Periodic Reporting for period 4 - PlasmaCellControl (Transcriptional control of plasma cell development and function)
Okres sprawozdawczy: 2022-07-01 do 2023-12-31
Plasma cells are essential for the acute response to infection and the long-term protection by providing humoral immunity through secretion of antibodies that recognize an almost unlimited number of pathogens. Activation of B cells by pathogens leads to short-lived plasma cells in lymphoid organs and long-lived memory plasma cells in the bone marrow that provide life-long protection by continuously secreting antibodies directed against previously encountered pathogens.
The differentiation of B cells to plasma cells is accompanied by massive reprogramming of gene expression. Some of the relevant key transcription factors are known, although their directly regulated target genes are so far elusive. The objective of the ERC project was to elucidate the gene-regulatory role of these regulators in the development and maintenance of plasma cells. Our experiments provided new molecular insight into the generation and function of plasma cells, which also contributed to a better understanding of how deregulated gene expression in plasma cells can lead to the development of disease such as multiple myeloma. By performing CRISPR-Cas9 knockout screens, we identified novel essential regulators of early plasma cell development. We also unraveled the molecular mechanism that is responsible for creating a broad antibody repertoire that is responsible to the generation of highly specific antibodies to fight all possible pathogens.
Plasma cells are the ultimate effector cells of humoral immunity that defend us against infection by a plethora of pathogens. Moreover, the memory plasma cells are responsible for the success of any vaccination strategy. In this sense, our basic research discoveries made important contributions to further our molecular understanding of plasma cells, which are of high medical importance, as they safeguard our health.
The differentiation of B cells to plasma cells is accompanied by massive reprogramming of gene expression. Some of the relevant key transcription factors are known, although their directly regulated target genes are so far elusive. The objective of the ERC project was to elucidate the gene-regulatory role of these regulators in the development and maintenance of plasma cells. Our experiments provided new molecular insight into the generation and function of plasma cells, which also contributed to a better understanding of how deregulated gene expression in plasma cells can lead to the development of disease such as multiple myeloma. By performing CRISPR-Cas9 knockout screens, we identified novel essential regulators of early plasma cell development. We also unraveled the molecular mechanism that is responsible for creating a broad antibody repertoire that is responsible to the generation of highly specific antibodies to fight all possible pathogens.
Plasma cells are the ultimate effector cells of humoral immunity that defend us against infection by a plethora of pathogens. Moreover, the memory plasma cells are responsible for the success of any vaccination strategy. In this sense, our basic research discoveries made important contributions to further our molecular understanding of plasma cells, which are of high medical importance, as they safeguard our health.
Generation of a broad antibody repertoire.
B cells express a broad repertoire of B-cell antigen receptors (BCRs), that can recognize almost any pathogen, and, upon pathogen encounter, develop into plasma cells secreting high-affinity antibodies to neutralize the pathogen. This broad BCR repertoire is generated by V(D)J recombination of the immunoglobulin heavy-chain (Igh) and kappa light-chain (Igk) genes in pro-B and pre-B cells, respectively. It was unknown how the V genes at these large loci (3 Mb) can participate in V(D)J recombination. In 2020 (Hill et al., Nature 584, 142-147), we published that the Igh locus undergoes extended cohesin-mediated loop extrusion across its entire length in pro-B cells, as these extra-long loops can be generated due to the Pax5-dependent downregulation of the cohesin-release factor Wapl. These extended loops allow all V genes across the Igh locus to participate in V-to-DJ recombination.
In a follow-up study (Hill et al., 2023, Nat. Commun. 14, 2316), we showed that only small loops can be generated in pre-B cells due to high Wapl expression. Consequently, the Igk locus undergoes contraction by folding into many small loops, which facilitates the participation of all V genes in V-J recombination at the Igk locus. These studies unraveled the molecular mechanisms that are responsible for generating a highly diverse antibody repertoire.
Conditional analysis of transcription factor function.
As Bhlha15 is not essential for plasma cells, we used conditional mutagenesis to demonstrate that Bhlha15 restrains the expression of the key regulator Blimp1 in plasma cells (Wöhner et al., 2022, Front. Immunol. 13, 859598).
