B cell antigen receptor (BCR) diversification in early mouse BM B cells is known to involve combinatorial gene rearrangements mediated by V(D)J recombination mechanisms. We have now found that V(D)J rearrangement is not the only mechanism for BCR diversification in early mouse B cells. Our results show that the AID is expressed in BM pre-B and immature B cells of normal wild-type mice. AID expression in these cells leads to active CSR and SHM as part of the normal, T-cell independent, developmental program. AID levels in developing B cells differ between various mouse strains; these differences suggest genetic factors that affect the level and timing of AID expression in various B-cell stages. BCR and TLR are involved in the expression of AID in early B cells, suggesting that signaling through these receptors is important for the induction of AID expression.
It is important to note that several observations indicate that small numbers of BM-residing plasma or memory B cells cannot account for our findings of AID-expression in early B cells. First, plasma cells are substantially larger than developing B cells and are gated out of our early B cell populations by size. Second, our developing B cell populations are AA4.1+
but BM plasma cells do not express surface AA4.1 or B220 (Kallies et al., 2004
; McKearn et al., 1984
). Third, Blimp-1 is a master transcription factor that is required for formation and maintenance of long-lived plasma cells and pre-plasma memory B cells in BM (Shapiro-Shelef et al., 2003
; Shapiro-Shelef et al., 2005
). Yet, high levels of Blimp-1 expression are restricted to rare cells in lymphoid tissues (from 0.1 to 0.5%) and suppress the expression of both B220 and AID (Kallies et al., 2004
), indicating that plasma or memory B cells are unlikely to be present in our AA4.1+
populations. Fourth, comparable levels of AID expression and ongoing CSR are found in early B cells from nude and wild-type littermate controls mice even though nude mice lack GC reactions, plasma cells, or memory B cell formation. Finally, B cell-specific Blimp-1-deficient mice that lack plasma cells show no significant differences in AID expression in both pre-B and immature B cells when compared to wild-type littermates (). Taken together these considerations provide compelling evidence against any significant contamination of mature B cells in our BM-derived pre-B and immature B cell populations.
A recent study using mutant mice carrying reporter gene constructs designed to detect AID-expressing cells by flow cytometric analysis shows no AID reporter expression in bone marrow cells in vivo
even though detectable AID expression could be found by real-time PCR analysis (Crouch et al., 2007
). However, we find clear AID expression and AID function in developing bone marrow B cells, suggesting that the reporter construct analyses may be insufficiently sensitive to detect lower levels of AID expression even when these are capable of both CSR and SHM. Kelsoe and his colleagues also recently reported that AID is expressed in mouse developing B cells in vivo
, and that splenic immature (or transitional) B cells can switch isotypes and differentiate to plasma cells upon in vitro
TLR stimulation (Ueda et al., 2007
), consistent with our findings that developing B cells can be activated by self or external TLR ligands and can express functional AID .
Our results indicating that active CSR can occur in early developing mouse B cells alters the prevailing paradigm of CSR limited to mature B cells stimulated in the periphery and usually in GCs. Our findings could also account for the unexpected CSR that has been found in mutant mice (μMT mice) that cannot produce membrane-bound μ-chains. The μMT mutant mice have a pro-B cell developmental block resulting in a lack of peripheral B cells with surface IgM (Kitamura et al., 1991
). Despite this defect in B cell development, μMT mice have been found to have normal levels of serum IgA (in a C57BL/6 background) (Macpherson et al., 2001
) or serum IgG and IgA (in a BALB/c background) (Orinska et al., 2002
; Hansan et al., 2002
). The ability of pre-B cells to undergo CSR, and thereby produce membrane-bound IgG or IgA to circumvent the pro-B block, would provide a straightforward explanation for IgG and IgA expression in the μMT mice. The differential Ig isotype expression in C57BL/6 and BALB/c μMT mice could reflect genetic differences affecting CSR in these mouse strains; this is reminiscent of the genetic background effects we find for CSR in pre-B and immature B cells in normal mice.
