IL-10–secreting CD8 cells suppress CD4 and CD8 T-cell growth and activation in a contact-dependent fashion. (A) Fresh CFSE-labeled OT-2 cells were stimulated with OVA²⁵⁷ + OVA³²³ ± TGF-βR-Fc neutralizing reagent (10 μg/mL), anti–IL-10 (5 μg/mL), TGF-β (10 ng/mL), or IL-10 (50 ng/mL). OT-1 cells primed for 4 days with OVA²⁵⁷ (Tc1 cells) or with IL-4/IL-12/DEX (Tregs; as in Figure 1) were added at a ratio of 1 Tc1/Treg to 2 targets. After 3 days, cells were stained for CD4/CD25. Gated CD4⁺ events (OT-2 targets) are shown. Top left quadrants indicate activated CD25⁺ CD4 cells which have divided. Total numbers of undivided targets recovered from each sample were 32 900 ± 4920 in unstimulated controls and 20 800 ± 7510 in the presence of Tregs. (B) Dose response of suppressive activity using OT-1 Tc1 cells or Tregs added to fresh CFSE-labeled OT-1 cultures at various doses. Experiment was analyzed as in panel A, but targets were distinguished by CFSE positivity alone. The percentage of suppression of the number of divided targets in a fixed volume of culture is shown. (C) Suppression by Tregs is contact dependent. CFSE⁺ OT-2 targets were cultured as in panel A but placed above and below a transwell membrane. OT-1 Tregs were added below the membrane. Cells were harvested after 3 days from above (no contact) and below (contact) the membrane and analyzed as in panel A. Total numbers of undivided targets recovered were 24 900 ± 8040 in unstimulated controls and 24 800 ± 6990 in the presence of Tregs. (D) Alloantigen-stimulated CD8 Tregs exhibit an anergic/suppressive phenotype. Tc1 cells/Tregs, generated in MLRs as in Figure 1C, were added to fresh BALB/c CD8 T cells + C57BL/6 DCs. Proliferation was measured by thymidine incorporation after 3 days. All data are representative of 3 independent experiments with similar results.