Regulatory DC-maintained long-lived CD4 T cells suppress antigen-specific CD4 T-cell activation and proliferation both in vitro and in vivo. (A-C) The suppressive effect of the long-lived CD4 T cells (Thy1.1⁻, Longeval CD4 T cells) on proliferation and CD25 expression of Thy1.1⁺ naive CD4 T cells activated by mDCs for 3 days in the presence of 200nM OVA_323-339. On day 3, Thy1.1⁺ CD4⁺ 7AAD⁻ viable T cells were counted (A) and gated for analyzing CD25 expression (C) by flow cytometry. For analyzing the influence of the long-lived CD4 T cells on CD4 T-cell division initiated by mDCs and OVA_323-339, Thy1.1⁺ CD4 T cells were labeled with CFSE, and Thy1.1⁺ CD4⁺ 7AAD⁻ viable T cells were gated for analyzing CFSE content (B). (D) Schematic of the experimental design of the DTH response. (E) BALB/c × C57BL/6J F1 mice were sensitized with OVA/CFA on day 0. On days −1, 3, and 6, the long-lived CD4 T cells (3 × 10⁶ cells/mouse) were adoptively transferred into the immunized mice via intravenous injection. On day 7, the mice were challenged with heat-aggregated OVA, and then DTH responses were assessed over the following 24 hours. The mice of the unimmunized control group (Ctrl) were challenged with heat-aggregated OVA. The DTH responses are expressed as the mean ± SDs of the increase in footpad thickness for 5 mice per group. *P < .05; **P < .01. Similar results were obtained in at least 3 independent experiments.