Heat-Shock Protein 70 as a Tumor Antigen for in vitro Dendritic Cell Pulsing in Renal Cell Carcinoma Cases

Abstract

Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed toefficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro.HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose,PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC).Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in thepresence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypicallycharacterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclearcells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primarycultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitrowith tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markersthan those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysateplus TNF-α. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented thehighest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cellmaturation in the absence of TNF-α. Conspecific HSP70 may effectively be used as a tumor antigen to pulsedendritic cells in vitro.