A Study of the Effect of Activated Peripheral Blood Mononuclear Cell Derived Cytokines Upon Monocyte Mediated Tumour Cytotoxicity

[摘要] Cytokine activated peripheral blood mononuclear (PBMC) cells, sometimes called 'Lymphokine Activated Killer (LAK)' cells, have been shown to mediate tumour cell cytotoxicity in vitro but their mode of action in vivo is poorly understood. LAK cell therapy in humans on the whole has been unsuccessful with only minor success against melanomas and renal cell carcinomas. However the mode of tumour regression in these few instances is still unknown. In vitro tumour cytotoxicity by LAK cells does occur but LAK cells do not traffic to, accumulate around or infiltrate the site of tumours in vivo. Intravenous infusion of LAK cells into peripheral circulation causes the cells to traffic to the liver and spleen for clearance. The spleen is a lymphoid cell reservoir and thus interaction with T cells and monocytes would be common. Therefore these cellular effector cells may be causing tumour cell regression via an indirect route, meaning they are activated by secondary cytokines produced by the LAK cells. This project investigates the hypothesis that cytokines known to be produced by LAK cells can induce an anti-tumour response upon monocytes in vitro. The aim of this project was firstly to investigate the effect of cytokines secreted from activated (PBMC) upon monocyte mediated tumour cell cytotoxicity. More specifically it was to determine the exposure time and concentration of mitogen, which would cause LAK cells to optimally secrete secondary cytokines which in turn would induce monocyte tumour cell cytotoxicity. Analysis of cytokine secretion allowed the dose and exposure time of stimulus (either IL-2 or anti-CD3 antibody) which generated the cytokine rich supernatants to be defined. Three cytokines were examined, in the supernatants, to determine whether they had any effect on monocyte tumour cytotoxicity, using a 48hr 3H-Uridine cytotoxicity assay. Their concentrations were determined using bioassays and ELISA's. The cellular mechanism of monocyte tumour cytotoxicity was examined by observing adhesion molecule upregulation (in particular LFA-1 and Mac-1), MHC II cell surface density and also TNF-alpha secretion by the monocytes. This was carried out to create a model of how these secondary cytokines were actually destroying the tumour cells. In conclusion, recombinant IL-2 and soluble monoclonal anti-CD3 both induced LAK activity and cytokine secretion in normal PBMC. Furthermore, secondary cytokines, particularly IL-1 and IFN-gamma, produced by the IL-2 stimulated cells induced both an increased TNF-alpha secretion and tumour cytotoxicity by monocytes. These two parameters were found to be directly proportional to one another. These results imply that secondary cytokine production by IL-2 activated PBMC causes increased tumour cell killing by monocytes in vitro. The cytokines IL-1 and IFN-gamma were found to be important in the upregulation of monocyte cytotoxicity and the cytokine TNF-alpha was found to be important in the direct mechanism of tumour cell killing. The adhesion molecules examined did not appear to have a role in the tumour cell killing. However other cytokines, mitogens and/ or adhesion molecules may also be involved and further study would be required to determine if this were the case.

[发布机构] University:University of Glasgow

[关键词] Molecular biology