SERIAL KILLING BY CYTOTOXIC T-LYMPHOCYTES - T-CELL RECEPTOR TRIGGERS DEGRANULATION, RE-FILLING OF THE LYTIC GRANULES AND SECRETION OF LYTICPROTEINS VIA A NON-GRANULE PATHWAY

CD8(+) cytotoxic T lymphocyte (CTL) clones begin to synthesize the lyt ic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secreto ry granules which are released after cross-linking of the T cell recep tor (TcR) upon target cell recognition. During lytic granule biogenesi s granzyme A protein synthesis can be detected between 2 and 10 days a fter allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A syn thesized is secreted directly from the CTL. TcR triggering of degranul ation also results in new synthesis of the lytic proteins, which can b e inhibited by cycloheximide (CHX). Some of the newly synthesized lyti c proteins can be stored in the cell and refill the granules. But up t o one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymat ic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal, Perforin i s also secreted via the constitutive pathway, both from the natural ki ller cell line, YT, and from CTL clones after TcR cross-linking. Const itutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delive red via a non-granule route. These results demonstrate that the perfor in/granzyme-mediated lytic pathway can be maintained while CTL kill mu ltiple targets. We show that CTL not only re-fill their granules durin g killing, but also secrete lytic proteins via a non-granule-mediated pathway.