Figure 6
Generation of SP epitopes of PPI is independent of TAP.
(A) To examine TAP dependency of PPI15–24 presentation by K562 cells, the CTL clone (1E6) recognizing PPI15–24 restricted by HLA-A2 was used in cytotoxicity assays in the presence of the varicellovirus-derived TAP inhibitor UL49.5 (19). As a control, we used the HA-2 CTL clone 5H17 (53), specific for an endogenously expressed, TAP-dependent minor histocompatibility antigenic epitope (sequence YIGEVLVSV derived from a diallelic gene encoding a novel human class I myosin protein) presented by HLA-A2 (54). Target cells for clone 1E6 were K562-PPI-A2 cells or K562-PPI-A2 cells additionally transfected to express UL49.5. Target cells for clone 5H17 were K562-A2 cells or K562-A2 cells additionally transfected to express UL49.5. Data are expressed as percent killing (see Methods for calculation of specific cytotoxicity using DELFIA assay); bars represent means and error bars, SEMs. In the presence of the 1E6 PPI15–24–specific CTLs, killing of K562-PPI-A2 cells cotransfected with UL49.5 (black bars) was comparable to that of untransfected K562-PPI-A2 cells (white bars), indicating that TAP inhibition by UL49.5 does not reduce PPI15–24 presentation. In contrast, killing of target cells by the HA-2–specific CTL 5H17 that recognizes a TAP-dependent epitope is markedly inhibited in the presence of UL49.5 (black bars). (B) To examine proteasome dependency of PPI15–24 generation, MS analyses of immuno- and constitutive proteasome digests of PPI1–30 were performed. Results are shown in Table 2. In the same experiment, several other fragments of PPI1–30 were generated by proteasome digestion. Sequences marked with an asterisk were generated by the constitutive proteasome only. For reference, the 10-mer PPI15–24 peptide eluted from surrogate β cells is indicated by the black rectangle.
