Heat-shock protein 70 as a tumor antigen for in vitro dendritic cell pulsing in renal cell carcinoma cases.

Fan-Dong Meng,You-Hong Jiang,Yan Li,Cheng-Guang Sui,Yun-Peng Liu,Xin Tian,Chun-Ming Yang,Ping Ma

doi:10.7314/apjcp.2014.15.20.8947

Abstract

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.

Highlights

Heat-shock proteins (HSPs) are important molecular chaperons that play essential roles in the regulation of protein synthesis, folding and vesicular trafficking
T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were 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
We improved the purifying method of higher purity immunocompetent HSP70, and we demonstrate the ability of HSP70 from conspecific Renal cell carcinoma (RCC) specimens to be used as a tumor antigen for pulsing dendritic cells (DCs) in vitro

Summary

Introduction

Heat-shock proteins (HSPs) are important molecular chaperons that play essential roles in the regulation of protein synthesis, folding and vesicular trafficking. They are ubiquitously expressed at a basal level but are induced in response to various stress conditions such as elevated temperature (Srivastava et al, 2002). HSP70 induces specific cytotoxic T lymphocyte (CTL) responses by delivering chaperoned antigens into dendritic cells (DCs) which are recognized by CD8+ T cells (Srivastava et al, 2002;Calderwood et al, 2005). We improved the purifying method of higher purity immunocompetent HSP70, and we demonstrate the ability of HSP70 from conspecific RCC specimens to be used as a tumor antigen for pulsing DCs in vitro

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