Immunological functions of
heat shock proteins (HSPs) have long been recognized. In this study we aimed to efficiently 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 the presence of
IL-4 and
GM-CSF and exposed to
tumor HSP70. After 24 hours, dendritic cells were phenotypically characterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primary cultured
renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitro with
tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and
HLA-DR maturation markers than those pulsed with
tumor cell lysate and comparable to that of dendritic cells pulsed with
tumor cell lysate plus TNF-α. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented the highest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70 as the
tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cell maturation in the absence of TNF-α. Conspecific HSP70 may effectively be used as a
tumor antigen to pulse dendritic cells in vitro.