Mechanism of suppression used by CD8 Tregs. (A) Suppression is reversed by TCR or CD28-related signals but not IL-2. Suppression assays were performed as in Figure 2A by using CFSE-labeled OT-2 cells ± OT-1 Tc1/Treg, stimulated with OVA²⁵⁷ + OVA³²³ (Ag) ± IL-2 (10 ng/mL), anti-CD28 (1 μg/mL), PMA (10 ng/mL) + ionomycin (400 ng/mL), or immobilized anti-CD3 alone (1 μg/mL). The percentage of suppression of divided targets generated in a fixed volume of culture (means ± SEMs from 3 independent experiments) is shown. (B) Direct T/T suppression in the absence of APCs. CFSE⁺ OT-1 CD8 cells depleted of all APCs (< 0.1%) were labeled with OVA²⁵⁷–H-2K^b Pentamer. OT-1 Tc1 cells/Tregs were added at a 1:2 suppressor-to-target ratio, and CD25 was analyzed in CFSE⁺-gated targets after 2 days. One of 3 independent experiments with similar results is shown. (C) Tregs possess equivalent cytotoxicity to Tc1 cells. OT-1 Tc1cells/Tregs were mixed with ³H-thymidine–labeled EG-7 cells, and CTL activity was measured after 4 hours. The percentage of DNA degradation in labeled targets is shown. (D) Tc1 cells/Tregs do not kill OT-2 targets in a suppression assay. OT-2 cells were stimulated with OVA²⁵⁷ + OVA³²³ ± OT-1 Tc1/Treg (1:1) for 6 or 24 hours, then stained with anti-CD4, annexin V, and propidium iodide (PI). Levels of apoptotic (annexin V⁺ PI^–) and necrotic (annexin V⁺ PI⁺) CD4 cells are shown. Similar results were obtained in 3 independent experiments. (E) CD8 Tregs do not express Foxp3. RT-PCR for Foxp3 (top) and HPRT control (bottom) were performed on OT-1 Tc1 cells/Tregs after 3 days and from CD4⁺CD25– or CD4⁺CD25⁺ cells stimulated with anti-CD3/anti-CD28/IL-2 as a positive control. Molecular weight markers (right-hand lane) confirmed correct size of PCR products. Data are representative of 3 independent experiments. (F) In vivo suppression of T-cell activation is abrogated by costimulation. CFSE⁺ OT-2 cells were transferred into C57BL/6 mice with or without unlabeled OT-1 Tc1 cells or Tregs (3:1), and mice were challenged with OVA ± anti-CD28 intranasally as indicated. Target cell activation in draining lymph nodes (CD4⁺ CFSE⁺ gated events) is shown after 2 days.