AbstractThe direction of the B cell response determines the quantity and quality of antibody produced, a balance of which is essential for an effective immune response. Quality of secreted antibody is regulated by the germinal center (GC) reaction, in which germinal center B cells (GCBs) undergo proliferation, somatic hypermutation of the BCR, and selection, collectively known as affinity maturation, to ultimately increase GCB affinity to antigen and thus improve antibody quality. Understanding these processes and the transcriptional networks involved in their control are essential to better understanding selection itself. The transcription factor IRF4 is an immediate early gene induced upon BCR and CD40 signaling and is required for activated B cell proliferation and differentiation into GCBs or plasma cells, situating it as a master regulator of B cell fate decisions. Despite its critical role in control of early B cell fates, the requirement for IRF4 in GCBs has been less understood. We hypothesized that, given its importance in early B cell fate decisions, IRF4 also functions during the GC response to control efficient selection of GCBs. By halving Irf4 gene copy number in an antigen-specific BCR transgenic mouse model, we were able to study the requirement for IRF4 in GC processes. Here, we show that IRF4 is expressed in GCBs at levels greater than naive B cells throughout the course of the GC response, and levels of IRF4 expression directly regulate acquisition of high affinity mutations. While GCB phenotype, GC zonation, proliferation, class-switching, BCR expression, and rates of somatic hypermutation were comparable to wild-type cells, Irf4 haploinsufficient GCBs were impaired in the generation of high affinity mutations. Further, in a competition setting, Irf4 haploinsufficient cells were disadvantaged for GC entry, indicating a role for B cell-intrinsic IRF4 in the acquisition of T cell help. We propose a mechanism whereby IRF4 acts to promote effective T cell interactions during both the GC entry and GCB selection checkpoints, consequently regulating affinity maturation.

Alternative polyadenylation (APA) is a type of post-transcriptional regulation that occurs throughout development and cellular differentiation to create multiple isoforms from one gene. Over 70% of mammalian genes are regulated by APA, but the regulatory mechanisms and relevance in different cellular contexts is not fully understood. An attractive model system to gain insight into this process is immunoglobulin (Ig) expression. B cells can synthesize either a secretory or membrane Ig heavy chain (IgH) from the same transcript through competing alternative splicing and APA. B cells predominantly produce the isoform encoding membrane IgH, but during plasma cell (PC) differentiation, switch to utilizing the proximal poly (A) site of the primary IgH transcript. This generates a shorter isoform encoding secretory IgH, also known as antibody. Although the differential expression of these isoforms has been known for over 40 years, the exact molecular switch that directs cell stage-specific APA of the IgH transcript in PCs remains enigmatic. Utilizing novel, fluorescently labeled probes that hybridize to the membrane (µM) or secretory (µS) IgM transcripts, I track their expression at the single-cell level after various perturbations. Measurement of µM and µS in Irf4-/- and Blimp Null lipopolysaccharide stimulated B cells clarified that Irf4 acts first to increase total IgM abundance while Blimp-1 is necessary for the isoform switch to favor µS. CRISPR knock-out experiments of candidate regulators of IgM APA in Cas9+ plasmablasts identified CstF2, Ell2, OBF1, and Symplekin as factors that promote µS poly(A) site usage. Treatment with the CPSF3 inhibitor JTE-607 recapitulated the phenotype seen from CstF2 and Symplekin deletion, leading to increased µM probe signal. Intriguingly, inhibition of the super elongation complex by the small molecule inhibitor KL-1 did not affect µM/µS ratios, suggesting that Ell2’s role in regulating µ poly(A) site choice occurs independently of its role in the super elongation complex. Through further experiments we hope to better understand the mechanisms regulating antibody secretion in PCs, a process critical for neutralizing pathogens yet detrimental in autoimmune diseases, and gain insight into the global regulation of APA.

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