Regulatory DC-maintained long-lived CD4 T cells suppress CD4 T-cell response via cell contact-dependent IL-10 production. (A-C) In Transwell experiments, the long-lived CD4 T cells (CD4 Tm2) were separated from the mDC/naive CD4 T cell/OVA_323-339 coculture system. On day 3, Thy1.1⁺ CD4⁺ T-cell proliferation (A) and CD25 expression (B) were assayed by flow cytometry. The supernatants were collected for assay of cytokines by ELISA (C). (D) Various numbers of Thy1.1⁻ CD4 Tm2 were added into the mDC/naive CD4 T cell/OVA_323-339 coculture system. On day 3, supernatants were collected for assay of cytokines by ELISA. (E-G) Thy1.1⁺ CD4⁺ T-cell proliferation (E-F) and CD25 expression (G) were assayed when the supernatants from mDC/CD4 T, mDC/Tm2, or Tm2/mDC/CD4 T, or neutralizing antibodies (anti–IL-4, anti–IL-10, anti–TGF-β, anti–B7-H1, or anti–CTLA-4), or arginine analog L-NAME were added into the coculture system. Numbers in the histograms indicate the geometric mean fluorescence of the test samples. *P < .05; **P < .01. Similar results were obtained in at least 3 independent experiments.