB/c mice that had rejected A20-CD20 or naive BALB/c mice (right). Naive SCID mice and naive BALB/c mice as the control. The graphs are representative of at least 5 experiments, each of which showed similar results.