Analysis of the Adhesion Step in the Herpes Simplex Virus Antibody-Dependent Cellular Cytotoxicity System

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The lysis of herpes simplex virus-infected tissue culture cells by antibody-dependent cellular cytotoxicity (ADCC) requires a preliminary step in which effector cells adhere to the immunoglobulin G antibody-coated targets. To study the adhesion step, we made use of two observations: (i) some of the mononuclear cells in human blood form rosettes with antibody-coated target cells, and (ii) most ADCC effector cells can be removed by allowing mononuclear cells to adhere to monolayers of antibody-sensitized tissue culture cells. The effect of various experimental conditions on the adhesion step was assessed in ADCC cultures both at unit gravity and after centrifugation. At unit gravity both rosette formation and monolayer adhesion were partially reduced at 4°C as compared to 37°C. Both were also partially inhibited in glucose-free medium containing sodium azide and 2-deoxyglucose but were unaffected in glucose-free medium containing only one of these energy inhibitors. In contrast, after centrifugation neither reaction was inhibited at 4°C or in glucose-free medium with sodium azide and 2-deoxyglucose. Cytochalasin B but not colchicine suppressed both reactions. Inhibition by cytochalasin B could not be reversed by centrifugation. Both reactions were independent of extracellular Ca2+ and Mg2+ and were unaffected by rendering mononuclear cells cytotoxically inactive by brief heat shock. These findings indicate that the adhesion step in ADCC directed against virus-infected or uninfected tissue culture cells is only modestly dependent on effector cell energy generation, that centrifugation greatly reduces this dependence, and that microfilaments but not microtubules are necessary. The modest ambient temperature and energy requirements, independence of extracellular divalent cations, lack of sensitivity to colchicine, and relative resistance to supraphysiological temperature serve to distinguish the adhesion step from the lytic step in ADCC.

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