A second aim of this study was to identify HLA-A2-restricted epitopes derived from GPC-3. When we analyzed the amino acid sequence of human GPC-3, 6 sequences were identified that were predicted both to bind to HLA-A2 and to be processed by the proteasome. We used flow cytometry analysis of T2 cells, which are TAP deficient, to measure the half-life of peptide binding to HLA-A2

and identified 4 peptides with prolonged, high affinity binding for HLA-A2. Of these, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted CTL epitope because: i) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and ii) HLA-A2 positive, monocyte-derived DC loaded with the peptide stimulated proliferation in autologous T DUB inhibitor cells and generated CTL that lysed HLA-A2 and GPC-3 positive tumour SCH727965 molecular weight cells. One of the peptides GPC-3169-177 ELFDSLFPV predicted to have strong binding to HLA-A2 was found to rapidly dissociate from HLA-A2 in the present

study and DC loaded with this peptide did not stimulate autologous T cells in HLA-A2 positive subjects, a finding confirmed by Nishimura and colleagues who found that DC loaded with GPC-3169-177 ELFDSLFPV were unable to induce CTL or T cells producing interferon-gamma [34]. Previously, Komori et al used HLA-A2.1 transgenic mice to identify HLA-A2 (A*0201)-restricted GPC-3 epitopes but found no evidence that CTL were generated

against GPC-3522-530 FLAELAYDL in animals immunized with DC pulsed with a mixture of peptides because, after spleen cell harvest, only CD4- T cells stimulated in vitro with the peptide GPC-3144-152 FVGEFFTDV produced high levels of interferon-γ[31]. These findings suggest that the epitope GPC-3144-152 might be immunodominant in this system or, alternatively, CTL reactive to GPC-3522-530 these may not have been generated in HLA-A2.1 transgenic mice due to differences in the T cell selleck screening library repertoire between mice and humans, resulting in some HLA-A2-restricted epitopes being recognized only by human T cells. Non-dominant epitopes, although having a weaker affinity to MHC, can still induce reactive CTL with cytotoxic activity and thus be applicable for immunotherapy [35]. Indeed, T cells responding to such epitopes are often better represented in the peripheral T cell repertoire because those responding to self-epitopes with strong MHC binding are more likely to be deleted in the thymus during the ontogeny of the immune system [36].