Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

The role of BAFF in B cell self tolerance was examined by tracking the fate of anti-HEL self-reactive B cells in BAFF transgenic mice using four different models of self-reactive B cell deletion. BAFF overexpression did not affect the development of self-reactive B cells normally deleted in the bone marrow or during the early stages of peripheral development. By contrast, self-reactive B cells normally deleted around the late T2 stage of peripheral development were rescued from deletion, matured, and colonized the splenic follicle. Furthermore, self-reactive B cells normally selectively deleted from the marginal zone repopulated this compartment when excess BAFF was present. Self-reactive B cells rescued by excess BAFF were not anergic. BAFF overexpression therefore rescued only self-reactive B cells normally deleted with relatively low stringency and facilitated their migration into otherwise forbidden microenvironments. This partial subversion of B cell self tolerance is likely to underlie the autoimmunity associated with BAFF overexpression.

B cells responding to T-dependent antigen either differentiate rapidly into extrafollicular plasma cells or enter germinal centers and undergo somatic hypermutation and affinity maturation. However, the physiological cues that direct B cell differentiation down one pathway versus the other are unknown. Here we show that the strength of the initial interaction between B cell receptor (BCR) and antigen is a primary determinant of this decision. B cells expressing a defined BCR specificity for hen egg lysozyme (HEL) were challenged with sheep red blood cell conjugates of a series of recombinant mutant HEL proteins engineered to bind this BCR over a 10,000-fold affinity range. Decreasing either initial BCR affinity or antigen density was found to selectively remove the extrafollicular plasma cell response but leave the germinal center response intact. Moreover, analysis of competing B cells revealed that high affinity specificities are more prevalent in the extrafollicular plasma cell versus the germinal center B cell response. Thus, the effectiveness of early T-dependent antibody responses is optimized by preferentially steering B cells reactive against either high affinity or abundant epitopes toward extrafollicular plasma cell differentiation. Conversely, responding clones with weaker antigen reactivity are primarily directed to germinal centers where they undergo affinity maturation.

The long-standing hypothesis that tolerance to self antigens is mediated by either elimination or functional inactivation (anergy) or self-reactive lymphocytes is now accepted, but little is known about the factors responsible for initiating one process rather than the other. In the B-cell lineage, tolerant self-reactive cells persist in the peripheral lymphoid organs of transgenic mice expressing lysozyme and anti-lysozyme immunoglobulin genes, but are eliminated in similar transgenic mice expressing anti-major histocompatibility complex immunoglobulin genes. By modifying the structure of the lysozyme transgene and the isotype of the anti-lysozyme immunoglobulin genes, we demonstrate here that induction of anergy or deletion is not due to differences in antibody affinity or isotype, but to recognition of monomeric or oligomeric soluble antigen versus highly multivalent membrane-bound antigen. Our findings indicate that the degree of receptor crosslinking can have qualitatively distinct signalling consequences for lymphocyte development.