The destruction of target cells by cytotoxic T lymphocytes (CTL) represents an important host response in malignant disease as well as in viral infection. Clarification of the mechanisms underlying CTL-mediated lysis may reveal methods to directly modulate CTL function, and lead to the development of therapeutically important pharmacologic or immunoregulatory agents for patients with malignancy or progressive viral infection. Recently, cytotoxic effector cells were found to contain cytotoxic granules that directly mediate cytotoxicity. These granules contain perforin, as well as large quantities of a novel serine esterase (or esterases), the role of which is unknown. A systematic analysis of CTL serin esterase structure and function is planned to clarify whether (a) a single moiety is ubiquitously present in diverse CTL, (b) whether different homologous proteins are expressed by different clones, and (c) whether different esterases are present within a single CTL clone. CTL serine esterases will be purified from cytotoxic granules, affinity - labeled with H- diisopropylfluorophosphate (DFP), and studied by several enzymatic and gel electrophoretic techniques to study dimeric linkage, charge properties, glycosylation, and effects of a variety of protease inhibitors. Structural analysis to confirm the relatedness of different purified esterases will be focused on the single tritiated peptide recovered after tryptic digestion of H- DFP labeled esterase. Peptide fingerprinting and sequence analysis will be used to assign serine esterases to one or more of the putative CTL-specific serine esterase cDNA sequences. The purified esterases will be used as immunogens to produce polyclonal and rat monoclonal antibodies. These reagents will define private and shared epitopes by Western blotting, but will also be used to block esterase function during granule and CTL- mediated lysis, and to study the fate of esterase during cytolysis by immunoelectron microscopy. The approaches developed for the study of murine CTL esterase will be extended to initiate similar analyses in human CTL clones.
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