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Post-transcriptional Regulation of Germinal Center B Cell Responses in Immunity and Disease

Periodic Reporting for period 3 - GCB-PRID (Post-transcriptional Regulation of Germinal Center B Cell Responses in Immunity and Disease)

Reporting period: 2019-09-01 to 2021-02-28

Our immune system efficiently protects us against the daily onslaught of foreign pathogens. However, the targeting, strength and extent of immune responses have to be tightly controlled. Breaches of these control mechanisms can result in autoimmune diseases. Antibodies secreted by B cells of the adaptive immune system establish an essential barrier against bacteria and viruses and their presence is the hallmark of protective vaccinations. B cells are licensed for their tasks during germinal center (GC) reactions and differentiation into antibody-secreting plasma cells. Unfortunately, B cell-derived autoantibodies and proinflammatory cytokines can cause or significantly contribute to autoimmune diseases.
Cellular fates and responses are largely controlled by transcription factors, who control transcription and thereby the production of mRNAs encoding for specific proteins. Major transcription factor networks regulating protective (or pathogenic) germinal center B (GCB) cell responses were identified and characterized in the past. However, accumulating evidence suggests that global protein abundance is determined to a large extent at the translational stage, highlighting the importance of post-transcriptional gene regulation (PTGR). The human genome encodes for >1500 RNA-binding proteins (RBPs) and >690 of them bind mRNA with various RNA recognition motifs. Little is known about the role of post-transcriptional regulation by RBPs in terminal (protective or autoimmune) B cell differentiation. We postulate that RBPs exercise critical post-transcriptional control over GCB and plasmacytic cell physiology and we aim to identify and molecularly characterize these regulatory mechanisms.

In recent years the incidence of autoimmune diseases has risen, underscoring the need to uncover disease pathomechanisms thereby paving the way for novel therapies. RPBs contain their RNA-binding domain as an obvious target for therapeutic intervention. Therefore, a basic understanding of the roles of RBPs in protective and autoimmune B cell responses should give rise to novel therapeutic target structures.

To this end, we will complement the analysis of sophisticated genetic mouse models with experiments using novel cell culture systems. We will monitor RBP activity with fluorescent sensors and use proteomics to reveal RBPs regulating the protein abundance of critical mediators of GCB and plasmacytic cell fates. In addition, we will conduct genetic screens to uncover relevant functions of a short list of candidate RBPs, whose protein expression we found to differ significantly between GCB and mantle zone B cells. Ultimately, we will use cellular immunology and RNA biochemistry to elucidate how these RBPs exert their post-transcriptional control. Through the integrated power of our multi-disciplinary approach we will thus pinpoint and investigate the functions of key RBPs regulating the biology of GCB and plasmacytic cells. GCB-PRID promises to uncover profoundly new insights into post-transcriptional regulation of adaptive immunity.
To date, we extended and optimized plasmablast generation in the induced germinal center B cell (iGB) culture system, which recapitulates key aspects of plasma cell differentiation in the mouse in vitro. These cells can be efficiently infected with lentiviral and retroviral vectors for genome and gene expression manipulation. We continue to optimize various aspects of the iGB cell system for screens and RBP functional analyses. We built a large repertoire of hematopoietic progenitor cell pools (Hoxb8FL) of different genotypes and are actively optimizing Hoxb8FL differentiation protocols into B lineage cells in vitro and in vivo to the germinal center and plasma cell states. Furthermore, we established various gene editing protocols in these cells and are validating in vivo differentiation potential after each manipulation. We obtained a retroviral libary to globally screen for RBP functions. We established an additional system to expand hematopoietic precursor cells in vitro for adoptive transfer in vivo that does not require expression of exogenous transgenes. We made progress in determining the roles of individual RBPs in terminal B cell differentiation. For validation purposes, we established and characterized a collection of mouse B cell lines resembling mature B cells, B cells bearing features of germinal center B cells and plasma cells. We established genome manipulation protocols in these cell lines and in vivo transfer.
We established mass spectrometry-based interaction proteomics using mRNA representing UTR fragments and we are optimising procedures for intermediate throughput in 96 well plates. In addition, we established RBPome analysis in cell lines resembling GC B cells and plasma cells. In these experiments we identify polyA mRNA binding proteins in a global and largely unbiased fashion.
Regarding the Ptgr reporter assays we are using transposon based reporter systems. We are optimising Ptgr reporter delivery by lentiviral and retroviral vectors in order to be able to infect iGB cells and Hoxb8FL cells for adoptive transfer.
We established the culture and genetic manipulation of human germinal center B cells from tonsils and adenoids for cross-species validation purposes.
We expect to pinpoint critical roles for RBPs during germinal center reactions and plasmacytic differentiation. Furthermore, we aim to identify RPBs binding to 3prime UTRs of the mRNA of critical mediators of germinal center reactions and plasmacytic differentiation by mass-spectrometry based proteomics. We want to uncover the functional significance of the underlying regulation.
In vitro plasmacytic differentiation