Murine Models of Human Immune Defects
Creators: name: Boast, Brigette
Publisher: name: The Australian National University
Year: 2021

description: Due to the considerable overlap between mammalian genetics and physiology, the use of lab rodents has allowed for great advancements in the understanding of human diseases and implementation of novel therapies, particularly within the cancer and immunology fields. Through the use of both forward and reverse genetics approaches, we have developed three mutant mouse strains, all with mutations in genes that are essential for correct immune development and function. The first two mutant strains have mutations in Pax5 and Ikzf1 and were discovered through ENU mutagenesis. The third strain has a mutation in Arpc1b and was discovered through the identification of a cohort of primary immunodeficiency (PID) patients with ARPC1B deficiency. Through ENU mutagenesis we discovered a strain of mice with a novel mutation in Pax5 (Y351*) that truncates the protein just before translation of the inhibition domain, reducing protein function yet still allowing for some B cell development to occur. A promiscuous and pluripotent population of CD19+B220- B cells develops from a very young age in the bone marrow of homozygous mice. Importantly, Pax5Y351*/Y351* mice survive well into adulthood and spontaneously develop malignant precursor B-ALL with a median onset of 239 days. Whole exome sequencing of 14 of these tumours revealed recurrent mutations in Jak3 and Ptpn11, confirming synergy with PAX5 mutations and the JAK/STAT and ERK signalling pathways in driving tumorigenesis. Also through ENU mutagenesis, a second strain of mice were discovered to have defects in B cell development. Sequencing of founder mice identified a missense mutation (L132P) in Ikzf1 (IKAROS) located in zinc finger 1 (ZF1) of the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P) conserves overall protein expression, and has a B cell-specific phenotype with no effect on T cell development. Mice have reduced antibody responses to immunization and show a progressive loss of serum immunoglobulins compared to wildtype littermates. RNAseq revealed a total loss of Hsf1 expression in follicular B cells providing an explanation for the humoral immune response defects observed in Ikzf1L132P/L132P mice. Lastly, mutations in Actin Related Protein 2/3 Complex Subunit B (ARPC1B) have been identified as a cause of PID in a cohort of 14 patients globally. ARPC1B is a subunit of the Arp2/3 complex, which is essential for F-actin polymerisation. Arp2/3 and is activated through the engagement of WASP, which is downstream of DOCK8. Similar to DOCK8 deficiency, ARPC1B patients have elevated levels of serum IgE and IgA. To gain an understanding of how the lack of ARPC1B leads to immunodeficiency and immune dysregulation, we have performed the first functional study of ARPC1B deficient mice (Arpc1bKO) and contrast results with murine DOCK8 deficiency (Dock8KO). Strikingly, naive Arpc1bKO have a hyper-responsive phenotype with decreased surface BCR expression, increased formation of germinal centres (GC) and spontaneous class switching. Interestingly, this contrasts with Dock8KO mice, which have normal BCR expression and are unable to form persistent GCs even after SRBC immunisation. Whilst both ARPC1BKO and DOCK8KO patients have hyper IgE, only Arpc1bKO mice have elevated IgE. As Arpc1bKO mice do not share a phenotype with Dock8KO mice, this indicates that Arp2/3 activity in resting B cells can occur independently of DOCK8. Conversely, the DOCK8-specific absence of GC B cells suggests a previously unknown role of DOCK8 independent from its function as an activator of the Arp2/3 complex through WASP. The use of murine models to study human diseases allows for a more in-depth understanding of the underlying pathomechanism and paves the way for future therapeutic treatments. This thesis describes three different mutant mice all modelling three different human diseases; B-ALL, CVID, and PID.