title: Generation of functional CD8+ T cells by human dendritic cells expressing glypican-3 epitopes.
abstract: 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 degrees 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 microg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 microF 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-3522-530 FLAELAYDL, 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-3522-530 FLAELAYDL, 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.
authors.affiliation: Department of Liver Studies & Transplantation, Kings College London, Denmark Hill Campus, Bessemer Road, London, UK.
authors.forename: James
authors.initials: J
authors.lastname: O'Beirne
authors.forename: Farzin
authors.initials: F
authors.lastname: Farzaneh
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Harrison
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology* / Q000378:metabolism; D053673:Glypicans / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015789:HLA-A2 Antigen / Q000276:immunology / Q000378:metabolism; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D018984:Epitopes, T-Lymphocyte; D053673:Glypicans; D015789:HLA-A2 Antigen; D012333:RNA, Messenger
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