T-LYMPHOCYTE AGGREGATION WITH IMMOBILIZED ANTI-TCR-ANTIBODIES IS DEPENDENT UPON ENERGY AND MICROFILAMENT ASSEMBLY

An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the ε{lunate} chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the Vβ-encoded determinant of the α β heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin ([125I]BSA). The labeled, Ab-coated beads were mixed at 4 °C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 °C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 ε{lunate} chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 μg of 2C11 Ab was used to coat 106 beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-d-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation. © 1990.