The purpose of these experiments was to determine the degree of reduction in the number of antigen-binding B lymphocytes in the spleens of mice that had been rendered tolerant in the perinatal period. Newborn or pregnant mice were injected with fluorescein (Flu) coupled onto human gamma globulin, and the spleen cells of the neonatally injected mice, or of the offspring of the pregnant mice, were analyzed 1-6 weeks later. Tolerogen doses were chosen so as to achieve either a two-thirds reduction (low dose) in the number of anti-Flu B cells capable of yielding anti-hapten plaque-forming cell clones after in vitro stimulation, or as representing a supra-optimal tolerogenic stimulus (high dose). Antigen-binding B cells were studied by a two-cycle procedure, namely an initial cycle of binding to Flu-gelatin thin layers, followed by analysis of the binding cells in the fluorescence-activated cell sorter (FACS) after suitable staining with Flu-protein conjugates. With the high dose of tolerogen, a modest diminution in Flu-binding cell numbers down to 56-71% of control values could be induced. When these residual Flu-specific B cells were analyzed in the FACS to quantitate their spectrum of Flu-binding avidities, profiles identical to those of controls were obtained. The reduction proved transient in nature, binding cell numbers having returned to 80% of normal by 2 weeks and to normal by 6 weeks. Nevertheless, the Flu-specific B cells were incapable of responding to antigen or mitogen by antibody formation. With the low dose of tolerogen, despite the desired degree of functional silencing of Flu-specific B cells, the numbers and avidity spectra of antigen-binding cells were entirely normal in both the neonatally injected and in utero-injected groups. The results indicate that tolerance induced amongst immature B lymphocytes is not due to a physical elimination of the relevant B cell clones or to a modulation or blockade of their surface Ig receptors. Rather, it is due to the recognition and storage of negative signals amongst cells that continue to display a normal complement of receptors. We therefore propose that the term "clonal anergy" is a more accurate description than either "clonal deletion" or "clonal abortion."
