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GCB-PRID Report Summary

Project ID: 682435
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - GCB-PRID (Post-transcriptional Regulation of Germinal Center B Cell Responses in Immunity and Disease)

Reporting period: 2016-09-01 to 2018-02-28

Summary of the context and overall objectives of the project

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, whose actions determine the amount of mRNA for a specific protein mediator is produced. Major transcription factor networks regulating protective (or pathogenic) GCB cell responses have been 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 RNA-binding proteins (RBP) in terminal (protective or autoimmune) B cell differentiation. We postulate that RBPs exercise critical post-transcriptional control over germinal center B (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 sophisticated genetic mouse models with 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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

To date we 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. Using reporter mouse stains that express the fluorescent protein YFP under expression control of the critical plasma cell transcription factor Blimp1 (Blimp1-eYFP reporter mice) we can demonstrate the generation of over 80% Blimp1-high IRF4-high plasmablasts within 12 days of culture. These cells can be efficiently infected with lentiviral and retroviral vectors for genome and gene expression manipulation. We continue to optimize the iGB cell system, including aspects of antigenic selection and Fas-induced apoptosis. We significantly extended our available repertoire of hematopoietic progenitor cell pools (Hoxb8FL) and are actively optimizing Hoxb8FL differentiation protocols in to B lineage cells in vitro and in vivo. Furthermore, we generated novel viral vectors for CRISPR/Cas9-mediated gene editing in these cell culture systems, which will be of great value for targeted screens of RNA binding proteins.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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.

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