Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes

Background: glypican 3 (gpc-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (hcc). since it is a potential target for t cell immunotherapy,we investigated the generation of functional,gpc-3 specific t cells from peripheral blood mononuclear cells (pbmc). methods. dendritic cells (dc) were derived from adherent pbmc cultured at 37°c for 7 days in x-vivo,1% autologous plasma,and 800 u/ml gm-csf plus 500 u/ml il-4. immature dc were transfected with 20 g of in vitro synthesised gpc-3 mrna by electroporation using the easy-ject plus system (equibio,uk) (300 v,150 f and 4 ms pulse time),or pulsed with peptide,and subsequently matured with lipopolysaccharide (lps). six predicted gpc-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to hla-a2.1 molecules using the cell line t2. results: dc transfected with gpc-3 mrna but not control dc demonstrated strong intracellular staining for gpc-3 and in vitro generated interferon-gamma expressing t cells from autologous pbmc harvested from normal subjects. one peptide,gpc-3522-530flaelaydl,fulfilled our criteria as a naturally processed,hla-a2-restricted cytotoxic t lymphocyte (ctl) epitope: i) it showed high affinity binding to hla-a2,in t2 cell binding assay; ii) it was generated by the mhc class i processing pathway in dc transfected with gpc-3 mrna,and iii) hla-a2 positive dc loaded with the peptide stimulated proliferation in autologous t cells and generated ctl that lysed hla-a2 and gpc-3 positive target cells. conclusions: these findings demonstrate that electroporation of gpc-3 mrna is an efficient method to load human monocyte-derived dc with antigen because in vitro they generated gpc-3-reactive t cells that were functional,as shown by interferon-gamma production. furthermore,this study identified a novel naturally processed,hla-a2-restricted ctl epitope,gpc-3522-530flaelaydl,which can be used to monitor hla-a2-restricted ctl responses in patients with hcc. further studies are required to investigate whether anti-gpc-3 immunotherapy has a role in the treatment of gpc-3 dependent tumours,such as hcc. © 2010 o'beirne et al; licensee biomed central ltd.