O'Beirne et al. Journal of Experimental & Clinical Cancer Research 2010, 29:48 http://www.jeccr.com/content/29/1/48 Open Access RESEARCH BioMed Central © 2010 O'Beirne et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes James O'Beirne 1,3 , Farzin Farzaneh 2 and Phillip M Harrison* 1 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°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 μg of in vitro synthesised GPC- 3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF 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-3 522-530 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-3 522-530 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. Background Increasing evidence suggests that immune responses play an important role in the control of cancer and manipula- tion of the immune system to recognize and attack cancer cells may be a valuable form of therapy [1]. Hepatocellu- lar carcinoma (HCC), which is the third most common cause of cancer death world-wide [2], is a potential target for immunotherapy [3] because there are numerous doc- umented cases of spontaneous regression [4] and the presence of cytotoxic tumour infiltrating lymphocytes (TIL) at histological examination is associated with a bet- ter prognosis after liver resection or transplantation [5]. Infusion of T lymphocytes, activated with anti CD3 and interleukin 2 (IL2), improved disease-free survival after HCC resection, suggesting a role for T cell immunother- apy in this setting [6]. However, current methods of isola- tion and in vitro expansion of T lymphocytes are cumbersome and expensive, and the durability of any anti-tumour immune response induced by administra- tion of non-antigen specific, in vitro expanded T cells is unknown [7]. * Correspondence: phillip.harrison@kcl.ac.uk 1 Department of Liver Studies & Transplantation, Kings College London, Denmark Hill Campus, Bessemer Road, London, SE5 9RS, UK Full list of author information is available at the end of the article