Mechanisms of antibody formation: II. Use of DNP-Ficoll in studies of hapten-specific B-cell responses

Mice injected with 10 μg N ϵ -dinitrophenyl- l-lysine-Ficoll (DNP-F) preparations ranging widely in epitope density (DNP 52-F to DNP 11.5-F) generated longlasting (6 months) anti-TNP IgM and IgG responses, as measured by the number of spleen PFC produced against sRBC-TNP or hRBC-TNP. The immunogenicity of DNP-F correlated directly with the epitope density; DNP 1.5-F was nonimmunogenic. When mice immunized with DNP-F and actively producing antibodies were challenged with 2 mg DNP-F at different time intervals after sensitization, a rapid and long-lasting reduction in the formation of anti-TNP IgM and IgG PFC occurred. Mice were primed in vivo with sRBC, and spleen cultures were initiated in the presence of sRBC-TNP. Addition of a large dose of DNP-F at the start of the cultures or at 1 and 2 days later greatly reduced but did not completely eradicate the formation of anti-TNP IgM PFC. The production of anti-sRBC PFC was not affected. This specific tolerogenic effect persisted when the cells were washed after a suitable period of exposure to DNP-F, and were then recultured. Generally, exposure of the cells to DNP-F for 8 hr sufficed to reduce the number of TNP PFC observed on subsequent days; exposure for 2 hr was inadequate. These observations indicate that DNP-F does not have to be continuously present in the culture medium to exert its tolerogenic effects. The concentrations of DNP-F capable of exerting tolerlogenic effects do not act by killing the TNP-PFC. Rather, this effect appears to have been a consequence of the restriction of clonal expansion of hapten-specific B-cells by DNP-F. The results also suggest that DNP-F may exert a tolerogenic effect after clonal expansion. In this instance, antigen-antibody complexes may have formed on the surface of hapten-specific B cells, with deamplification of antibody gene transcription.