Periodic Reporting for period 4 - GCB-PRID (Post-transcriptional Regulation of Germinal Center B Cell Responses in Immunity and Disease)
Période du rapport: 2021-03-01 au 2023-02-28
Our immune system is an efficient protector against the daily onslaught of foreign pathogens, but the strength, targeting, and extent of immune responses must be tightly regulated. When these control mechanisms are breached, autoimmune diseases can occur. B cells of the adaptive immune system produce antibodies, which form a barrier against bacteria and viruses, and are key mediators of protective vaccinations. During their terminal differentiation, B cells are licensed for their tasks, beginning with germinal center reactions followed by the production of antibody-secreting plasma cells. B cell-derived autoantibodies and proinflammatory cytokines cause or significantly contribute to autoimmune diseases. The rising incidence of these diseases in recent years highlights the need to better understand disease pathomechanisms in order to tailor novel therapies.
Cellular fates and responses are largely controlled by transcription factors, which control the production of mRNAs that encode specific proteins. Major transcription factor networks regulating protective germinal center B cell responses were identified and characterized in the past. However, growing evidence suggests that global protein abundance is determined to a great degree at the translational level, emphasizing the importance of post-transcriptional gene regulation. There are over 1500 human RNA-binding proteins, with 690 of them binding mRNA through various RNA recognition motifs. Little is known about the role of post-transcriptional regulation by these proteins in terminal (protective or autoimmune) B cell differentiation. We hypothesized that RNA-binding proteins play critical roles in germinal center B cell and plasmacytic cell physiology. To identify and characterize these regulatory mechanisms, we combined the analysis of sophisticated genetic mouse models with experiments using novel cell culture systems, as well as genetic, molecular biological, and biochemical approaches. A better understanding of the roles of RNA-binding proteins in both protective and autoimmune B cell responses could open up novel possibilities for therapeutic targets.
We described in detail how B cell-specific expression changes of TNFAIP3/A20 and c-Rel, whose proteins levels are regulated by post-transcriptional gene regulation, impact terminal B cell differentiation and autoimmunity. We employed interaction proteomics to identify novel proteins binding to the untranslated regions of mRNAs encoding for critical mediators of germinal center B cell and plasmacytic cell fates, including Bcl6 and c-Rel. The regulatory functions of these RNA-binding proteins were validated by CRISPR-mediated knockout in germinal center B cell-like cell lines. In addition, we conducted genetic screens to uncover RNA-binding proteins whose loss enhanced or decreased germinal center B cell generation in vivo. We then used cellular immunology and RNA biochemistry techniques to elucidate how individual RNA-binding proteins or protein families exert their post-transcriptional control. Through the integrated power of our multi-disciplinary approach ,we thus pinpointed functions of key RNA-binding proteins regulating the biology of germinal center B cell and plasmacytic cells. Through GCB-PRID we thus uncovered new insights into post-transcriptional regulation of adaptive immunity.
Cellular fates and responses are largely controlled by transcription factors, which control the production of mRNAs that encode specific proteins. Major transcription factor networks regulating protective germinal center B cell responses were identified and characterized in the past. However, growing evidence suggests that global protein abundance is determined to a great degree at the translational level, emphasizing the importance of post-transcriptional gene regulation. There are over 1500 human RNA-binding proteins, with 690 of them binding mRNA through various RNA recognition motifs. Little is known about the role of post-transcriptional regulation by these proteins in terminal (protective or autoimmune) B cell differentiation. We hypothesized that RNA-binding proteins play critical roles in germinal center B cell and plasmacytic cell physiology. To identify and characterize these regulatory mechanisms, we combined the analysis of sophisticated genetic mouse models with experiments using novel cell culture systems, as well as genetic, molecular biological, and biochemical approaches. A better understanding of the roles of RNA-binding proteins in both protective and autoimmune B cell responses could open up novel possibilities for therapeutic targets.
We described in detail how B cell-specific expression changes of TNFAIP3/A20 and c-Rel, whose proteins levels are regulated by post-transcriptional gene regulation, impact terminal B cell differentiation and autoimmunity. We employed interaction proteomics to identify novel proteins binding to the untranslated regions of mRNAs encoding for critical mediators of germinal center B cell and plasmacytic cell fates, including Bcl6 and c-Rel. The regulatory functions of these RNA-binding proteins were validated by CRISPR-mediated knockout in germinal center B cell-like cell lines. In addition, we conducted genetic screens to uncover RNA-binding proteins whose loss enhanced or decreased germinal center B cell generation in vivo. We then used cellular immunology and RNA biochemistry techniques to elucidate how individual RNA-binding proteins or protein families exert their post-transcriptional control. Through the integrated power of our multi-disciplinary approach ,we thus pinpointed functions of key RNA-binding proteins regulating the biology of germinal center B cell and plasmacytic cells. Through GCB-PRID we thus uncovered new insights into post-transcriptional regulation of adaptive immunity.
