Mechanism of Lymphocyte-Mediated Cytotoxicity

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The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of preformed cytotoxic mediators into the synapse-like space between the cytotoxic lymphocyte and its bound target. The generally accepted cytotoxic mediators are perforin and the granzyme proteases. However, mRNA encoding a novel cathepsin protease, cathepsin W, has been shown to be expressed exclusively in NK and cytotoxic T lymphocytes with no meaningful current information as to its function. We hypothesize that it is either a granule cytotoxic mediator like granzymes or that is a perforin processing enzyme. In order to test these models, we expressed human procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots of extracts of NK cells and CTL, some of these mAb react with a single 25-30kD band, where processed, catalytically active cathepsin W is expected. Other mAb react with a 45kD band, consistent with procathepsin W. Using these mAbs to follow the fate of cathepsin W after activation, stimulation of the human NK cell line NK92 with PMA and ionomycin to trigger granule exocytosis causes depletion of cathepsin W from the cells and its release into the medium within 3 hours in both cases. Similar results were seen when human CD8+ CTL were treated with anti-CD3. In both cases cat W release paralleled degranulation as measured by granzyme A release. Fluorescence microscopy with anti-cathepsin W mAbs show that the processed protein is expressed in a vesicular/granular pattern within the cytoplasm, while procathepsin W is seen in a more diffuse pattern consistent with endoplasmic reticulum. We are also examining the functional status of granule exocytosis in nave, memory, and effector subpopulations of human blood T lymphocytes phenotypically defined by surface markers. These studies show that effector subsets of both CD4+ and CD8+ T cells express granule markers most strongly, memory subsets are also positive for most granule markers, but nave T cells show negligible expression of these markers. When granule exocytosis is assessed by surface expression of CD107a after activation, only effector subsets appear to have the ability to exocytose.