Mice undergoing cGVH develop a lupus disease similar to mice with spontaneous lupus. cGVH mice develop the same spectrum of autoantibodies and similar kidney pathology. We have extended the comparison between these models to include the genetic control and etiology of anti-DNAs. The analogy remains compelling: the mouse models of SLE such as MRL/lpr
have two phases of autoantibody induction. The first phase is characterized by polyclonal, unmutated IgM Abs (38
) and appears to result from nonspecific activation. The second phase is characterized by oligoclonal, IgG Abs and resembles a typical, Ag-driven immune response. Members of clones have somatic mutations that yield amino acids such as arginine and asparagines that play a role in DNA–protein interaction (39
cGVH in the anti-DNA site directed transgenic mice also undergoes both types of activation. All B cells have increased levels of activation markers in 3H9(+) after cGVH, even though only a subset of B cells are anti-DNA (20
). A similar phenotype was found in preliminary data from bm12→56(+) (excepting CD23). This indicates a nonspecific activation phase (20
). This study also shows that the IgM B cells are highly diverse i.e., polyclonal. Most, but not all, IgM hybridomas bind DNA, and the anti-DNAs have different avidities. In addition, most of these B cells are independently derived. Even though the majority express the sd-tg and the Vκ38c L chain, they can be shown to be of independent origin by several criteria: they utilize different Jκ segments or they have unique Vκ-Jκ junctions. Within the entire subset of IgM anti-dsDNA hybridomas, at most 4/18 (22%) might be clonally related as compared with 8/11, or 73%, in the set of IgG anti-DNA (see below).
That cGVH also causes an Ag-driven immune response was first suggested by the narrow spectrum of the specificities of the IgG autoantibodies expressed in these mice (12
). If autoimmunity in GVH were entirely due to nonspecific activation, then a much broader spectrum would be expected and would include many other autoantibodies seen in other murine models of autoimmunity. This is not the case and instead just autoantibodies typical of SLE are expressed. The transgenic models allows us to reach a similar conclusion: whereas nonspecific activation should yield edited anti-DNAs or anti-ssDNA Abs, the majority of monoclonal IgGs bind dsDNA. Serum anti-DNA titers also show that anti-dsDNA increases disproportionately to anti-ssDNA (). In sum, the restricted production of B cells that recognize a single Ag and the bias toward anti-dsDNA point toward a stage of Ag activation during cGVH.
The IgG anti-DNAs have other properties of Ag-driven immune responses, in that they are members of expanded clones and are mutated. In our study of eleven randomly selected IgG2a anti-dsDNAs, two sets had highly homologous VH and VH CDR3 regions, suggesting that each set represents an expanded clone ( and ). Additionally, some mutations among members are shared, a property that usually results from their accumulation during clonal expansion. Taken together, the homologous CDR3 sequences and the hierarchical pattern of shared mutations provide strong evidence that members of these sets have descended from a single B cell precursor. As discussed above, the V gene usage and pattern of mutations indicate that the IgG anti-DNA B cells were selected for and driven by DNA.
Even though the sd-tg is associated with relatively high affinity anti-DNAs, it appears to be excluded in the DNA-driven phase of the 56R model. Although every B cell at the initial stage of development inherits the 3H9/56R sd-tg, none of the GVH induced IgGs are encoded by the sd-tg. In the anti-dsDNA IgG subset, all cells used an upstream VH gene that replaced the sd-tg or a VH derived from the untargeted allele. How these arise could be explained in three different ways (see ) :
(a) A leaky H-STOP control. It is thought that a functional H chain, presumably in combination with surrogate L chain, stops further rearrangement at the H locus. This STOP signal explains why the majority of B cells are incompletely rearranged (DJ) on the silent allele. Accordingly, an H chain tg should prevent rearrangement on the untargeted allele. Nevertheless, H chain rearrangement is found in H chain transgenics, either at the untargeted allele or in the form of VH replacement of the tg VH. The frequency of such rearrangements varies; in the anti-DNA sd-tgs LPS-activated B cells 2% (3H9), 14% (3H9/56R), and 38% (3H9/56R/76R) have one or the other form. The site at which these secondary VH rearrangements occur could be in the pro-B cell, because the sd-tg does not completely stop rearrangement, or later because of editing. At least some VH replacement must occur early because N addition is found at the V-D-J junctions. Thus, the tg− B cells may be selected from a subpopulation of B cells with a VH repertoire encoded by endogenous genes. Alternatively, TdT could be reengaged in mature B cells under cGVH.
(b) Mutation correction. Endogenous VH genes may be introduced after Ag selection of the 3H9/56R B cells. We and others have noted that V gene replacement occurs during clonal expansion. In the 3H9/Vκ8 MRL/lpr
, L chain rearrangement followed (and rescued) a nonsense-mutated L chain (28
). VH replacement is seen in expanded clones from RA synovium (40
). As noted above, the clones observed in GVH-induced anti-DNAs have unusually high frequencies of somatic mutations. The R:S of these mutations in certain FRs is low, showing that many lethal mutations have occurred during clonal expansion. Thus, it is conceivable that inactivation of the sd-tg followed by replacement or rearrangement at the untargeted allele explains the shift to endogenous VH gene usage. This model implies that rearrangement can be reinitiated after Ag-selection. This may be special to those cells in a clone with a lethal H or L chain mutation.
