Identification of novel regulators of B lymphocyte biology

Abstract

The differentiation of B cells into antibody secreting cells (ASCs) and the production of protective antibodies is a critical part of the adaptive immune response to infection. ASCs are also important for the formation of immunological memory which provides protection against reinfection and the generation of ASCs is the goal for almost all current vaccination strategies. Despite the importance of these cells, we still lack a complete understanding of the factors that control B cell differentiation into ASCs, ASC survival and antibody secretion, all of which must be tightly regulated to ensure an optimal immune response.

Here, I have developed a CRISPR/Cas9 mediated arrayed screening approach for the identification of novel positive and negative regulators of primary mouse B cell proliferation, survival, differentiation into ASCs and antibody secretion. By interrogating multiple gene sets I have identified all elements within the ASC gene signature that are essential for the in vitro generation of ASCs. I have also identified several novel negative regulators of the B cell differentiation process (AB124611, Arhgef18, A430078G23Rik, Fam43a, Pold1, Ripk3, Rnf130 and Rps6ka5). This work has also uncovered a novel role for 6 genes, (Cdv3, Hspa5, Sec61a1, Selk, Sumo2, Vcp) in driving the proliferation of B cells. One of these genes, Cdv3, has no previous association with proliferation in any cell type and presents an exciting new candidate for further investigation.

I have demonstrated that within the ASC gene signature there are 35 genes which are essential for efficient antibody secretion. Interestingly, many of these genes are components of the ER protein processing pathway, however, not all elements of this pathway appear to be essential for antibody secretion. These results raise the possibility of there being a specific pathway for antibody secretion, or that the genes identified in this thesis may represent weak links in the ER protein processing pathway which could potentially be exploited therapeutically to inhibit antibody secretion in disease settings.

Finally, I have used an Irf4 deficient mouse model to uncover a novel role for Irf4 in the development of the peritoneal B-1a population. I have shown that Irf4-/- mice lack peritoneal B-1a cells and by examining multiple stages of B-1a cell development I have demonstrated that in the absence of Irf4, B-1a cell development is blocked at the transitional B-1a stage. By employing RNA sequencing to analyse the transcriptional profiles of the remaining Irf4-/- B-1 cells and analysis of previously published ChIP sequencing data, I have revealed a potential role for Irf4 in directly activating the expression of Bhlhe41, a transcription factor that is required for B-1a cell development and homeostasis.

Together, the results from this thesis build upon decades of previous work on the genetic regulation of B cell biology. Integrating the novel regulators of B cell proliferation and differentiation that I have identified in this thesis into the current model of ASC generation will improve our understanding of how the decision between undergoing differentiation or maintaining the B cell fate is made. A detailed understanding of how this fate decision is made has far reaching implications for human health and disease as this information can be used to inform vaccine design, reveal the causes of immunodeficiencies or highlight novel avenues for targeting pathogenic ASCs in autoimmunity and cancer.