SUSTAINED SIGNALING LEADING TO T-CELL ACTIVATION RESULTS FROM PROLONGED T-CELL RECEPTOR OCCUPANCY - ROLE OF T-CELL ACTIN CYTOSKELETON

Using antigen-specific T cell clones and peptide-pulsed antigen-presen ting cells (APCs) we investigated the mechanisms that lead to sustaine d signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton pl ays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble an tibodies. First, addition of antibodies to the major histocompatibilit y complex molecules recognized by the TCR aborts the ongoing intracell ular calcium concentration ([Ca2+](i)) increase in preformed T-APC con jugates, indicating that the sustained signaling requires the continuo us occupancy of TCR. Second, time-lapse image recording shows that T l ymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2](i) increase, which is accompanied by the formation of a large and ch anging area of contact between the two opposing membranes. Third, drug s that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clost ridium botulinum toxin induce a rapid block of [Ca2+](i) rise, coincid ent with a block of the cyclic changes in T cell shape. Finally, the a ddition of Cytochalasin D or of anti-MHC antibodies to preformed conju gates inhibits interferon gamma production in an 1-antigen dose- and t ime-dependent fashion. These results identify T cell actin cytoskeleto n as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells eq uipped with low affinity TCR can be triggered by a small number of com plexes on APCs.