Cytotoxic T lymphocytes (CTLs) kill targets by releasing cytotoxic agents from lytic granules. Killing is a multi-step process. The CTL adheres to a target, allowing its T-cell receptors to recognize antigen. This triggers a signal transduction cascade that leads to the polarization of the microtubule cytoskeleton and granules towards the target, followed by exocytosis that occurs specifically at the site of contact. As with cytokine production by helper T cells (Th cells), target cell killing is absolutely dependent on Ca2+ influx, which is involved in regulating both reorientation and release. Current evidence suggests that Ca2+ influx in CTLs, as in Th cells, occurs via depletion-activated channels. The molecules that couple increases in Ca2+ to reorientation are unknown. The Ca2+/calmodulin-dependent phosphatase calcineurin, which plays a critical role in cytokine production by Th cells, is also involved in lytic granule exocytosis, although the relevant substrates remain to be identified and calcineurin activation is only one Ca2+-dependent step involved. There are thus striking similarities and important differences between Ca2+ signals in Th cells and CTLs, illustrating how cells can use similar signal transduction pathways to generate different functional outcomes.

CTLs kill virus-infected, tumor, and transplanted targets via secretion of lytic agents including perforin and granzymes. Knowledge of the signals controlling this important process remains vague. We have tested the idea that protein kinase C (PKC)theta, a member of the novel PKC (nPKC) family, which has been shown to play a preferential role in critical Th cell functions, plays a similar, preferential role in CTL lytic granule exocytosis using T acute lymphoblastic leukemia-104 (TALL-104) human leukemic CTLs as a model. We provide evidence consistent with the idea that PKC activity is important for the degranulation step of lytic granule exocytosis, as opposed to upstream events. In contrast with previous work, our results with pharmacological agents suggest that conventional PKCs (cPKCs) and nPKCs may participate. Our results suggest that stimulation with soluble agents that bypass the TCR and trigger granule exocytosis activates PKCalpha and PKCtheta, which can both accumulate at the site of contact with a target cell, although PKCtheta did so more often. Finally, using a novel assay that detects granule exocytosis specifically in transfected, viable cells, we find that overexpression of constitutively active mutants of PKCalpha or PKCtheta can synergize with increases in intracellular [Ca(2+)] to promote granule exocytosis. Taken together, our results lend support for the idea that PKCtheta does not play a preferential role in CTL granule exocytosis.

We have tested the idea that calcineurin, a calcium-dependent phosphatase that is critical for activating cytokine gene expression in helper T cells, plays a role in lytic granule exocytosis in cytotoxic T lymphocytes (CTLs). We used TALL Immunopharmacology 47, 119 -125), JNK is not required for lytic granule exocytosis, but we were not able to exclude a role for P38. Exocytosis could be inhibited by expressing GFP fused to a C-terminal fragment of CAIN (cabin 1), but not by expressing VIVIT-GFP. Finally, expressing either full-length or truncated constitutively active mutant calcineurin A enhanced lytic granule exocytosis. However, the mutant calcineurin was unable to support exocytosis when cells were stimulated in the absence of Ca 2؉ influx. Taken together, our results support the idea that activation of calcineurin is required for lytic granule exocytosis but suggest that it is not the sole Ca 2؉ -dependent step.

Cytotoxic T lymphocytes kill targets via secretion of lytic agents including perforin and granzymes. Recently, new methods have been developed to monitor cytotoxic T lymphocyte degranulation. These include detecting the appearance of lysosome-associated membrane protein on the cell's surface, and monitoring decreases in cellular perforin content. We have combined these methods with microscopy and flow cytometry to provide the first analysis of how single cytotoxic T cells degranulate. We used TALL-104 human leukaemic cytotoxic T cells as a model system, and stimulated them with thapsigargin and PMA, soluble agents that mimic the two major signalling pathways activated by T cell receptor cross-linking. Our results indicate that essentially every TALL-104 cell responds to maximal stimulation by releasing about half of its lytic granule complement. This reflects complete release of the contents of half the cell's granules, rather than partial release of the contents of all of the granules. Sub-maximal stimulation reduces both the fraction of cells that respond and the magnitude of single cell responses. We find that individual cells respond to maximal stimulation with a variable latency, and provide evidence that, once it starts, degranulation is a slow process taking tens of minutes.