Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice

Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the V-H-D-H-J(H) and V kappa-J kappa regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse V-H and the KI gene, suggesting that receptor editing through V-H replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.

Automated summary

Anti-dsDNA antibodies are a marker of systemic lupus erythematosus and their high-affinity reactivity is believed to be the result of somatic hypermutation. This study investigated whether low-affinity precursors of these antibodies are subjected to immune tolerance. The researchers generated a mouse model carrying germline-reverted sequences of high-affinity anti-dsDNA antibodies and found that multiple tolerance checkpoints prevented these low-affinity precursors from undergoing clonal expansion and affinity maturation in germinal centers.