862 CD3 negative large granular lymphocytes recognize an immunogenic determinant associated with the 72 KD heat shock protein (HSP) on human sarcoma cells

Abstract

Traditionally, heat shock proteins (HSP) are believed to be located intracellularly, where they perform a variety of chaperoning function. However, recent publications have demonstrated that under certain circumstances malignant cell types express HSP on the cell surface. Our studies confirm this finding and correlate HSP72 cell surface expression, induced by nonlethal heat shock, with increased tumorigenicity against CD3– natural killer cells (NK). A monoclonal antibody (mAb, RPN1l97) directed against the major heat inducible 72 kD heat shock protein (HSP72) binds to the cell surface of tumor cells (i. e. human Ewing's sarcoma cells or osteosarcoma cells), but not to normal cells (i.e. PBL, fibroblasts, PHA blasta, B-LCL) after single nonlethal heat shock (41.8°C, 200 min) followed by a recovery period at 37°C (4 h). Despite a decrease in the MHC class I cell surface expression after heat shock a marked increase (2-fold) in tumorigenicity as compared to untreated tumor cells was found. Analysis of cytotoxic activity of CD3– large granular lymphocytes (NK cells), CD3+ MHC restricted CTL and unseparated effector cells in a cell mediated lympholysis assay (CML), demonstrated that the CD3–NK effector cell population and not the GD3+CTL population, is responsible for the recognition of heat shocked tumor cells. By antibody inhibition (using this HSP72 specific MAb, RPN1197) an immunogenic HSP72 determinant, which is expressed only on the cell surface of tumor cells after nonlethal heat shock could be determined as the relevant recognition structure for CD3–NK cells. As a control, blocking of MHC class I restricted recognition (using either MHC class I specific MAb W6/32 on the target cells or a/β TCR WT31 on effector cells) had no inhibitory effect on the lysis of heat shocked tumor cells. In summary, our data indicate that CD3– NK cells recognize a heat inducible HSP72 related immunogenic epitope, on the cell surface of sarcoma cells, but not on normal cells. Supported by grant M90/91-Is1 from the Deutsche Krebshilfe and EC-Project CHRX-Ct93-0260 Traditionally, heat shock proteins (HSP) are believed to be located intracellularly, where they perform a variety of chaperoning function. However, recent publications have demonstrated that under certain circumstances malignant cell types express HSP on the cell surface. Our studies confirm this finding and correlate HSP72 cell surface expression, induced by nonlethal heat shock, with increased tumorigenicity against CD3– natural killer cells (NK). A monoclonal antibody (mAb, RPN1l97) directed against the major heat inducible 72 kD heat shock protein (HSP72) binds to the cell surface of tumor cells (i. e. human Ewing's sarcoma cells or osteosarcoma cells), but not to normal cells (i.e. PBL, fibroblasts, PHA blasta, B-LCL) after single nonlethal heat shock (41.8°C, 200 min) followed by a recovery period at 37°C (4 h). Despite a decrease in the MHC class I cell surface expression after heat shock a marked increase (2-fold) in tumorigenicity as compared to untreated tumor cells was found. Analysis of cytotoxic activity of CD3– large granular lymphocytes (NK cells), CD3+ MHC restricted CTL and unseparated effector cells in a cell mediated lympholysis assay (CML), demonstrated that the CD3–NK effector cell population and not the GD3+CTL population, is responsible for the recognition of heat shocked tumor cells. By antibody inhibition (using this HSP72 specific MAb, RPN1197) an immunogenic HSP72 determinant, which is expressed only on the cell surface of tumor cells after nonlethal heat shock could be determined as the relevant recognition structure for CD3–NK cells. As a control, blocking of MHC class I restricted recognition (using either MHC class I specific MAb W6/32 on the target cells or a/β TCR WT31 on effector cells) had no inhibitory effect on the lysis of heat shocked tumor cells. In summary, our data indicate that CD3– NK cells recognize a heat inducible HSP72 related immunogenic epitope, on the cell surface of sarcoma cells, but not on normal cells. Supported by grant M90/91-Is1 from the Deutsche Krebshilfe and EC-Project CHRX-Ct93-0260