The role of granule serine proteases in CTL lytic function was probed by treatment of intact CTL with PMSF in combination with agents which raise the pH of acidic intracellular compartments, and then compared the residual granule BLT esterase activity with the residual lytic activity. PMSF alone caused losses of about 50% in lytic activity and 90% in BLT esterase, while PMSF and ammonium chloride caused losses of about 70% in lytic activity and 99% in BLT esterase. These results suggest that serine esterases in low pH compartments are unlikely to be directly involved in the lytic function. One of the major CTL granule proteases, a BLT esterase, has been useful as a granule marker for measuring exocytosis. With mouse CTL, the enyzme is secreted after stimulation by target cell antigen or immobilized antibodies against the T cell receptor complex, and with human CTL, immobilized anti-T3 stimulate. In both these systems, anti LFA-1 antibodies block secretion, showing this can act directly on effector cells rather than necessarily blocking a target cell binding. Using in vivo generated CTL, BLT esterase was found in granules by the same approaches used for cloned CTL. Secretion of this enzyme was triggered specifically by tumor target cells and preceded cytotoxicity. Cytolysin activity was found in dense granules of these CTL, but was about 100 x lower than found in cloned CTL. Cloned helper T cells also contain granules with high levels of BLT esterase which reacts with antibodies to the dimeric """"""""major"""""""" LGL serine protease. Using BLT esterase release as a marker, granule exocytosis from these cells has been triggered by antigen after processing by class 2 matched presenting cells, by Con A, by PMA and calcium ionophore, and by immobilized anti-T cell receptor antibodies. In the latter situation, secretion can be measured within on hour, and secretion of the lysosomal enzyme beta-N-acetyl hexosaminidase is also detected. EM studies of these helper cells have revealed cytoplasmic granules containing striking and unusual lamellar figures undergoing exocytosis. Using beta-N- acetyl hexosaminidase release as an assay, granule exocytosis was not observed from normal T cells stimulated by anti-T3 coated beads.
