To identify proteins (enzymes) involved in T cell-receptor (TCR)- triggered mechanobiochemical activities of cytotoxic T-lymphocytes (CTL), we study the functional organization of cell surface receptors and underlying membrane matrix/cytoskeleton, as well as biochemical events that follow occupation of functionally important surface proteins by appropriate crosslinking ligands, antigens, or mAb. Biochemistry of CTL activation: We developed a novel functional assay (granule exocytosis assay) for CTL activation and characterized activating and inhibitory biochemical pathways. These pathways re implicated in "ON" and "OFF" signalling which forms a basis for cyclic activities of CTL. Protein kinase C and calmodulin-binding proteins (CaM-BP) are part of TCR-triggered activating pathway, while cAMP-dependent protein kinase which counteracts both early and late events of activating pathway, represents the key enzyme of inhibiting pathway. The biochemical studies of T-cells suggested that CaM-dependent phosphatase (calcineurin) is the main beneficiary of TCR-induced increases n intracellular Ca++and pointed to the dephosphorylation reactions as the major response to TCR crosslinking. Plasma membrane organization: During functional studies of organization of surface proteins we came to the conclusion that inhibition of CTL activation by anti-CD8 mAb is most likely explained by physical proximity of CD8 with TCR. However, near-neighbor analysis studies with chemical cross-linking reagents unexpectedly revealed that CD8 is most easily crosslinked to class I MHC proteins on the surface of CTL. The experiments designed to isolate and characterize functional domains in CTL plasma membrane suggest the existence of a subset of surface proteins which are associated with membrane matrix and demonstrated TCR-dependent changes in their organization. Immunopharmacological implications: A novel strategy is suggested for manipulation of immune response by taking advantage of newly-found knowledge of biochemistry of lymphocyte activation. Non-tumor promoting protein kinase C activators (e.g. compounds of Bryostatin family) and antagonists of cAMP (e.g. Rp- cAMPS) are proposed to be used as immunoenhancing drugs.
NIH ExPorter
NIH ExPorter