Rosette Formation in Relation to Active Synthesis of Antibody

Abstract

Abstract Studies were done on the requirement for an active synthetic process for the production of rosettes by mouse spleen cells, and on the relation of rosette-forming cells (RFC) to plaque-forming cells (PFC). Evidence for an active process was obtained by injection of mice with actinomycin D on the day of antigen injection. This prevented the appearance of both RFC and PFC. In other approaches, the inhibiting agents were applied in vitro to suspensions of cells producing rosettes. Rosette formation was inhibited on incubation at 37°C, in contrast with 4°C, by inhibitors of protein synthesis, cycloheximide and puromycin, and by anti-metabolites, iodoacetate and arsanilic acid. The most direct evidence that rosettes and plaques can be produced by the same cells was obtained by differential density centrifugation. Following bovine plasma albumin density gradient centrifugation of original spleen cell suspensions, both rosettes and plaques were found to be produced by cells of the same, relatively low, density. When such suspensions were first incubated for rosette formation, however, the RFC, now in rosettes, were found in a fraction of higher density, intermediate between that of white blood cells and red blood cells. The number of plaques produced by this higher density fraction was found to be markedly increased over the analogous fraction from original spleen cell suspensions, in which rosettes had not been formed. The nature of the receptors on the cell surface involved in rosette formation was examined in several ways. It was shown that rosette formation could be inhibited by exposure of the cells to an excess of antigen or to anti-IgG serum or its Fab fragments. The rosettes were partially sensitive to complement, indicating that IgM molecules as well as IgG molecules can be involved in rosette formation.