Dysfunctional Activation and Apoptotic Fragility in Hem1-Deficient B Cells

Abstract

Loss-of-function mutations in NCKAP1L and its associated protein, HEM1, cause severeprimary immunodeficiency (PID) in children associated with mature B lymphopenia and impaired humoral immune response to foreign antigens, but also with B cell hyperactivation and production of autoantibodies. However, the mechanism by which loss of HEM1 causes this phenotype in B cells is poorly understood. In this study, we examined increased activation and metabolic activity in Hem1-deficient murine B cells from a transgenic conditional knockout mouse model. By fixing the B cell receptor repertoire using MD4 (IgHEL), we were able to confirm higher activation in Hem1-deficient B cells independent of BCR specificity. We demonstrate that this hyperactivated phenotype is directly associated with vulnerability to apoptosis, where risk of cell death rises with increasing strength of BCR activation. We observed increased mitochondrial mass and ROS in Hem1-deficient cells, which may both increase signal strength and promote cytotoxicity leading to apoptosis. Our results are supported by RNASeq validated by RT-qPCR, which showed upregulation of genes associated with growth, metabolism, proliferation, and DNA synthesis following disruption of Hem1.