CD8 Tregs display a unique cell-surface phenotype which becomes stable after extensive culture. (A) OT-1 Tc1 (filled histograms) and Treg (open histograms) effectors were stained with a panel of antibodies. Positive staining relative to negative control is indicated by the markers. Similar results were obtained in 6 independent experiments. CD86 was barely detectable on either subset (not shown). (B) OT-1 cells fail to down-regulate CD45RB and up-regulate CD44 during culture in Treg-inducing conditions. CFSE-labeled OT-1 cells cultured in Tc1 or Treg conditions were analyzed after 3 days. CD8-gated events are shown. Boxed regions (% divided) indicate the similar extent of cell division observed under Tc1 and Treg conditions. Mean fluorescence intensities (MFIs) in these regions are indicated. Similar results were obtained in 3 independent experiments. (C) Conversion of naive to memory phenotype is inhibited by IL-4 + IL-12 in CD8 but not CD4 populations. The percentage of CD45RB^hi CD44^lo(naive phenotype) and CD45RB^lo CD44^hi (memory phenotype) cells was determined in CD8/CD4 BALB/c populations and after 7 days stimulation with C57BL/6 DC (MLR CD4/CD8) ± IL-4/IL-12/DEX. All cultured cells were CD62L negative (not shown). Means ± SEMs from 3 experiments are shown. (D) Both naive and memory CD8 T cells segregate into CD44^hi and CD44^lo populations after culture in Tc1- or Treg-inducing conditions, respectively. OT-1 CD8 cells were separated into CD44^lo (naive) and CD44^hi (memory) populations (left), mixed with DCs (20:1 ratio) and stimulated in Tc1- or Treg-inducing conditions as in panel B. CD44 expression of cells that were initially naive (top right) or memory (bottom right) phenotype was determined after 4 days. OT-1 Tc1/Treg cells were maintained in culture for 4 weeks using repeated restimulation with DCs, OVA²⁵⁷, and cytokines ± IL-4/IL-12/DEX. Intracellular cytokine analysis (E; as in Figure 1), or surface marker staining (F; as in Figure 4A) was performed. Filled histograms indicate Tc1 cell line; open histograms, Treg cell line. (G) Tregs were generated over a period of 4 days as in Figure 1 (Treg effectors) or 4 weeks as in panel E (Treg cell line). Washed cells were split and placed in secondary cultures with IL-15 + IL-7 + IL-4/IL-12/DEX (Treg conditions) or with IL-15 + IL-7 only (Tc1 conditions) for a further 4 days (effectors) or 7 days (cell lines). Treg cell lines also received DCs + OVA^257-264 for the final 7 days. Finally, cells were reanalyzed for cytokine profile and phenotype. The percentage of cells positive for IFN-γ and IL-10 are shown (central histogram), and levels of CD44/CD45RB are indicated on the right. Data are representative of 3 independent experiments.