(A) CFSE-labeled naive and (B) in vitro–derived effector Thy1.2⁺ TCR_gagCblb^+/+ and TCR_gagCblb^–/– cells were incubated with irradiated Thy1.1⁺ syngeneic splenocytes pulsed with 5 μg/ml of gag peptide, with and without anti-mouse CD25 (20 μg/ml) and anti-mouse IL-2 (20 μg/ml). After 4 days, CFSE dilution of Thy1.2⁺ CD8⁺ cells was determined by FACS. The numbers in each box indicate the percentage of proliferating transgenic cells. (C) CFSE-labeled CTLL-2 cells were incubated with r-mIL-2 (20 ng/ml) with or without anti-mouse CD25 (20 μg/ml), anti-mouse IL-2 (20 μg/ml), or a combination of these antibodies. After 4 days, proliferation was measured by CFSE dilution by FACS. Percentage blockade was calculated by dividing the percentage of CTLL-2 cell proliferation in the presence of antibody(s) by the percentage of CTLL-2 cell proliferation in the absence of antibody. (D) Proliferation of CFSE-labeled in vitro–derived effector TCR_gagCblb^+/+, TCR_gagIl2^–/–, TCR_gagCblb^–/–, and TCR_gagCblb^–/–Il2^–/– cells was determined 5 days after activation with antigen. Filled histograms indicate no antigen control. (E) The number of effector TCR_gagCblb^+/+, TCR_gagIl2^–/–, TCR_gagCblb^–/–, and TCR_gagCblb^–/–Il2^–/– cells 7 days after stimulation with antigen with or without r-h-IL-2 (20 U/ml). The dashed line indicates the number of effector cells that were plated at time 0. (F) In vitro–derived effector CFSE-labeled Thy1.2⁺TCR_gagCD28^–/– and TCR_gagCD28^–/–Cblb^–/– cells were incubated with irradiated Thy1.1⁺ syngeneic splenocytes pulsed with 5 μg/ml of gag peptide, with or without anti-mouse CD25 (20 μg/ml) and anti-mouse IL-2 (20 μg/ml). After 4 days, CFSE-dilution of Thy1.2⁺ CD8⁺ cells was analyzed. The numbers in each box indicate the percentage of proliferating transgenic cells.