Fig. 4.

Peptides Notch-1 and Numb-1 expanded antigen-specific CD8⁺ cells. a Notch-1-TCR⁺ CD8⁺ cells expanded by incubation with 5 μg Notch-1 (2112–212)and b Numb-1-TCR⁺ CD8⁺ cells expanded by incubation with 1 μg Numb-1 (87–95). TCR^hi, TCR^med, and TCR^lo, populations are shown in gates R2, R3 and R4. Numbers in each box indicate the percentage of Ag-specific cells in the entire population. In the absence of Ag, IL-2 induced expansion of Ag-specific cells, was: Notch-1-TCR⁺ cells = (TCR^hi: 0.1%, TCR^med: 0.1%, TCR^lo: 1.0%). Numb-1-TCR⁺ cells = (TCR^hi: 0.3%, TCR^med: 1.0%, and TCR^lo: 0.4%) in PBMC from the same donor cultured with IL-2. c SK-OV-3.A2 cells present Numb-1peptide to Numb-1 peptide-activated PBMC. d Presentation of Numb-1 peptide to Numb-1 peptide-activated cells is dependent on [P]-lation by protein-Ser/Thr-kinases PI3 K does not appear to be involved in peptide presentation as shown by lack of effect of wortmannin. The MAPK-kinase inhibitor SB20380 had a weak inhibitory effect. IFN-γ was quantified at 24 h (Closed symbols) and at 48 h (Open symbols). Numb-1 peptide-activated PBMC produced more IFN-γ than Notch peptide-activated PBMC. At 48 h the amount of IFN- γ produced by two Notch peptide-activated cell lines was similar with the amount produced by the IL-2-activated cell lines. Only Notch-1 peptide can be presented by HLA-A2 antigens after Notch digestion by proteasome according to the program paproc.de