Th17 cells are more resistant to AICD than Th1 cells in vitro and in vivo. (A) Th1 and Th17 cells were generated from ovalbumin-specific TCR-transgenic T cells in vitro, as described in “Methods.” Expression levels of intracellular interferon-γ and IL-17 on live CD4⁺ cells are shown for Th1 and Th17 cells 4 days. IL-4⁺ cells were < 1% in either subset (data not shown). (B) Polarized Th1 and Th17 cells were restimulated in the presence of anti-CD3 mAb at the indicated concentrations. Cells were stained with annexin V and DAPI to detect early and late apoptotic cells, respectively. Data represent 1 of 3 similar experiments. (C) Survival of Th1 and Th17 cells in mixed culture in vitro. Th1 cells were generated from Thy1.1 OT-II mice, and Th17 cells were from Thy1.2 OT-II mice. Mixed Th1 (Thy1.1⁺) and Th17 (Thy1.2⁺) cells were cultured at a ratio of 1:1. Apoptosis was analyzed with (right panel) or without (left panel) anti-CD3 mAb treatment for 20 hours. Data represent 1 of 3 replicate experiments. (D) Survival of Th1 and Th17 cells in allogeneic recipients in vivo. Th1 cells generated from normal B6 mice and Th17 cells from Ly5.1 B6 mice were together injected intravenously into lethally irradiated BALB/c recipients at a ratio of 1:1. The percentages of live Th1 and Th17 cells were determined (left panel) in the recipient spleen 4 days after cell transfer. Apoptosis was determined by DAPI and annexin V staining (middle panel). Cell division was determined by 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester staining (right panel).