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IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.

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Systemic lupus erythematosus and numerous other autoimmune diseases are characterized by affinity-matured, class-switched autoantibodies to nuclear antigens. Such antibodies are generally thought to arise in germinal centers (GCs). Several strategies to block GC formation and progression are currently being explored clinically. However, recent studies have suggested a key role for extrafollicular responses in driving the early events in autoimmune development. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a lupus murine model, where we could genetically block the GC pathway. We find that a B cell intrinsic block in GC formation accelerates extrafollicular responses and exacerbates autoimmune progression. The manifestations included higher levels of circulating, class-switched autoantibodies, as well as antibody- and complement-deposition in the kidney glomeruli. GC B cell cultures in vitro showed that loss of the GC transcription factor Bcl-6 prevents cellular expansion and accelerates plasma cell differentiation. This suggests that the in vivo phenotype was a direct consequence of rewiring of B cell intrinsic transcriptional programming. In a competitive scenario in vivo, in autoreactive mixed bone marrow chimeras, B cells harboring the genetic GC block contributed disproportionately highly to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune pathogenesis and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response.Highlights-Genetic GC block exacerbates autoimmune progression in a lupus model-An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells in vitro-B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive setting in vivo-Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterparts

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family with HNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.