We have attempted to identify proteins and enzymatic activities involved in T cell-receptor (TcR)-triggered cytotoxic T lymphocyte (CTL) activation and effector functions. Several independent complementary approaches were used. A novel functional assay for CTL activation was developed by taking advantage of granule location of an easily detectable enzymatic activity. It is demonstrated that intensity of response (exocytosis of granules) can be manipulated by changing surface density of an immobilized anti-TcR mAb. Using such an assay, we found that TcR-triggered exocytosis is not constitutive and is regulated through cross-linking of TcR by antigen or anti-TcR mAb. The results of experiments utilizing different specific inhibitors and chelators are consistent with a model in which TcR-triggered exocytosis in CTL is mediated by protein kinase C activation, external Ca++ translocation through plasma membrane Ca++- channels and modulation of activity of Ca++, calmodulin (CaM)- dependent enzymes. Detailed studies of TcR-triggered exocytosis allowed us to identify an inhibitory biochemical pathway in CTL. We have found that cAMP - raising toxins and drugs as well as cAMP-dependent protein kinase activators inhibit TcR-triggered exocytosis, thus functioning as a possible """"""""OFF"""""""" signalling pathway. Work is now in progress to identify surface antigens which are coupled to this inhibitory pathway. It is shown that inhibition of CTL activation by anti-Lyt-2 mAb is not due to the triggering of a biochemical inhibitory pathway. The powerful effects of protein kinase C activators on lymphocyte physiology prompted us to consider them as potential immunomodulating drugs. We have found that compounds of the Bryostatin family (polycyclic lactone with protein kinase C activating and anti- cancer properties) are not lymphotoxic and can express both immunostimulating and immunosuppressive activities.
| Kojima, H; Toda, M; Sitkovsky, M V (2000) Comparison of Fas- versus perforin-mediated pathways of cytotoxicity in TCR- and Thy-1-activated murine T cells. Int Immunol 12:365-74 |