For both normal and μMT mice, culturing B220+
BM cells in vitro
with IL-7 has been reported to lead to AID expression, IgG germline transcription, and the detection of PSTs (Seagal et al., 2003
). These results are consistent with our findings of AID expression and CSR even in unstimulated BM early B cells. However, because μMT/lpr mice showed an increase in the production of IgG-producing B cells in these in vitro
cultures, it was suggested that IgG-positive B cells developing in the BM in vivo
are deleted by a Fas/FasL-mediated pathway (Seagal et al., 2003
). However, μMT mice exhibit nearly normal levels of serum IgG and IgA, indicating that, in vivo
, IgG- and IgA-expressing BM B cells are not necessarily deleted even in the presence of an intact Fas/FasL pathway. Rather, CSR in the early stages of in vivo
B cell development might provide a protective mechanism for young, pre-immune animals by generating IgG- or IgA-producing cells. As suggested previously, CSR at the level of pre-B cells could also represent an alternative backup pathway for B cell development (Macpherson et al., 2001
Abelson murine leukemia virus (Ab-MuLV) infection of mouse BM cells can lead to establishment of transformed tumor cell lines that have a pre-B cell phenotype (Burrows et al., 1981
; DePinho et al., 1984
; Sugiyama et al., 1986
). Some of these pre-B cell-lines exhibit CSR of γ2b or γ3 heavy chain constant region genes, discordant with the notion that CSR is limited to mature B cells. These findings have generally been ascribed to inappropriate CSR regulation in these transformed cell lines. Our demonstration of CSR in normal pre-B cells could explain the active CSR found in Ab-transformed pre-B cell-lines, but a recent report showing that Ab-MuLV infection of BM pre-B cells can greatly enhance AID expression provides an alternate mechanism to account for CSR in these cell-lines (Gourzi et al., 2006
). Interestingly, Ab-MuLV induction of AID in infected pre-B cells inhibits tumorigenicity and it has been suggested that AID-induced DNA damage, leading to decreased proliferation and increased NK cell targeting, might be involved in this inhibition (Gourzi et al., 2006
). It would appear that such effects of AID expression on proliferation and NK cell targeting are limited to virus-infected cells because (1) normal mature B cells proliferate and expand greatly after stimulations that induce AID expression and CSR (Muramatsu et al., 2000
), and (2) as our results indicate, AID-expressing BM pre-B cells in normal mice appear to differentiate normally into immature B cells that express AID at higher levels. Thus, AID expression appears to play at least two roles in early B cells; induction in virally-infected pre-B cells leading to inhibition of the tumorigenicity of these infected cells, and induction in normal early B cells to provide receptor diversification during B cell development.
AID expression in immature B cells also mediates SHM. A previous report had shown AID expression, but no SHM of λ light chain, in immature B cells from wild-type C57BL/6 mice (Mao et al., 2004
). We now find that AID expression can, in normal mice, lead to SHM of Vκ4 in the BM pre-B and immature B cells. We do not know why SHM was not detected previously in λ light chain of C57BL/6 mice but this could reflect a lower SHM frequency in λ relative to Vκ4 or might be due to a process that selectively amplifies early B cells carrying mutations in Vκ4 (see below).
What roles might SHM be playing in early B cells? Obviously this mechanism could provide additional pre-immune repertoire diversity in mature B cells to supplement the diversity from V(D)J recombination. AID expression in early B cells could also be involved in receptor editing of self-reactive BCR using SHM as a mechanism. Previous studies have indicated that B cells expressing rearranged Vκ4 genes are frequently negatively selected, perhaps because they are autoreactive (Kalled and Brodeur, 1990
). We propose that the Vκ4 hypermutation that we have detected in immature B cells might be linked to receptor editing during the maturation of these cells. TLR dependency of AID expression during B cell development could be related to this hypothesis. In mature B cells, synergistic engagement of TLR/BCR can activate quiescent or autoreactive B cells in vitro
(Leadbetter et al., 2002
) and we suggest that autoreactive developing B cells can be activated by the same synergistic engagement of TLR/BCR. Thus, expression of AID during B cell development could both provide Ig diversity and serve as an additional mechanism of receptor editing in autoreactive B cells.
When SHC, which is known to also require AID, was first found in immature B cells in the chicken bursa (Reynaud et al., 1987
), it was a notable deviation from B cell diversification by V(D)J recombination in the bone marrow of mice and humans. However, our results, showing AID-mediated Ig diversification in the mouse bone marrow, suggest that AID expression and function are also a part of the normal early B cell developmental program in mice, as in chickens, sheep, and other species (Cooper, 2002
). Our findings lessen the appearance of evolutionary differences in B cell development between these species.