There is compelling evidence that B cells play a critical role in the pathogenesis of many of the organ-specific and systemic autoimmune diseases. Systemic autoimmune disorders with a known B cell component include systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disorder and scleroderma, among others. Many of the hallmark antibodies associated with these disorders are against nuclear components such as DNA, ribonuclear proteins and DNA repair proteins, antigens not readily exposed to the cells of the immune system. It is not understood why antinuclear antibodies develop but defective apoptosis, defective clearance of apoptotic material, excess necrosis from ongoing chronic damage, defects in toll receptor signaling and a breakdown in B and T cell tolerance mechanisms have been suggested as potential culprits. Ultimately, these autoantibodies can trigger an autoimmune cascade characterized by inflammation and tissue destruction.
One way B cells contribute to autoimmune disorders is by secreting autoreactive antibodies. However, it appears this is not the only way B cells can trigger or exacerbate autoimmunity. Recently, it was demonstrated that autoimmune mice with B cells but lacking secreted antibodies, still develop a milder form of lupus nephritis, while mice completely lacking B cells did not (Chan et al., 1999). This suggested an antibody-independent contribution of B cells to autoimmunity. Subsequent studies provided evidence that B cells contribute to autoimmunity by activating autoreactive CD4+ T cells, likely as antigen-presenting cells. Therefore, B cells contribute to autoimmune disease through the production of autoreactive pathogenic antibodies, and as antigen-presenting cells to autoreactive T cells. Given the importance of antibody and B cell receptor (BCR) specificity to self-antigens in autoimmune disorders, it is of great significance to delineate the differential contribution of the various mechanisms that contribute to diversity, specificity and function of the BCR and of antibodies. The three main mechanisms responsible for generating diversity and increasing specificity of the B cell repertoire are V(D)J recombination -which generates the pre-immune naïve repertoire - and the antigen-driven processes, SHM and CSR. AID is critical to two of these mechanisms: SHM and CSR. It is through its requirement for both of these processes that AID plays a major role in B cell-mediated autoimmunity (Fig. 9.2).
Figure 9.2. The two main pathways of potential AID contribution to B cell mediated autoimmunity.
Figure 9.2. The two main pathways of potential AID contribution to B cell mediated autoimmunity.
AID triggers SHM and CSR during secondary B cell responses by deaminating cytosines in the variable and switch regions of the immunoglobulin locus (Rada et al., 2002). It is absolutely required for both of these reactions: B cells from organisms lacking AID activity can only secrete unmutated, germline IgM antibodies (Muramatsu et al., 2000; Revy et al., 2000). Therefore, mouse models wherein AID is lacking in an autoimmune background are powerful tools to examine the contribution of autoreactive germline antibodies to autoimmunity. Isolating the contribution of SHM vs. CSR to autoimmunity is more difficult since no molecule has been described to date whose deficiency eliminates one of the mechanisms and not the other. However, through genetic manipulation of AID expression in various backgrounds, we have generated a series of mice that either lack SHM, CSR or are characterized by a very limited naïve B cell repertoire, in order to examine the differential contribution of these processes to the antibody-dependent and antibody-independent pathways by which B cells contribute to autoimmunity.
