ACTIVATION AND TRIGGERING OF CYTOXIC CELLS

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We have previously used redirected cytotoxicity experiments to study and define triggering molecules on cytotoxic cells. While the TcR and FcR are the principal triggering molecules on human leukocytes, we have recently found that adhesion molecules can also serve as cytotoxic triggers on some cell types. In this project we have used anti-CD44 containing bispecific antibodies to show that CD44 can trigger cytotoxic responses in activated NK cells and in fresh PMN. In NK cells, we found that CD44 becomes a cytotoxic trigger after activation with IL-2. Since CD44 is present before and after activation, we asked what causes activation. No changes in CD44 isoform or protein molecular weight were detected by PCR and immunoprecipitation analysis but activation did require protein transcription. A tyrosine- phosphorylated protein was found to co-precipitate with CD44 from activated NK cells. By adding inhibitors during the effector phase of lysis, we found that PI 3-kinase is required for CD44-dependent lysis and that protein kinase C and the cytoskeleton modulate lysis. No increase in intracellular Ca2+ or release of BLT esterase was observed as a result of CD44 crosslinking, whereas CD16 (Fc RIII) induced both these responses. In addition to CD44, we have found that CD38, CD69, and CD56 are also cytotoxic triggers on activated NK cells. In PMN, CD44 triggers a cytotoxic response as measured in an 18 hr assay. Unlike NK cells, fresh, unactivated PMN mediate this response. PMN recognize and kill target cells coated with hyaluronic acid (HA), the principal ligand of CD44, and this activity is blocked by anti-CD44 antibodies. This is in contrast to NK cells that do not lyse HA coated targets. Thus CD44 and perhaps other adhesion molecules, appear to be important to PMN and NK cell cytotoxic function. In other studies involving effector cell activation, we found that in mice bearing mammary tumors, there is a progressive loss in cytolytic function of T cells that correlates with tumor growth. In looking for the source of this phenomenon, we found that there is a selective loss in STAT5 protein and RNA during tumor growth. Other STAT proteins were unchanged during tumor growth. STAT5 is used in the signaling of some cytokines, including IL-2, 3,5,7,9 and others, and thus may be a target for regulating immune function in vivo.