Multiple tolerance checkpoints suppress low-affinity germline precursor B cells of high affinity anti-dsDNA antibody-producing cells in BCR knock-in mice

Abstract

Abstract Previously, we revealed that disease-associated dsDNA-reactive high-affinity antibodies evolve from ssDNA-reactive low-affinity germline precursors through clonal expansion and affinity maturation in acute systemic lupus erythematosus (SLE) patients. Our findings raise questions whether low-affinity precursors of anti-dsDNA antibody producing cells are subjected to immune tolerance, and under what conditions they expand and undergo affinity maturation in vivo. To address these questions, we generated G9gl, a site-directed knock-in (KI) mouse line carrying reverse mutated forms of VH-DH-JH and Vk-Jk regions of an acute SLE patient-derived high-affinity anti-dsDNA antibody. G9gl heterozygous mice had reduced numbers of B cells in their periphery, and half of peripheral B cells expressed endogenous IgH from non-KI allele due to receptor editing. G9gl-expressing B cells exhibited several anergic phenotypes, including reduced surface BCR and shorter life span. Yet, they underwent class-switching and produced antibodies following CD40 and IL-4 in vitro stimulation. G9gl B cells responded to ssDNA, but not dsDNA in vitro, and were selectively excluded from germinal center reaction in several conditions such as Treg depletion and DNA-conjugated mBSA immunization in vivo. However, some of G9gl-expressing B cells differentiated to somatically mutated IgG+ memory B cells when immunized with bacterial DNA-conjugated mBSA. Therefore, multiple tolerance checkpoints prevent low-affinity anti-DNA precursor B cells from undergoing clonal expansion and affinity maturation in germinal centers. However, G9gl-expressing B cell might undergo class-switching and somatic hypermutation through the extra-follicular pathway.