Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.

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