Lfng overexpression enhances proliferative expansion of DN3 and DN4 thymocytes on OP9-DL1 and OP9-DL4. (A) Proliferative expansion of DN3 and DN4 thymocytes on OP9-DL1. Graphs display the fold expansion of Lfng Tg⁺ (diamonds) versus Lfng Tg⁻ (squares) DN3 or DN4 progeny at each time point relative to the number of cells seeded on day 0. (B) Proliferative expansion of DN4 thymocytes from Lfng Tg⁺ (diamonds) versus Lfng Tg⁻ (squares) mice on OP9-DL1 versus OP9-DL4 stromal cells. Graphs display the -fold expansion at each time point relative to the number of cells seeded. Similar results were obtained in 3 independent experiments. (C) Prolonged proliferative expansion of Lfng Tg⁺ DN3 and DN4 progeny in OP9-DL1 stromal cell cultures. DN3 and DN4 thymocytes from Lfng Tg⁻ (top row) and Lfng Tg⁺ (bottom row) mice were cultured on OP9-DL1 stromal cells for 8 days. BrdU was added to each culture 2 hours before harvesting cells and staining with anti-BrdU–fluorescein isothiocyanate (FITC) and anti-CD4–allophycocyanin (APC) plus anti-CD8–APC. Histograms depict percentage of BrdU⁺ DN and DP cells generated from cultures initiated with DN3 (left) or DN4 (right) thymocytes. Similar results were obtained in 2 independent experiments.