Evidence for multipotentiality of antibody synthesizing cells

Immunization of mice with two unrelated antigens or antigenic determinants regularly results in the appearance of hemolytic plaque forming cells (PFC) of each specificity and of some PFC (1-4%) reacting to both determinants. Micromanipulation of individual double PFC appearing after immunization with trinitrophenyl conjugated sheep erythrocytes (TNP-SRBC) into media containing indicator erythrocytes (native SRBC and TNP-horse RBC) and a soluble specific inhibitor (TNP-BSA or soluble SRBC antigen), showed that the specific inhibitor suppressed the lysis of the corresponding indicator but did not interfered with the lysis of the unrelated indicator. Persistance of one specific activity in spite of complete inhibition of the other indicated that these double PFC synthesize two different antibody molecules. The destiny of double cells was studied in individual cell cultures by micromanipulating them into wells containing heavily irradiated normal mouse spleen cells and both determinants (TNP-SRBC). Those double cells which divided they generated in 24 hours monospecific daughter PFC (either anti-TNP or anti-native SRBC). Individually cultured monospecific PFC from mice immunized with one antigen (native SRBC) generated daughter PFC of the same specificity. By contrast, monospecific PFC from mice immunized with TNP-SRBC generated PFC of either the same specificity of (more frequently) of the one the other specificity. Thus, immunization with substituted erythrocytes resulted in the development of three clonotypes: two clones each composed only of cells of either specificity (amphispecific clonotype). For the maintenance of this amphispecific clone, continuous presence in the culture media of both antigenic determinants was necessary. Removal of the one for 48 hours (but not for 24 hours) abolished the generation of daughter cells of the same specificity.