To measure the interaction between immobilized anti-TCR Ab and T lymphocytes, characterize the phenomenon in terms of its biochemistry and function, and investigate the role of cytoskeleton in receptor-mediated signalling. Perturbation of the TCR with anti-TCR-antibodies (Ab) is followed by a sequence of biochemical and biological responses similar to those observed subsequent to T-cell interaction with antigen (Ag) . Using fluoresceinated Ab, discrete clustering of the TCR in defined areas of the cell surface (""""""""patching"""""""") , followed by coalescence into a unitary aggregate (""""""""capping"""""""") have been observed. Receptor endocytosis usually follows. Ab immobilized onto solid matrices have been used as an alternative to perturbation of the TCR with polyvalent, soluble Ab. Immobilized Ab may represent a model of TCR perturbation similar to physiologic conditions, since normal Ag/TCR interaction occurs in the context of the cell surface MHC of APC. As opposed to soluble Ab, the rigidity of a solid matrix would effectively prevent receptor mobility and internalization. Consequently, receptor perturbation by immobilized Ab might occur independently at multiple sites of the cell surface. Preventing this process of TCR internalization may have biochemical and biological consequences. As a first step toward understanding these phenomenon, we have defined conditions and requirements for the interaction of T lymphocytes with immobilized anti-TCR Ab. Preliminary findings suggest that specific binding of anti-TCR Ab-coated beads to 8-5-5 T cells (a CD3*, CD4*, murine T-cell clone) was temperature dependent, augmented by increasing extracellular (Ca2+) , and followed a time-course parallel to signal generation, measured as inositol phospholipid (InsPL) hydrolysis. Cytochalasin pretreatment (to disrupt microfilament assembly) inhibited conjugation in a dose-dependent manner, but enhanced InsPL hydrolysis induced by treating 2Cll-coated cells with soluble anti-hamster Ab's. Vinca alkaloids, which disrupt microtubules, were ineffective. Therefore, signal transduction mediated by InsPL hydrolysis takes place independently of conjugation, and may be negatively affected by microfilament assembly.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Basic Sciences
United States
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