Capping of human T cell specific determinants: kinetics of capping and receptor re-expression and regulation by the cytoskeleton.

The present study was undertaken to characterize the capping mechanism of normal human T lymphocytes. Monoclonal antibodies directed against three T cell specific surface glycoproteins were utilized as specific probes of membrane function. At 37°C, T cells bound with divalent monoclonal antibody (OKT-3, -4, or -8) and FITC-F(ab')2 goat anti-mouse IgG (FITC-anti-antibody) initiated a prompt sequence of patching (1 to 2 min), capping (2 to 30 min), and endocytosis (>15 min) of ligand-receptor complexes that was virtually completed by 30 min. The T cell-specific receptors capped independently and were randomly distributed throughout the membrane. The time to achieve half-maximal capping was 4.6 to 7.1 min. By comparison, T cells bound only with fluoresceinated monoclonal antibody (FITC-OKT-8) capped the ligand at a rate significantly slower than T cells bound with OKT-8 and FITC-anti-antibody. However, when bound with monoclonal antibodies alone, T cells failed to cap and, after 24 hr in culture, the ligand remained on cell surfaces. The rate of T cell capping was regulated by the cytoskeleton. Colchicine, podophyllotoxin, and vinblastine, agents that inhibit microtubule assembly by binding to tubulin subunits, significantly augmented the rate of capping of each of the three receptors during the initial 10 min incubation period, whereas lumicolchicine, the photoinactivated congener of colchicine that cannot bind to tubulin, did not alter the kinetics. By contrast, cytochalasin B, an agent that inhibits the polymerization of actin filaments, blocked the induction of capping. Upon clearing the cell surface of receptor molecules by capping and endocytosis, 16 to 18% of cells regenerated new receptors by 2 hr, 43 to 59% by 14 hr, and more than 92% at 24 hr. The cytoskeletal components appeared to regulate the rate of insertion of new receptor molecules into the cell membrane because colchicine significantly enhanced the kinetics of re-expression of each of the receptor molecules at 2 hr and 14 hr whereas cytochalasin B impaired their generation, particularly at 14 and 24 hr. In conclusion, this study characterized human T cell capping utilizing monoclonal antibodies as specific probes of membrane function, and demonstrated that a) the kinetics of capping of the T cell-specific glycoprotein receptors are similar; b) the rate of capping appears to depend upon the extent of receptor cross-linking; c) T cell capping is regulated by both microtubule- and microfilament-associated proteins; and, d) after endocytosis of ligand-receptor complexes, receptor regeneration is completed within 24 hr and is regulated by cytoskeletal components.