Sperm protein 17 (Sp17) is a novel cancer-testis antigen. We previously reported the successful generation of Sp17-specific HLA-A1-restricted cytotoxic T-lymphocytes (CTLs) from the peripheral blood of a healthy donor using a recombinant Sp17 protein. These CTLs were able to kill not only target cells pulsed with the recombinant protein but also fresh Sp17؉ tumor cells. In the present study, we have identified a nonapeptide sequence within the Sp17 protein that is predicted to have a high binding affinity for the HLA-A1 molecules. We generated the synthetic nonapeptide and successfully propagated a peptide-specific CTL line. We confirmed that peptide Sp17 Sperm protein 17 (Sp17) is a spermatozoa-specific protein of unknown function, although it has been implicated in acrosome reactions during fertilization 1 and appears to bind heparan sulfate. 2 It is a highly immunogenic protein in vivo because a high proportion of vasectomized males spontaneously develops anti-Sp17 antibodies. We have identified Sp17 as a novel cancer-testis (CT) antigen in multiple myeloma 3 and ovarian cancer. 4 Sp17 expression is found at both the mRNA and protein levels and its normal tissue expression is limited to only testis and not any other normal tissues. The restricted normal tissue expression of Sp17 has now been confirmed independently, 5 suggesting that an Sp17-based tumor vaccine will be tumor-specific with very limited toxicities to normal tissues.The clinical relevance of our findings has been supported by further work demonstrating the ability to generate Sp17-specific cytotoxic T-lymphocytes (CTLs) that could kill Sp17-positive fresh autologous or HLA-matched tumor cells from healthy donors 6,7 and cancer-bearing patients. 4,8 Through these works, we have successfully generated an Sp17-specific HLA-A1-restricted CTL line from a healthy donor. 6 Since delineation of the CTL epitope may provide the tool for the future study and characterization of CD8 T-cell function in vivo and the generation of epitope-specific therapeutic strategies, we have in the present study used this T-cell line to determine the Sp17 CTL epitope restricted by the HLA-A1 element. MATERIAL AND METHODSPeripheral blood mononuclear cells (PBMCs) were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. Dendritic cells (DCs) were generated from peripheral blood monocytes as previously described. 9 Briefly, PBMCs were seeded into 6-well culture plates containing 3 ml of RPMI 1640 and 10% FCS at 5-10 ϫ 10 6 per well. After 2 hr at 37°C, nonadherent cells were removed and the adherent cells were cultured at 37°C in RPMI 1640 supplemented with 10% FCS, 800 IU/ml GM-CSF and 1000 IU/ml IL-4. After 7 days of culture, DCs were harvested for pulsing with the synthetic peptide and used as antigen-presenting cells for the generation of Sp17 peptide-specific CTLs. Successful generation of DCs was confirmed in flow cytometry.Following culture, the DCs were washed twice and added to 50 ml polypropylene tubes. The DCs were pulsed with...