Regulatory DCs maintain survival of antigen-specific CD4⁺ T cells in vitro for > 30 days via OX-40L. (A) Naive CD4⁺ T cells were activated by mDCs in the presence of antigen OVA_323-339 for 4 days, and then the effector CD4⁺ T cells were purified to coculture with diffDCs at a ratio of 10:1 for various times in the presence of 50% ESSC supernatant and 10nM OVA_323-339. In some experiments, anti–OX-40L or anti–4-1BBL blocking antibody (5μg/mL) was added into the coculture system. Viable CD4⁺ T cells were counted by flow cytometry. (B) The Transwell system was used to separate effector CD4 T cells from diffDCs and then viable CD4 T cells were counted. (C) OX-40L and 4-1BBL expression on diffDCs and mDCs were detected by flow cytometry. Numbers in the histograms indicate the geometric mean fluorescence of the test samples. (D) The purified effector CD4⁺ T cells labeled with 5μM CFSE were cocultured with diffDCs at a ratio of 10:1 for 3 or 6 days in the presence of 50% ESSC supernatant and 10nM OVA_323-339 with or without anti–OX-40L blocking antibody. Viable CD4⁺ T cells were gated for analyzing CFSE content of CD4 T cells by flow cytometry. **P < .01. Similar results were obtained in at least 3 independent experiments.