Peptide-MHC complexes acquired by DN Treg cells induce apoptosis and suppress proliferation of Ag-specific CTLs. DN T cells from an HLA-A2^- donor (donor A) were cocultured with HLA-A2⁺ mature DCs (donor B) pulsed with either Melan-A or gp100 (control) peptides overnight. DN T cells were then separated from DCs by cell sorting and used as effector cells against Melan-A–specific CTLs generated from donor B. Induction of apoptosis of Melan-A–specific T cells was measured by combined Melan-A–multimer, annexin V, and PI staining on CD8-gated T cells. (A) Dot plot of annexin V-FITC/Melan-A multimer staining of CD3⁺CD8⁺-gated Melan-A–specific CTLs after 4 hours of coincubation with DN T cells stimulated either with Melan-A–pulsed DCs (left panel) or gp100-pulsed DCs (right panel) at different E/T ratios from 0 to 5:1. Numbers in the cross represent percentages of cells in the different quadrants within CD8⁺ T cells. One of 3 independent experiments is shown. (B) Percentage of different apoptotic target cell subpopulations (▪, DN-A2–Melan-A; ○, DN-A2–gp100) calculated from annexin V/PI staining. (C) Melan-A–specific CTL (CTL_resp) were cultured either with peptide-pulsed DCs or Melan-A–primed or unprimed DN T cells at a ratio of 1:1. [³H]TdR incorporation was measured after 24 hours of culture. Panels show one of at least 3 different experiments; bars represent the means (± SD) of triplicate wells.