9.3.1 Potential novel mouse models designed to distinguish the role of SHM, CSR and the naïve repertoire in autoimmunity
Often, pathogenic antibodies in autoimmunity are IgG, strongly implicating a role for CSR in autoimmunity. Consistent with this, mice deficient for the activating receptors FcR and FcRIII experienced reduced kidney damage while mice deficient in the inhibitory receptor FcRII, experienced increased severity (Sylvestre and Ravetch, 1994; Takai et al., 1996; Ravetch and Bolland, 2001). In addition, there is good evidence that some isotypes are more frequently associated with pathogenic antibodies than others (Takahashi et al., 1991). SHM also plays an important role in systemic autoimmunity. Most autoantibodies derived from SLE patients and in MRL/lpr mice, a mouse model for SLE, are hypermutated (Shlomchik et al., 1990; Shlomchik et al., 1987; Radic et al., 1989; van Es et al., 1991; Winkler et al., 1992; Wellmann et al., 2005). In MRL/lpr mice, there was a correlation with autoreactive antibodies and specific mutations, particularly those introducing arginines into the CDRs (Radic et al., 1989). It is likely that because SHM is random in relation to affinity, new mutations could be introduced that increase affinity to self-antigens generating autoreactive antibodies. In a normal individual, these mutated B cells bearing autoreactive BCR's and capable of secreting autoreactive antibodies, are eliminated. However, in autoimmune-prone individuals peripheral tolerance checkpoints may be defective, resulting in the recruitment of these cells into the memory compartment. Such disruption can be accomplished by, among others, defects in B or T cell signaling in germinal centers, defective apoptosis or even disruption of the germinal center environment such that SHM can occur outside of germinal centers without proper monitoring of newly-generated autoreactive B cells (William et al., 2002). Finally, the naïve repertoire also contributes to autoimmunity. Newly-generated B cells, each bearing a different and unique BCR, may harbor autoreactivity-enhancing amino acids in their CDR3s formed during V(D)J recombination. This is particularly true for large, positively charged amino acids such as arginines, which can be either encoded in the D or J elements or formed during N-region addition by terminal deoxynucleotide transferase or TDT. These cells are normally modified by receptor editing or deleted during central tolerance in the bone marrow. In most autoimmune mice, however, these cells readily survive into the periphery and may become the precursors to high-affinity autoreactive B cells. Consistent with the formation of autoreactive receptors during V(D)J recombination is the finding that mice deficient in TDT experienced a significant decrease in glomerulonephitis (Molano et al., 2003; Robey et al., 2004).
In order to distinguish the role of these various mechanisms that contribute to antibody diversity and versatility, we crossed various strains with B cell defects into the AID-deficient MRL/lpr background. For example, muS-/-.MRL/lpr mice (with a defect in the exon encoding the secretory domain for IgM) have a full naïve repertoire, can undergo SHM and CSR, but cannot secrete IgM (Boes et al., 2000). They also have elevated levels of IgG autoantibodies to dsDNA, increased deposition of immune complexes in glomeruli, severe glomerulonephritis and early onset of lupus-like syndrome. Crossing the strain to AID-deficient MRL/lpr resulted in mice lacking any secreted antibodies, or
SHM, but still maintaining a full naive repertoire of B cells with surface expression of IgM. These mice (lacking any secreted antibodies) can be compared to AID.MRL/lpr mice (secreting only IgM) to isolate the impact of secreted IgM in the lupus syndrome of MRL/lpr mice.
JHT.MRL/lpr mice completely lack B cells and their products and are similar to mice generated by Shlomchik and colleagues (Chan et al., 1999). These mice have a very mild manifestation of the MRL/lpr syndrome. Rescuing B cells in these mice by introducing a transgene-encoding membrane IgM but incapable of secreting antibodies (JHT.mIg.MRL/lpr), restores some of the phenotype, in particular the infiltration of mononuclear cells into the kidneys and other tissues, suggesting an antibody-independent pathway for B cells in autoimmunity. However, these mice express a single VH (186.2), which is not normally autoreactive. This led us to speculate that the relevant B cells became autoreactive through SHM and affinity maturation against self-antigen. We are testing this hypothesis by crossing the JHT.mIg.MRL/lpr to AID.MRL/lpr mice. The resulting offspring lack secreted antibodies, have B cells from a limited repertoire and lack SHM. These mice can be used to compare to AID wild-type JHT.mIg.MRL/lpr, and will provide clues to the significance of SHM and affinity maturation to the antibody-independent contribution of B cells to autoimmunity.
Another possible strategy to isolate the role of CSR from SHM, is by altering the amino-acid sequence of the AID protein through the generation of specific AID-knocked-in mice. Loss of the C-terminal nuclear export signal region of AID results in normal SHM but impaired CSR (Ta et al., 2003; Barreto et al., 2003). This possibility, however, needs more clarification since it is unclear if AID-mediated mutation in these mice is targeted to Ig loci, considering that loss of the NES is expected to increase AID levels in the nucleus (Brar et al., 2004), which may cause widespread AID-mediated deamination.
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