B cell lymphocytosis and reprogramming due to bi-allelic CARD11 mutations

ABSTRACT

Adaptive immune responses are tightly controlled by proteins including CARD11 that regulate signaling events downstream of the T and B cell receptors. Germline mutations in CARD11 cause several distinct monogenic inborn errors of immunity (IEI) with early childhood onset and potentially fatal prognoses. Somatic CARD11 gain-of-function (GOF) mutations are associated with B cell malignancies. Precisely how various CARD11 mutations culminate in unique clinical entities, and the mechanisms of CARD11-driven B cell proliferation, are not fully understood.We sought to identify the genetic basis of disease and characterize immune cell phenotypes and functions in a patient with apparent BENTA (B cell Expansion with NF-κB and T cell Anergy) disease and a history of sibling death in early childhood.We used whole exome sequencing, flow and mass cytometry, whole blood mRNA analyses, in vitro T cell proliferation and B cell differentiation assays, and single cell RNA sequencing of patient-derived samples to identify the genetic basis of disease and define its molecular and cellular mechanisms. We also ectopically expressed wild type (WT) and mutant forms of CARD11 in T and B cells and assessed their functional impacts on NF-κB-dependent responses.We report a surprising new genetic basis of BENTA caused by homozygosity for the novel CARD11 mutation R331P and characterized by massive expansion of B cells with a naïve surface phenotype and aberrant transcriptional program. This autosomal recessive form of BENTA features exaggerated B cell lymphocytosis relative to mono-allelic BENTA. Further, we have identified patterns of gene expression that distinguish autosomal recessive BENTA patients' B cells from those of healthy controls, and from mono-allelic BENTA. We found ectopic CARD11 R331P expression induced constitutive NF-κB activity in T and B cells. These data suggest R331P is a gain-of-function mutation and causes BENTA in homozygosity.These results define a novel autosomal recessive form of BENTA disease. Additional analysis of mutation-driven changes in B cell function may shed light on the mechanisms of B lymphomagenesis in patients with germline or somatic CARD11 variants.