This paper describes an in vitro system for the study of the factors that control and regulate the cellular events in the immune response. It was reported earlier (1) that dissociated cell suspensions obtained from the spleens of normal mice could be immunized in vitro to heterologous erythrocytes. The response is primary in the sense that it follows the first experimental exposure to the erythrocyte antigen. The response was measured by the increase in the number of hemolytic plaque-forming cells and by assay of antibody in the culture supernatants. The critical conditions for culture and immunization of the spleen cells included low oxygen tension, gentle agitation of the cultures, the inclusion of fetal bovine serum in the medium, adequate spleen cell density, and daily feeding of the cultures with a nutritional mixture. This paper presents a more detailed account of the experimental system and some comparisons between in vitro and in vivo responses under a variety of experimental conditions. The in vitro response closely parallels that seen in vivo with respect to size, early kinetics, effect of antigen dose, and the inhibitory effect of passive antibody. These findings encourage the belief that observations made with the in vitro system are relevant to our understanding of the in vivo response. The in vitro differs from the in vivo response in that it appears to show a greater capacity to discriminate between different homologous erythrocyte antigens and that it shows no limitation or termination of the increase in 19S antibody-forming cells 4 or 5 days after the initiation of the response. These differences have been characterized in the hope that their further study may uncover underlying regulatory mechanisms involved in the response. * This is publication No. 217 from

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Dissociated cells from the spleens of unimmunized mice were cultured with and without various mammalian erythrocytes. Spleen cell suspensions cultured with heterologous red cells developed levels of hemolytic plaque-forming cells only one log(2) less than those seen in vivo. The reaction is specific for the in vitro immunizing erythrocytes. Antibody was demonstrated in the culture fluids.