Figure 1.

Kinetics of AICD of Th1 and Th2 effectors restimulated with antigen. Effectors specific for PCCF were generated in vitro from naive CD4⁺ cells of H-2^k/k AND TCR Tg mice (Vβ3/Vα11; see Materials and Methods) After 5 d, Th1 and Th2 effectors were harvested, and low density cells obtained by Percoll gradient separation were restimulated at 5 × 10⁵/ml with PCCF (5 μM) and APC (1.7 × 10⁵/ml). (a) Kinetics of Th1 and Th2 AICD. Effector cells restimulated for different times as above were stained with TdT and dUTP for detection of nicked DNA (TUNEL method), followed by biotinylated UTP, and then FITC-streptavidin. Gated CD4⁺ cells were analyzed for DNA strand breaks (TdT⁺ cells) by FACScan^® analysis. The results are the representative of six experiments. (b) Longterm kinetics of AICD. Effectors were harvested at 1, 2, 3, and 4 d after restimulation, and apoptosis in live cells determined by TdT staining as described above. (c) Kinetics of cell recovery. The cultures harvested in Fig. 1 b were also analyzed for number of viable effector cells determined counting trypan blue excluding cells. Results are expressed as the ratio of viable CD4 T cells recovered per culture per CD4 T cells plated at time of restimulation. (d) Effector polarization. Cytokines were measured in the supernatants collected 18–24 h after restimulation of effector cells generated from Tg mice. IL-2 titers (filled bar) were determined by bioassay. IFN-γ, IL-4, and IL-5 titers were determined by ELISA as described in Materials and Methods.