Generation of specific cytotoxicity T lymphocytes induced by tumor-derived heat shock protein 90-peptide complexes in vitro

Abstract

To investigate the specific induction of cytotoxic lymphocyte (CTL) by tumor-derived heat shock protein 90-peptide complexes (HSP90-PC). Heat shock protein 90-peptide complex (HSP90-PC) was isolated and purified by liquid chromatography after precipitation by 50% - 70% (NH4) 2SO4 saturation from 10 specimens of renal carcinoma resected from 10 patients aged 40 - 60 during operation. The component containing HSP90-PC was filtered and sterilized. The molecular weight and the identity of the purified HSP90-PC were confirmed by SDS-PAGE and Western blotting. 10 - 15 ml peripheral blood was extracted from these patients. T cells were amplified. Flow cytometry was used to detect the phenotype of dendritic cells (DCs). The DCs in the experimental group were cultured for 5 days and then HSP90-PC and tumor necrosis factor (TNF)-alpha was added into the culture. The HSP90-PC pulsed DCs were collected and co-cultured with auto-T cells for 72 hours. Flow cytometry was used to detect the content of CD8(+) T cells. The DC of the control group were mixed directly with auto-T cells and the content of CD8(+) T cells was examined by flow cytometry. The proliferation of T cells co-cultured with the HSP90-PC pulsed DCs was significantly remarkable and the content of CD8(+) CTLs was significantly more in comparison with the control DC (P < 0.01). HSP90-PC prepared from tumor tissues has strong immunogenicity and the DC sensitized thereby effectively induces the proliferation of CTL. Application of HSP90-PC provides a new approach in tumor immunotherapy.