Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Ig transgenic models to neo-self Ags have helped to classify two manifestations of B cell tolerance: clonal deletion and functional inactivation (anergy) (1–3). Recently, the distinction between these two has come into question as “anergized” cells have been shown to have a reduced lifespan and may be in a state of “delayed deletion” (4). Furthermore, the relative contribution of deletion versus receptor editing to the elimination of autoreactive B cells is being reevaluated (5, 6). Given that most autoimmune diseases are characterized by the presence of autoantibodies directed toward a discrete set of autoantigens, we are interested in determining whether the mechanisms described for the maintenance of tolerance to neo-self Ags apply to disease-associated autoantigens. Anti–double-stranded (ds)1 DNA Abs are one of the hallmarks of SLE and the MRL-lpr/lpr murine model for SLE, and rising titers of these Abs correlate with disease exacerbation (7). In the serum of nonautoimmune individuals, anti-dsDNA Abs are not present, suggesting that this specificity is regulated, yet the mechanism governing this regulation remains unclear. To follow the fate of anti-dsDNA B cells, we have used Ig transgenic mice. The transgene (tg) being studied encodes the VH3H9 H chain, originally isolated from anti-dsDNA Igs in diseased MRL-lpr/lpr mice, in combination with different L chains (8). Transfection studies have shown that this H chain can pair with a variety of different L chains to generate both anti–single-stranded (ss)DNA and anti-dsDNA Abs (9). As a tg, VH3H9 can pair with endogenous L chains to generate anti-ssDNA, anti-dsDNA, and non-DNA B cells, allowing us to study the regulation of anti-dsDNA B cells in the presence of B cells with other specificities (10, 11). Tracking anti-dsDNA B cells in a diverse repertoire is important because this more closely mimics the conditions present in SLE. In addition, precedent exists for a differential fate of autoreactive B cells in the context of a monoclonal versus polyclonal B cell repertoire (12, 13). There have been conflicting reports on the fate of anti-dsDNA B cells in nonautoimmune mice. Some state that anti-dsDNA B cells are deleted in the bone marrow; others report that these cells exit to the periphery, but do not secrete anti-dsDNA Ab due to endogenous Ig expression or B cell functional inactivation (14–17). These discrepancies may be a reflection of the degree to which individual Ig tgs are capable of inhibiting endogenous Ig rearrangement, which could rescue an otherwise autoreactive B cell. Whether endogenous Ig expression is due to the active induction of receptor editing or is the consequence of a defect on the part of the Ig tgs to inhibit rearrangement can be difficult to assess, particularly for L chain tgs (15–19). A more interesting possibility to explain these divergent outcomes is that they reflect the different specificities of the tgs used in these studies, which may in turn differ in their regulation (14–17, 20). Because anti-dsDNA Abs from SLE patients and lupus mice are heterogeneous, and the particular specificities which are significant in disease are not known, it will be important to understand these differences (21, 22). The VH3H9 tg offers an opportunity to study the regulation of a range of anti-DNA B cells. Initial studies using the VH3H9 H chain tg on the BALB/c background demonstrated that neither anti-ssDNA nor anti-dsDNA serum Abs were elevated over tg(−) BALB/c control sera (23). When hybridoma panels were generated from the spleens of VH3H9 tg mice, anti-ssDNA and non-DNA hybridomas were recovered, but not anti-dsDNA hybridomas (23, 24). Transfection studies clearly showed that this H chain has the capacity to generate anti-dsDNA B cells (9). Importantly, we have also recovered this specificity in hybridoma panels generated from VH3H9 MRL-lpr/lpr spleens (10). The absence of anti-dsDNA hybridomas from BALB/c-derived panels suggests, therefore, that they are either deleted in the bone marrow or, if present, cannot be rescued as hybridomas. To address the mechanisms governing the regulation of anti-dsDNA B cells in a diverse repertoire and to avoid the use of L chain tgs altogether, we relied on the fact that VH3H9 can pair with endogenous Vλ1 L chains to generate anti-dsDNA Abs (9). Thus, we were able to track the λ1-bearing anti-dsDNA B cells in the context of the diverse repertoire of the VH3H9 tg mouse. We have found that anti-dsDNA B cells are not deleted in the bone marrow, but instead exit to populate the spleen. Their unique surface phenotype suggests that they are both developmentally arrested and antigen experienced. These cells exhibit an increased in vivo turnover rate and are localized to the T–B interface of the splenic white pulp.