Impaired generation of memory Th1/Th2 cells from Bmi1^−/− effector Th2 cells. (A and B) Bmi1^+/+, Bmi1^+/−, or Bmi1^−/− effector Th2 (A) or Th1 (B) cells with DO11.10 Tg background were intravenously transferred into BALB/c nu/nu mice (n = 5). 5 wk later, the number of KJ1⁺ memory Th2 cells was determined by flow cytometry. Typical staining patterns of KJ1/CD4 and percentages of KJ1⁺ cells are shown. Four independent experiments were performed with similar results. (C) Bmi1^+/+, Bmi1^+/−, or Bmi1^−/− effector Th2 cells with DO11. 10 Tg background were intravenously transferred into BALB/c mice (n = 5). (D) Impaired semi-acute survival of Bmi1^+/− Th2 cells. Thy1.1 Bmi1^+/+ (Ly5.2 background) and Thy1.2 Bmi1^+/− (Ly5.2 background) effector Th2 cells were mixed (1:1) and transferred (3 × 10⁷ cells/mouse) into syngeneic C57BL/6 mice with a Ly5.1 background. The ratio of Thy1.2⁺/Thy1.1⁺ cells in Ly5.2⁺ cells is shown (n = 3). (E) Homeostatic proliferation of the splenic memory Th2 cells was assessed by the BrdU incorporation in vivo. Representative BrdU staining profiles of KJ1⁺ memory Th2 cells prepared 5 wk after cell transfer are shown. The percentages of BrdU⁺ cells with standard deviation are shown (n = 5). Three independent experiments were performed with similar results. (F) Apoptotic cell death of freshly prepared memory Th2 cells from the spleen was measured by a TUNEL assay. As a positive control, cells were treated with DNase I. Representative TUNEL profiles are shown with percentages of TUNEL⁺ cells. Two independent experiments were performed with similar results.