Ig repertoire expression of BALB/c primary and secondary B cell precursors specific for phosphorylcholine

To better understand the regulation of Ig repertoire expression and the generation of memory B cells, we have used the splenic focus assay to analyze primary and in vitro generated secondary phosphorylcholine (PC)-specific BALB/c B cell precursors. A quantitative analysis of the kinetics of appearance of these precursors over the time of the culture period was performed. The precursors were analyzed for TEPC15 Id expression, H chain isotype, relative affinity, and epitope specificity (PC vs p-nitrophenyl PC). In addition, we tested the hypothesis that primary and secondary precursors could be distinguished on the basis of the expression of the cell-surface Ag defined by the mAb JIId. We found that secondary in vitro PC-hemocyanin stimulation resulted in an increase in the anti-PC precursor frequency, relative to primary stimulation, of 37%. This increase was caused almost entirely by the secondary stimulation of non-TEPC15 expressing precursors. Relative to the primary precursors, the secondary precursors also: 1) expressed a lower percentage of IgM and a higher percentage of IgG, 2) had a higher relative affinity, and 3) expressed a lower level of JIId-defined Ag. Very little difference was observed in the epitope specificity of the primary vs secondary precursor repertoire. Based on an analysis of the kinetics of the response, together with JIId cell sorting and transfer experiments, our results support a model whereby a portion of the secondary anti-PC response is derived from secondary precursors arising from primary precursors, and the other portion is derived from distinct secondary precursors expressing a relatively low amount of the JIId Ag.