As high-affinity plasma cells develop from germinal center (GC) B cells, conditional mutagenesis of Bhlhe40 revealed that this regulator restrains the generation of GC B cells and plasma cells (Rauschmeier et al., 2021, J. Exp. Med. 219, e20211406).
B cell activation leads to the generation of memory B, GC B and plasma cells. We discovered that limited access of activated B cells to antigen drives the generation of early memory B cells, while restraining plasma cell differentiation (Glaros et al., 2021, Immunity 14, 2005-2023).
Analysis of Pax5 in B cells revealed an essential role of this regulator in PI3K signaling. Consequently, conditional inactivation of Pax5 resulted in the loss of both GC B cells and plasma cells (Calderón et al., 2021, Sci. Immunol. 6, eabg5003)
As Pax5 is not expressed in plasma cells, it became a dogma that the Pax5 repression is essential for plasma cell development to proceed. By ectopic overexpression of Pax5 in plasma cells, we could, however, demonstrate that functional plasma cells are still generated in the presence of Pax5 (Liu et al., 2020, J. Exp. Med. 217, e20200147).
Transcriptional control of plasma cells by Blimp1, Ikaros (Ikzf1), Aiolos (Ikzf3), E2A (Tcf3) and E2-2 (Tcf4). As these regulators are essential for plasma cell generation, we established the auxin-inducible degron (Aid) system to identifying direct target genes of these regulators in plasma cells by rapid protein degradation combined with nascent transcript analysis. We generated functional Blimp1(Aid), Ikzf1(Aid), Ikzf3(Aid), Tcf3(Aid) and Tcf4(Aid) alleles in addition to the Rosa26(Tir1-F74G) allele, which encodes the mutant E3 ligase adaptor Tir1-F74G that facilitates rapid protein degradation by the proteasome in mice injected with the auxin derivative 5-Ph-IAA. We next crossed the Rosa26(Tir1-F74G) allele into the Aid-tagged mouse strains. As we obtained the compound mutant strains late in 2022, we could perform the acute protein degradation and nascent transcript analyses only in 2023. These experiments revealed that Blimp1, Ikaros and Aiolos are dedicated repressors that silence B-cell-specific gene expression in plasma cells. In contrast, E2A and E2-2 function as transcriptional activators to induce plasma cell-specific gene expression (Fedl, Rauschmeier et al., 2024; Schwickert, Froussios et al., 2024, in preparation).
CRISPR-Cas9 screens to identify novel regulators of plasma cells. By performing a CRISPR-Cas9 knockout screen in in vitro differentiated plasma cells, we identified several genes with essential functions in plasma cells (Pinter et al., 2022, Front. Immunol. 13, 979606).
As plasma cells reside in vivo in specific survival niches, we next performed an ‘in vivo’ CRISPR-Cas9 screen by establishing an elaborate multi-step screening protocol. This ‘in vivo’ screen yielded new, exciting genes that play a critical role in the early phase of plasma cell differentiation (Calderón et al., 2024, in preparation).
B cells express a broad repertoire of B-cell antigen receptors (BCRs), that can recognize almost any pathogen, and, upon pathogen encounter, develop into plasma cells secreting high-affinity antibodies to neutralize the pathogen. This broad BCR repertoire is generated by V(D)J recombination of the immunoglobulin heavy-chain (Igh) and kappa light-chain (Igk) genes in pro-B and pre-B cells, respectively. It was unknown how the V genes at these large loci (3 Mb) can participate in V(D)J recombination. In 2020 (Hill et al., Nature 584, 142-147), we published that the Igh locus undergoes extended cohesin-mediated loop extrusion across its entire length in pro-B cells, as these extra-long loops can be generated due to the Pax5-dependent downregulation of the cohesin-release factor Wapl. These extended loops allow all V genes across the Igh locus to participate in V-to-DJ recombination.
In a follow-up study (Hill et al., 2023, Nat. Commun. 14, 2316), we showed that only small loops can be generated in pre-B cells due to high Wapl expression. Consequently, the Igk locus undergoes contraction by folding into many small loops, which facilitates the participation of all V genes in V-J recombination at the Igk locus. These studies unraveled the molecular mechanisms that are responsible for generating a highly diverse antibody repertoire.
