EXTRAFOLLICULAR B CELLS AND ANTI-SLIT DIAPHRAGM AUTOANTIBODIES UNDERLY A SHARED AUTOIMMUNE PATHOGENESIS IN CHILDHOOD AND ADULT IDIOPATHIC PODOCYTOPATHIES

Idiopathic podocytopathies, namely idiopathic nephrotic syndrome in non-biopsied children and the histological diagnosis of minimal change disease and primary focal segmental glomerulosclerosis, are characterized by severe cytoskeletal derangements in podocytes that lead to nephrotic syndrome. These entities were long seen as a disease spectrum mediated by circulating factors, but the exact mechanisms causing podocyte injury remained elusive. Recently, the discovery of autoantibodies against slit diaphragm proteins in patients, along with the demonstration of nephrotic syndrome induction in mice by immunization with slit diaphragm proteins Nephrin and CRB2, strongly suggested an etiology of anti-slit diaphragm autoimmunity. However, the exact autoantibody landscape in idiopathic podocytopathies, including the presence of anti-CRB2 autoantibodies, and the immune dysregulations responsible for autoantibody production remain misunderstood. Our study aimed to examine the relationship between anti-slit diaphragm autoantibodies and B cell dysregulation in children and adults with idiopathic podocytopathies.

Anti-CRB2 and anti-Nephrin autoantibodies were measured by ELISA in a multicenter cohort of 82 patients with idiopathic podocytopathies, 22 with other glomerulopathies and 28 healthy controls. Kidney biopsy samples were stained for IgG deposits and imaged with confocal microscopy and high-resolution structured illumination microscopy. Peripheral blood B cell phenotype was assessed by flow cytometry. B cell transcriptional signatures and clonality were evaluated by single-cell RNA-sequencing.

Anti-CRB2 autoantibodies were detected in all types of active idiopathic podocytopathies, with seropositivity significantly higher in adults (27/31 [87%] vs 23/51 [45%], P < 0.01) and titers significantly reduced in remission (51 vs 115 µg/mL, P < 0.01). Double seropositivity for anti-CRB2 and anti-Nephrin was seen in 14/82 (17%) patients. IgG deposits colocalizing with CRB2 were identified in the glomeruli of anti-CRB2 seropositive patients. The expansion of extrafollicular T-bet+ CD21low atypical B cells, a population associated with autoimmunity, was seen in both children and adults. In children, both autoantibodies correlated with atypical B cell expansion. In adults, elevated frequencies of classical memory B cells indicated a greater degree of B cell dysregulation than in children. Further profiling in children using single-cell RNA-sequencing revealed a broadly dysregulated B cell pool poised for activation and the preferential accumulation of autoimmunity-prone VH4-39+ B cell clonotypes within the repertoire. 

We discovered that, despite being more frequent in adults, anti-CRB2 autoantibodies were seen in all types of idiopathic podocytopathies and could co-occur with anti-Nephrin autoantibodies. Moreover, we showed that children and adults had a dysregulated, autoimmunity prone B cell pool characterized by the expansion of extrafollicular atypical B cells. Altogether, these findings position anti-slit diaphragm autoimmunity as central to the pathogenesis of both childhood and adult idiopathic podocytopathies.