Conditional Disruption of Hem-1 Results in Impaired B Cell Development and Aberrant Antibody Production

Abstract

Hematopoietic protein-1 is a hematopoietic cell specific member of the actin-regulatory WAVE complex, which acts downstream of multiple immune receptors to stimulate F-actin polymerization. Loss-of-function mutations in the gene encoding Hem-1, NCKAP1L, have recently been found to result in Primary Immunodeficiency Disease in humans, characterized by recurrent bacterial and viral respiratory infections, asthma, skin infections, bacteremia, atopy, and autoimmunity. However, the cellular and molecular mechanisms of how loss of Hem-1 results in PID are not known. In this study, we generated constitutive and B cell specific Nckap1l knockout mice using the Cre-LoxP system to dissect the importance of Hem-1 in B cell development and functions. We found that mice with B cell specific disruption of Hem-1 lack mature recirculating B cells in the bone marrow and lymph nodes, along with reduced peripheral B cell populations such as B1, follicular and marginal zone (MZ) B cells due to the decreased capability to efficiently migrate to different lymphoid tissues. Immunization with the T-independent antigens NP-ficoll and heat-killed S. pneumoniae (HKSP), failed to elicit antibody production due to a lack of the innate-like B1 and MZ B cell populations. Immunization with HKSP failed to produce antibody titers sufficient to protect Hem-1 deficient mice from a challenge with a lethal dose of the bacteria Streptococcus pneumoniae. In contrast to the poor antibody response to T-independent antigens, Hem-1 deficient mice produced pronounced IgM and IgG2c antibody titers following immunization with the T-dependent antigen NP-KLH. Hem-1 deficient B cells demonstrated hyperreactive states as shown by: decreased surface IgM expression, increased and sustained calcium influx following IgM stimulation, increased expression of activation markers and higher expression of phosphorylated molecules downstream of the BCR before and after IgM stimulation. These changes in Hem-1 deficient B cells are permissive to the formation of the unique T-bet+CD11c+ B cell population that form autoantibodies. These collective findings suggest that Hem-1 is essential for normal development of B cells, B cell migration and homing and regulating BCR signaling thus providing a mouse model that may provide further insight into the mechanism of autoimmunity in children with mutations in NCKAP1L.