A novel cytotoxic T lymphocyte activation assay: Optimized conditions for antigen receptor triggered granule enzyme secretion

Abstract

A method is described for the quantitative studies of cytotoxic T lymphocyte (CTL) activation. This functional assay is based on the measurements of secreted granule-associated enzymatic activity (BLT esterase (BLT-E)) after incubation of CTL with activating stimuli. Immobilized mAb against CTL's antigen receptor (anti-TcR mAb), concanavalin A or a combination of PMA and ionophore A23187, were able to trigger the secretion of enzyme in the absence of target cells. Soluble anti-TcR mAb alone did not activate CTL, but using their conjugate with immobilized rabbit anti-mouse Ig antibody (RAMIg) TcR-mediated secretion of BLT-E was detected. Use of non-ionic detergents Nonidet P-40 or Triton X-100 (0.0125–0.2%) did not affect measurements of BLT-E activity. The efficiency of CTL exocytosis triggering by anti-TcR mAb which were immobilized on the surface of different plasticware is compared and conditions for studies of small and large numbers of CTL are described. The intensity of CTL response varies markedly with changes in buffer system, culture medium, additions of proteins. The optimal conditions for TcR complex triggered activation of murine CTL are described. Intensity of secretion can be easily manipulated by changing the surface density of immobilized anti-TcR mAb, thereby providing the possibility to screen inhibiting or activating agents (drugs or mAb) at selected sub-optimal levels of CTL activation. The potential for the use of described assay in screening of hybridoma supernatants for the presence of activating or inhibitory mAb against CTL's surface proteins is discussed. Since BLT-E secretion reflects exocytosis of granules from CTL, the conditions described here could be used for the detection of secretion of other markers of granules in future modifications of granule exocytosis assay.