We generated and analyzed mouse models allowing the B cell- and germinal center B cell-specific overexpression of c-Rel, a transcription factor linked to human autoimmunity. Elevated c-Rel protein levels conferred a dramatic advantage to B cells to enter the germinal center and led to enhanced plasma cell production and antibody secretion upon immunization. We discovered that while naïve B cells contain more c-Rel mRNA than germinal center B cells, the latter produce more c-Rel protein. These findings from mice were confirmed in human tonsillar B and germinal center B cells and provide clear evidence for a central role for post-transcriptional gene regulation of c-Rel in immune responses and autoimmunity. Furthermore, we uncovered that enhancing c-Rel expression is sufficient to induce c-Rel nuclear translocation and transcriptional reprogramming in these cells. TNFAIP3/A20 is a central negative regulator of immune signals linked to several human autoimmune diseases. We investigated the consequences of haploinsufficiency of TNFAIP3/A20 specifically in B cells in combination with additional autoimmunity-predisposing variables, including elevated levels of the B cell survival cytokine BAFF and elevated levels of the anti-apoptotic protein BclxL. These combinations resulted in lethal autoimmune syndromes closely recapitulating many aspects of human systemic lupus erythematosus, including phospholipid syndrome. Furthermore, we showed that aberrant B cell activation has complex and in part contradictory effects on its immune microenvironment. Overall, our results clearly implicate TNFAIP3/A20 as a prime target for post-transcriptional gene regulation.
We extended and optimized plasmablast generation as well as genetic manipulation systems in the mouse induced germinal center B cell (iGB) culture system, which recapitulates key aspects of plasma cell differentiation in the mouse in vitro. Furthermore, we adopted a novel approach of culturing and manipulating tonsillar human germinal center B cells. For in vivo studies of terminal B cell differentiation, we built a large repertoire of hematopoietic progenitor cell pools (Hoxb8FL) of different genotypes (different Cre lines, conditional Cas9 expression) and established various gene editing as well as overexpression protocols in these cells. The potential for B cell differentiation of these cells pools was evaluated by adoptive transfer into recipient mice. These efforts produced a novel method to genetically interrogate terminal B cell differentiation in vivo in small to intermediate throughput, including genetic screens.
We established and optimized mass spectrometry-based interaction proteomics using mRNA representing UTR fragments. These efforts were paralleled by global identification of all polyA-containing mRNA-binding proteins (RBPome) in cell lines resembling germinal center B cells and plasma cells. We identified candidate RBPs validated to bind to the untranslated regions of Bcl6 and c-Rel and regulate their expression and are exploring their functions.
Finally, we identified critical functions of the Roquin1/2 and Staufen1/2 RNA-binding protein families in germinal center B cells and humoral immune responses.
We extended and optimized plasmablast generation as well as genetic manipulation systems in the mouse induced germinal center B cell (iGB) culture system, which recapitulates key aspects of plasma cell differentiation in the mouse in vitro. Furthermore, we adopted a novel approach of culturing and manipulating tonsillar human germinal center B cells. For in vivo studies of terminal B cell differentiation, we built a large repertoire of hematopoietic progenitor cell pools (Hoxb8FL) of different genotypes (different Cre lines, conditional Cas9 expression) and established various gene editing as well as overexpression protocols in these cells. The potential for B cell differentiation of these cells pools was evaluated by adoptive transfer into recipient mice. These efforts produced a novel method to genetically interrogate terminal B cell differentiation in vivo in small to intermediate throughput, including genetic screens.
We established and optimized mass spectrometry-based interaction proteomics using mRNA representing UTR fragments. These efforts were paralleled by global identification of all polyA-containing mRNA-binding proteins (RBPome) in cell lines resembling germinal center B cells and plasma cells. We identified candidate RBPs validated to bind to the untranslated regions of Bcl6 and c-Rel and regulate their expression and are exploring their functions.
Finally, we identified critical functions of the Roquin1/2 and Staufen1/2 RNA-binding protein families in germinal center B cells and humoral immune responses.
We pinpointed individual RNA-binding proteins as powerful regulators of germinal center B cell biology and the production of antigen-specific antibodies and defined their regulated proteome. Through interaction proteomics and fluorescent sensors we identified novel RBPs regulating aspects of terminal B cell differentiation. We built a novel, fast and efficient in vivo genetic interrogation system for terminal B cell differentiation and conducted a genetic screen for RNA-binding protein functions in germinal center B cells. We aim to deliver a global functional classification of RNA-binding proteins in germinal center B cells and a mechanistic elucidation of the roles of key members of this protein family.