Conditional analysis of transcription factor function.
As Bhlha15 is not essential for plasma cells, we used conditional mutagenesis to demonstrate that Bhlha15 restrains the expression of the key regulator Blimp1 in plasma cells (Wöhner et al., 2022, Front. Immunol. 13, 859598).
As high-affinity plasma cells develop from germinal center (GC) B cells, conditional mutagenesis of Bhlhe40 revealed that this regulator restrains the generation of GC B cells and plasma cells (Rauschmeier et al., 2021, J. Exp. Med. 219, e20211406).
B cell activation leads to the generation of memory B, GC B and plasma cells. We discovered that limited access of activated B cells to antigen drives the generation of early memory B cells, while restraining plasma cell differentiation (Glaros et al., 2021, Immunity 14, 2005-2023).
Analysis of Pax5 in B cells revealed an essential role of this regulator in PI3K signaling. Consequently, conditional inactivation of Pax5 resulted in the loss of both GC B cells and plasma cells (Calderón et al., 2021, Sci. Immunol. 6, eabg5003)
As Pax5 is not expressed in plasma cells, it became a dogma that the Pax5 repression is essential for plasma cell development to proceed. By ectopic overexpression of Pax5 in plasma cells, we could, however, demonstrate that functional plasma cells are still generated in the presence of Pax5 (Liu et al., 2020, J. Exp. Med. 217, e20200147).
Transcriptional control of plasma cells by Blimp1, Ikaros (Ikzf1), Aiolos (Ikzf3), E2A (Tcf3) and E2-2 (Tcf4). As these regulators are essential for plasma cell generation, we established the auxin-inducible degron (Aid) system to identifying direct target genes of these regulators in plasma cells by rapid protein degradation combined with nascent transcript analysis. We generated functional Blimp1(Aid), Ikzf1(Aid), Ikzf3(Aid), Tcf3(Aid) and Tcf4(Aid) alleles in addition to the Rosa26(Tir1-F74G) allele, which encodes the mutant E3 ligase adaptor Tir1-F74G that facilitates rapid protein degradation by the proteasome in mice injected with the auxin derivative 5-Ph-IAA. We next crossed the Rosa26(Tir1-F74G) allele into the Aid-tagged mouse strains. As we obtained the compound mutant strains late in 2022, we could perform the acute protein degradation and nascent transcript analyses only in 2023. These experiments revealed that Blimp1, Ikaros and Aiolos are dedicated repressors that silence B-cell-specific gene expression in plasma cells. In contrast, E2A and E2-2 function as transcriptional activators to induce plasma cell-specific gene expression (Fedl, Rauschmeier et al., 2024; Schwickert, Froussios et al., 2024, in preparation).
CRISPR-Cas9 screens to identify novel regulators of plasma cells. By performing a CRISPR-Cas9 knockout screen in in vitro differentiated plasma cells, we identified several genes with essential functions in plasma cells (Pinter et al., 2022, Front. Immunol. 13, 979606).
As plasma cells reside in vivo in specific survival niches, we next performed an ‘in vivo’ CRISPR-Cas9 screen by establishing an elaborate multi-step screening protocol. This ‘in vivo’ screen yielded new, exciting genes that play a critical role in the early phase of plasma cell differentiation (Calderón et al., 2024, in preparation).
Although it was known that Aid-dependent protein degradation can work in vivo in the mouse, we had to considerably adapt the protocol for the use of rapid degradation of 5 transcription factors in plasma cells in vivo (Fedl, Rauschmeier et al., 2024; Schwickert, Froussios et al., 2024, in preparation).
To identify novel physiologically relevant regulators of early plasma cell development, we designed a novel ‘in vivo’ CRISPR-Cas9 knockout screen in plasma cells by establishing an entirely new multi-step screening protocol (Calderón et al., 2024, in preparation).
To identify novel physiologically relevant regulators of early plasma cell development, we designed a novel ‘in vivo’ CRISPR-Cas9 knockout screen in plasma cells by establishing an entirely new multi-step screening protocol (Calderón et al., 2024, in preparation).