We have demonstrated here that VH
3609μ/Vκ21C-encoded ATA B cells are negatively selected in the major pathway of BM B cell development. The majority of ATA B cells in the spleen showed maturational arrest. They are short-lived, replaced rapidly from the BM, and are unlikely to be the major source of natural serum ATA. Instead, cells already established outside of conventional lymphoid organs, together with preexisting B-1 cells in the spleen (12
), appear to play a significant role in the self-antigen–dependent production of serum ATA. These positively selected B-1 cells could be revealed more readily in PerC of ATAμ-only Tg mice, due to a lower proportion of negatively selected cells, such that CD5+
B cells constitute a significant fraction of the PerC B cells, more similar to normal mice. In contrast, in ATAμκ Tg mice, forced expression of the ATA specificity by all BM immature B cells resulted in predominant negative selection, even in the PerC population, whereas the serum ATA level was intact, independent from negative selection and likely contributed by the presence of infrequent B-1 B cells. Recent reports describing normal or even elevated serum immunoglobulin levels in mice lacking most peripheral B cells due to blocked BM development resulting from absence of IL-7 (38
) or conditional deletion of Rag-2 (37
) are consistent with this notion. Taken together, BM-derived B cell development and selection in conventional lymphoid organs seems unlikely to produce the B-1 cells that participate in the daily production and maintenance of natural serum autoantibody in normal healthy individuals.
CD5 induction, commonly thought to mark B-1 cells, nonetheless occurred for BM-derived ATA immature B cells during negative selection as a result of self-antigen exposure. Thus, CD5 induction can occur during B cell development, but before cell fate is determined, either negatively as shown here, or positively as found with positive selection in T cell development (39
). It should be emphasized, however, that these CD5-induced B cells from BM are not equivalent to long-lived B-1 cells generated after positive selection as our serum ATA data indicated, and that the induction of CD5 is not a general consequence for all self-antigen–exposed tolerant B cells. Despite the expectation that negative selection will be frequent during BM B cell development (3
), CD5 induction is normally rare (19
) and typically absent from, or present at very low levels on, tolerant B cells (28
). Only B cells expressing the prototypic ATA κ chain became CD5+
in ATAμ Tg mice. Thus, CD5 induction from immature B cells appears to require specific types of antigen or a specific threshold of BCR signaling. Previously, it was considered that the paucity of CD5 induction during BM B cell development might be due to a relative decrease of B-1–associated specificities in the newly generated B cell repertoire by either increasing diversity in μ-heavy chain rearrangement (41
) or altered pre-BCR selection (21
). This study suggests that even if generated, B cells with such specificities will likely make little or no contribution to any mature B cell pool in normal mice.
The finding of a positive impact of self-antigen on serum autoantibody levels, first reported for the heavy-only 6C10-μ Tg line and shown here with ATA-μκ Tg mice, stands in sharp contrast to most other models of B cell autoreactivity where the presence of autoantigen has negative effects (4
). One other previously reported exception is an RF Tg model where the presence of antigen has little effect, either negative or positive, leading to the description of this Tg BCR as “clonally indifferent” in normal mouse background (46
). This has been attributed to the relatively low affinity of this BCR for antigen, as a higher affinity RF model shows strong antigen-mediated central tolerance in the presence of antigen (47
). The ATA BCR does not appear to exhibit a particularly low affinity for antigen, as the presence of high levels of Tg-encoded serum autoantibody is accompanied by arrested B cell development and/or elimination of the specificity in the spleen by receptor editing. This suggests that the ATA specificity, representative of a B-1–associated natural autoantibody, is regulated in a manner very distinct from previous models of autoreactivity associated with pathogenesis.
We have previously characterized B cell development in another Tg model expressing a prototypic CD5+
B-1–associated specificity to a phosphatidylcholine-associated cryptic determinant on erythrocytes revealed by treatment with the proteolytic enzyme bromelain (49
). Unlike the ATA specificity, however, B cells in this BR1-VH
9 model did not edit nor show obvious signs of anergy in the spleen, but instead adopted a typical B-1 phenotype and showed evidence of extended survival in culture (50
). Although a definitive explanation for this difference remains to be determined, recent analysis of a VH
11 knock-in mouse (unpublished data) indicates that physiological expression of this heavy chain results in accumulation of a fraction of VH
9 B cells in spleen showing the “arrested development” phenotype, as described here with ATA BCR animals. Quantitative differences in the level of BCR between these lines might be responsible for such a different outcome. In addition, differences in ATA versus phosphatidylcholine B cell development could arise from variations in the expression level of the antigens. These are issues that we are currently investigating.
There has been emerging interest on the origin of natural serum autoantibody and its relevance to antibodies to virus or commensal bacteria. The constitutive presence of neutralizing antibody in serum is essential, protecting against viral or bacterial replication to prevent potentially lethal disease progression, playing a role as a part of innate immunity (51
). Natural autoantibodies are often reactive to carbohydrate on self-antigens (16
), which might be shared with determinants on bacterial, viral, or tumor antigens. These observations have led to the hypothesis that positive selection of the autoreactive B cell repertoire might be an active process to provide protective immunity for immunological surveillance. Previously, it was speculated that these self-antigens might be unique, being inefficient in inducing tolerance (26
). However, our study shows that these natural autoantigens can function as tolerogens, similar to classical autoantigens. The transfer of fetal B cell progenitors, but not adult BM B cell progenitors, into the adult mouse environment resulted in B-1 cell positive selection, i.e., accumulation of CD5+
B cells (20
). This raises the additional possibility that differences of immature B cell responsiveness that specifically allow B-1 cell positive selection may also account for autoreactive B cell production from such fetal precursors.
Serum ATA is already detectable in the fetus and newborns of ATA Tg mice (17
) before BM B cell development is established, supporting the idea that B-1 cell positive selection occurs before birth. Some B-1 cells are maintained throughout life by self-renewal (12
) as a persisting fetal/neonatal B-1 cell population, although it is likely that some B-1 cells might be generated continuously from precursors in the BM as a minor divergent pathway. The intestinal lamina propria appears to serve as a major site for B-1 cell differentiation into antibody-secreting cells and class switching to IgA (13
) in normal mice. Interestingly, there is a significant level of endogenous (non-ATA) IgA secretion in ATAμκ Tg mice as found with other Ig Tg B cell mouse models (33
), suggesting a strong ongoing serum antibody production separate from the majority of BM B cell development. Unique IgA production in the gut lamina propria in mice deficient for the majority of B and T cells has been reported (59
). These findings suggest that there are B cell system(s) that actively provide protective immunity independent from the majority of B cell development in the adult. B-1 cell development may represent such a system, uniquely allowing positive selection to self- and/or commensal antigens. This is an important issue to address in future studies.