Dissecting Hem-1 functions in B lymphocyte Development and Primary Immunodeficiency Disease

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PROJECT SUMMARY Primary immunodeficiency diseases (PID) are group of inherited conditions where components of immune signaling pathways are either missing or are dysfunctional, resulting in severe recurring infections that often progress to autoimmune disease or cancer. Of the greater than 300 PIDs that have been identified so far, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 individuals (4 now deceased) from 4 independent families were identified with PIDs that were linked to mutations in NCKAP1L, which encodes for a conserved hematopoietic cell-specific actin regulatory protein called Hematopoietic protein-1 (Hem-1). Affected children presented with recurring respiratory and skin infections, otitis media, impaired antibody responses to pneumococcal immunization (characteristic of B cell immunodeficiency), dysregulated cytokine production, and autoimmunity. Although the cellular and molecular functions of Hem-1 orthologues in flies and worms are relatively well characterized, there is a critical knowledge gap regarding the cell specific functions of Hem-1 in primary mammalian immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific cellular and molecular functions of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to target Hem-1 in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, signaling, and protective immunity. Our Specific Aims are to: (1) utilize conditional Cre-LoxP mediated gene targeting in mice to define how B cell specific loss-of-function mutations in Hem-1 alter peripheral B cell development, antibody responses, and protective immunity to Pneumococcus and Influenza A virus, two important community acquired infections; (2) determine the impact of Hem-1 mutations on B cell signaling and transcription; (3) utilize CRISPR/Cas9 gene editing in “humanized mice” to assess the impact of loss-of-function mutations in Hem-1 on the dynamic development of primary human B lymphocytes. To demonstrate feasibility, we have generated mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient primary human hematopoietic stem cells. Utilizing these innovative mouse and human model systems, we will test our overall hypothesis that B cell specific expression of Hem-1 is essential for the development of marginal zone and B1 B cells, T- independent antibody responses, and protective B cell immunity. These studies are highly significant because they will utilize innovative approaches to define for the first time, the cellular and molecular mechanisms of how non-coding mutations in NCLAP1L disrupt B cell development, signaling, and protective humoral immunity, resulting in PID and potentially autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the disruption of Hem1 in cell-type specific manner is critical for dissecting the mechanisms of how mutations in Hem-1 result in PID and autoimmunity.