(c) Autoimmune V gene replacement. VH and/or VL rearrangement may be induced in autoimmunity. In fact, it has been reported that recombination activating gene levels are unusually high in SLE (41
). This would lead to “reediting.” The key distinction between editing and reediting is the setting in which the secondary rearrangement occurs. Editing refers to rearrangement that takes place in immature cells in the bone marrow. By contrast, reediting takes place in mature peripheral cells, and is associated with Ag-driven immune responses. In our bm12→56R fusion, a clear distinction between peripheral subsets provided evidence of reediting under cGVH. In the IgG cells, secondary H chain rearrangement occurred frequently (see above), whereas among IgM cells, most of which used an unmodified 3H9/56R H chain, it must have been rare. In addition, the IgG clones displayed characteristics typical of B cells that have undergone a secondary immune response, as indicated by clonally related sequences (discussed above) and a high frequency of somatic point mutations. In this subset, oligoclonality and hypermutation, along with isotype switching, clearly associates secondary VH rearrangment with Ag activation under cGVH, and indicates reediting. The increased frequency of such H chain rearrangement in anti-dsDNA IgG cells, and the fact that the anti-dsDNA IgG subset has been implicated in SLE pathology, implies that reediting during clonal expansion could play a key role in disease development. The mechanism of reediting is also more clearly established in light of these data. Within the anti-dsDNA IgG subset, very few cells derived from VH replacement in the sd-tg allele (see clone 2). This can be explained by the high percentage of such VH rearrangements that might result in nonfunctional joints (42
). Most interestingly, a high percentage of these cells (80%) used the second allele. Along with the fact that none of these cells expressed both alleles simultaneously, this strongly suggests that most VH reeditings result in deactivation of the first (here, the transgenic) allele. This in turn enables the more efficacious classical rearrangement process to occur at the untargeted allele.
Our data do not allow us to distinguish among these models. The reediting model or the lethal mutation model are appealing because they explain the shift from tg to endogenous gene usage. If we assume that DNA selects and drives anti-DNA B cells, then we would expect the response to include, indeed to prefer, the tg+
B cells. The alternative explanation for the shift would mean that the subpopulation of cells arising because of a leaky STOP signal has a selective advantage. Although this possibility may appear initially unlikely, further consideration of our sequence data could be supportive. The endogenous VH usage we found was highly restricted. This implies that certain endogenous VH genes may have DNA binding properties. In fact, most members of the anti-dsDNA IgG clones sequenced used the J558.B3, J558.B4 genes. These genes are unique to the B6 VH locus (30
) and have CDR1/CDR2 regions that are rich in asparagine residues. These residues form a pocket of positive and negative charges that should attract DNA/protein complexes. These features suggest that B cells carrying H chains with increased affinity for dsDNA may be preferentially selected and expanded. Additionally, in the nearby FWR2 area of these hybridomas, residues located between positions 24 and 30 sustained somatic mutations to arginine codons. These arginines occupy the space adjacent to the previously mentioned pocket of asparagine residues, suggesting that these mutations actively increase the tendency of this region to bind dsDNA. The VH characteristics of these clones strongly suggest a process of positive selection, with DNA as the Ag.
This analysis prompts us to consider the role of strain-specific loci in the development of disease (SLE susceptibility loci). These and other studies have shown that lupus autoantibodies may be determined by allotype-linked gene(s). For example, [MRL/lpr (Ighj
) × B6/lpr (Ighb
)]F1 mice express SLE-Abs mainly of the b allotype (43
). This bias is borne out in our study. We found that most IgG2a anti-dsDNA mAbs rescued in the bm12→56(+) were of the Ighb
allotype. These findings may provide clues to the cause of this allotype dependence. In our mouse model, which possesses a diploid number of ~150 VHJ558 (a and b), the VH germline repertoire used by the anti-dsDNA IgG Abs does not represent a random sample. Rather most (73%) of the monoclonal anti-dsDNA Abs sequenced were encoded by VH genes that shared the positively charged motif in the CDR2 region. This region, not found in most other J558 VH, represents a highly restricted population. Therefore, in this model, it is likely that the higher predisposition to form anti-dsDNA B cells on a B6 background would be due to availability of these VH germlines, and, specifically, that differences such as charge between the b and a allotype V regions would make the b-allotype more likely to bind charged Ag, such as DNA.
In summary, then, we have identified two stages of activation during cGVH: a stage of nonspecific activation represented by the IgM subset, and a stage of Ag activation represented by the IgG subset. In the former, allo-T cells provided signals that stimulated most B cells nonspecifically. In the latter, Ag selected and expanded specific B cell clones. The Ag is most likely dsDNA, as evidenced by the selection of cells carrying a dense, positively charged region in the CDR, and by arginine mutations proximate to this area. The fact that all members of clone 1 bound only DNA also makes it the likely driving molecule.