CHARACTERIZATION OF ANTI-SINGLE-STRANDED DNA B-CELLS IN A NON-AUTOIMMUNE BACKGROUND

The engagement of the surface Ig of a B cell can result in proliferati on and/or differentiation, deletion, or a state of unresponsiveness ch aracterized by lack of proliferation and/or Ab production defined as ' 'anergy'' (1-3). Early experiments using both Ag-specific (4-6) and no nspecific Ig receptor stimuli (7) have shown that animals can be rende red tolerant, or unresponsive, to subsequent stimulation by Ag. The pa rameters that influence the ultimate outcome may include the nature of the signals transduced by the B cell receptor, availability of T cell help, and the Ag's valency and concentration (reviewed in Refs. 8 and 9). Recent studies using Ig transgenic models have more clearly chara cterized clonal deletion and clonal anergy as mechanisms of self-toler ance in the B cell compartment. Although these studies have shown that autoreactive B cells can be deleted in the bone marrow (BM)(3) (10-12 ) or rendered functionally compromised (3, 13, 14), a precise definiti on of the anergic B cell and whether B cell anergy takes multiple form s remain to be established. It is still controversial, for example, wh ether anergic B cells. actually are in a state of ''delayed deletion,' ' and whether this is affected by competition from non-autoreactive B cells (15-19). We have been interested in the regulation of B cells be aring a disease-associated specificity, anti-DNA. Anti-DNA Abs are a d iagnostic marker for the autoimmune disease systemic lupus erythematos us (SLE) in humans and are present at a high frequency in the MRL-lpr/ lpr mouse model of SLE. Anti-DNA Abs, however, are heterogeneous with respect to their specificities for ssDNA, dsDNA, and DNA-associated pr oteins, and the relationship between pathologic and nonpathologic anti -DNA Abs remains controversial (20, 21). Those anti-DNA Abs that stain nuclei in a homogeneous pattern (that we define as ''anti-homogeneous nuclear (HN)'') have been clinically associated with SLE, while other anti-DNA Abs are transiently present in normal patients and mice, eit her in the context of a Viral infection (22) or after immunization wit h DNA (23-25). Our approach has been to use an Ig heavy chain transgen e (Tg) whose V-H was originally isolated from a diseased MRL-lpr/lpr m ouse (26): the VH3H9 IgM heavy chain Tg, either alone or mated to ligh t chain Tgs resulting in monoclonal B cell repertoires (27). Paired wi th endogenous light chains, VH3H9 generates non-DNA-binding B cells as well as an increased frequency of B cells with a range of anti-DNA sp ecificities: anti-ssDNA, anti-dsDNA, and a subset of the latter, which are also anti-HN (27, 28). This allows us to follow the fate of diffe rent anti-DNA B cells in the context of other specificities. We have p reviously suggested that VH3H9 Tg mice on a non-autoimmune genetic bac kground neg actively regulate anti-DNA B cells, since they do not have anti-DNA Abs in the serum (27). Moreover, the manifestation of reg ul ation differs depending on the specificity of the B cells for DNA: ant i-dsDNA/anti-HN B cells are not retrievable as hybridomas, while, in c ontrast, anti-ssDNA B cells are seen at hi,oh frequency in the spleen, both at the population level and in hybridoma analysis (27, 29). The lack of serum Ig, however, suggested that the anti-ssDNA B cells that were present might be functionally inactivated, or anergized. To obtai n a source of anti-ssDNA Tg B cells for functional studies, we used VH 3H9/V kappa 8 Tg mice, which are generated when VH3H9 Tg mice are mate d to mice transgenic for the V kappa 8 light chain (30). The VH3H9/V k appa 8 combination generates an Ig that binds both ssDNA and cardiolip in and has been observed in anti-DNA Igs from lupus mice (31). We have shown that mice bearing these Tgs have a B cell repertoire that is es sentially monoclonal for the Tg Ig (27). In VH3H9V kappa 8 Tg mice, an ti-ssDNA B cells dominate the peripheral B cell repertoire, but anti-s sDNA Abs are absent from the serum (27). The lack of serum anti-ssDNA Abs, despite the presence of Tg anti-ssDNA B cells, suggests that the Tg B cells present in VH3H9/V kappa 8 Tg mice may be functionally inac tivated. In this way, anti-ssDNA B cells seem to behave similarly to a nother Tg model of B cell tolerance, the anti-hen egg lysozyme (HEL) I g Tg B cells in the context of a soluble HEL Tg (3, 14). However, othe r scenarios could explain the lack of serum anti-ssDNA Ig titers in th ese mice. For example, anti-DNA Tg B cells could be actively secreting anti-ssDNA Ig in vivo, which forms immune complexes with DNA and is s ubsequently cleared from the circulation. In this paper, we have isola ted B cells from VH3H9/V kappa 8 Tg BALB/c mice and analyzed them phen otypically and functionally to determine whether anti-ssDNA B cells ar e indeed anergized, and if so, to begin to define this state. We demon strate that VH3H9/V kappa 8 Tg B cells are functionally and phenotypic ally distinct from normal resting B cells: they show a decreased abili ty to secrete Ig in response to both T-independent and T-dependent sti muli, show suboptimal proliferation to anti-IgM F(ab)'(2) fragments an d LPS, and have a two-to fivefold decrease in total surface Ig density . Despite their functionally compromised state, however, B cells from VH3H9/V kappa 8 Tg BALB/c mice have a turnover rate comparable to norm al resting B cells, suggesting that they are long lived in the periphe ry.