We also describe a novel naturally processed, immunogenic epitope, GPC-3522-530 FLAELAYDL, which this website is restricted to HLA-A2, a common class 1 allele in various ethnic groups, including Asians and Caucasians. Methods Cell lines T2 cells (HLA-A*0201) and the human

hepatocellular carcinoma cell line HepG2 (HLA-A*0201 and GPC-3 positive) were obtained from ATCC and expression of HLA-A2 and GPC-3 confirmed in the latter using flow cytometry, after staining with monoclonal antibodies against HLA-A2.1 (BB7.2, Dako, UK), and GPC-3 (Biomosaics Inc, Burlington, USA), respectively (data not shown). The cell lines were cultured in RPMI (Gibco, UK) or DMEM (Cambrex, UK), respectively, supplemented with 10% foetal calf serum (FCS) (Cambrex, UK) and antibiotics (penicillin G 100 IU/ml and Streptomycin 50 μg/ml). T2 binding assays The prediction tools SYFPEITHI [15] and HLAmotif [16] were used to reveal GPC-3 SBI-0206965 supplier peptide epitopes with predicted strong binding to HLA-A2. The top 30 peptides LY411575 molecular weight were reassessed using RankPep [17], which also predicts epitopes generated by the proteasome, and 6 peptide epitopes were selected (Table 1). These peptides were synthesized using standard f-moc technology (>95% purity, as determined by reverse phase HPLC; Sigma, UK), along with an AFP-derived, HLA-A2-binding peptide (GVALQTMKQ) [18], and a random,

non-HLA-A2 binding, control peptide (RGYVYQGL). The AFP peptide has only one anchor but is an “”immunodominant”" epitope [19] and its use was convenient because T cells reactive to this epitope have been shown to lyse HepG2 cells. Due to the hydrophobicity of peptides binding to HLA-A2, the lyophilized peptides

were resuspended in DMSO at 10 mM. Table 1 GPC-3 peptides predicted Sitaxentan to bind to HLA-A2 and be processed by the proteasome, and control peptides used in the study GPC-3 peptide Position Sequence 1 229-237 FLQALNLGI 2 522-530 FLAELAYDL 3 299-307 YILSLEELV 4 186-194 GLPSALDI 5 222-230 SLQVTRIFL 6 169-177 ELFDSLFPV AFP peptide   GVALQTMKQ Control peptide   RGYVYQGL The selected epitopes were tested for their binding affinity to HLA-A2.1 molecules using the cell line T2, which is deficient in TAP1 and TAP 2 (transporters associated with antigen processing 1 and 2) [20]. Although T2 cells express very low levels of HLA-A2.1 molecules under normal culture conditions, cell surface expression is upregulated when appropriate peptides bind and stabilize the HLA-A2.1 molecule. Thus, up-regulation of HLA-A2.1 expression in T2 cells by a peptide is regarded as an indication of it being an HLA-A2.1-restricted epitope [19]. HLA-A2.1 expression on the T2 cell surface was quantified by staining the cells with HLA-A2-specific antibody (1 μg/ml), as described [21].