Periodic Reporting for period 2 - PostTransGC (Post-transcriptional and post-translational mechanisms underlying B cell selection by T cells in germinal centers)
Okres sprawozdawczy: 2022-11-01 do 2024-04-30
Long-lasting protection against pathogens and robust responses to vaccination depend on the generation of high-affinity antibodies through cellular processes occurring in draining lymph nodes. Traditionally, investigations into these processes focus on two biological aspects: the analysis of genes influencing the formation of antibody-secreting cells and the examination of changes in cell location and movement using conventional immunological methods. In the context of this grant, our research examines events occurring post the changes in the cellular genetic program, establishing connections between genetic expression and cellular responses.
Given the critical role of antibody-mediated immune responses in establishing immunity against pathogens, understanding these molecular mechanisms becomes pivotal for designing more effective vaccines. Moreover, dysregulation in these processes can contribute to lymphoma development, and uncovering unknown checkpoints controlling the differentiation of antibody-forming cells could pave the way for new therapeutic drugs in clinical treatment. Notably, recent findings, including those from our group, reveal a correlation between the presence of antibody-forming cells in tumors and patient survival, though the functions of these cells remain unclear. The project's objective is to investigate events occurring after gene expression during the differentiation of antibody-forming cells. This encompasses alterations in RNA subsequent to its generation by specific genes and changes in proteins governing cell functions. Additionally, we integrate data on cellular localization with RNA modifications to establish links between immune niches and cell functions.
Given the critical role of antibody-mediated immune responses in establishing immunity against pathogens, understanding these molecular mechanisms becomes pivotal for designing more effective vaccines. Moreover, dysregulation in these processes can contribute to lymphoma development, and uncovering unknown checkpoints controlling the differentiation of antibody-forming cells could pave the way for new therapeutic drugs in clinical treatment. Notably, recent findings, including those from our group, reveal a correlation between the presence of antibody-forming cells in tumors and patient survival, though the functions of these cells remain unclear. The project's objective is to investigate events occurring after gene expression during the differentiation of antibody-forming cells. This encompasses alterations in RNA subsequent to its generation by specific genes and changes in proteins governing cell functions. Additionally, we integrate data on cellular localization with RNA modifications to establish links between immune niches and cell functions.
Aim 1. In-situ analyses of quantal gene transcription during B cell selection by T cells.
The first part focuses on the crucial process of B cell selection in germinal centers, where the generation of high-affinity antibodies is essential for effective protection against infections. While previous transcriptomic analyses and experiments in transgenic mice have revealed key genes involved in the germinal center response, they lack the temporal and spatial information required to understand gene expression dynamics in situ. To address this limitation, we developed a single-molecule fluorescence in situ hybridization (smFISH) approach that enabled us to precisely quantify changes in gene transcript levels within individual B cells during their interaction with T cells. This study is almost ready for publication.
Specifically, we report the following key findings:
Delivery of T cell help increases the number of Myc-positive cells over time, rather than the total amount of Myc transcripts per cell.
Furthermore, visualization of transcription start sites revealed that T cell help enhances the rate of Myc mRNA production through an increase in the frequency of transcriptional activity. Thus, the continuous burst activity of the Myc gene in response to T cell help controls B cell selection, rather than the total amount of Myc transcript at a given time point.
Combination of imaging and in situ gene expression analyses revealed that active Myc transcription primarily occurs at the edge of the light zone, a novel niche that we term the "light zone rim”.
Aim 2. Determine how post-transcriptional modifications through m6A machineries control GC B cell functions.
Our results establish the core principles of the m6A mechanism and functions in differentiated cells of the adaptive immune response under physiological conditions. (Two studies were published in the Journal of Experimental Medicine and Cell Reports)
1. Using three mouse strains in which the methyltransferase, Mettl3, was deleted at different time points during the immune response, we show that m6A is required for both early B cell activation and for a sustained germinal center reaction..
2. METTL3 functions are essential for expression of cell cycle-related genes, including Myc and MYC-related gene programs.
3. Myc mRNA is highly methylated, and METTL3 activity stabilizes Myc transcripts in GC B cells.
4. The typical m6A “reader” YTHDF2, which enhances degradation of methylated mRNAs, does not play a role in expression of Myc and MYC-related genes in the antibody immune response.
5. The atypical m6A binder IGF2BP3 was found to functionally phenocopy Mettl3 in the germinal center by maintaining effective levels of Myc transcripts and activating the Myc pathway.
Collectively, we conclude that Myc and Myc-related genes are controlled by specific m6A molecular modifications in the adaptive immune response.
In the second study, we show that:
1. Single-cell RNA-seq analyses of antigen-specific B cells revealed a pre-germinal center (GC) subpopulation that expresses high levels of RNA binding proteins, including YTHDF2, which degrades methylated mRNA.
2. YTHDF2-deficient B cells differentiate into pre-GC B cells in response to vaccination or viral infection but cannot differentiate into GC B cells.
3. RNA-seq and protein expression analyses revealed that YTHDF2-deficient pre-GC B cells express high levels of plasma cells genes, including IRF4 and XBP1, as well as high levels of secreted immunoglobulin transcripts.
4. Mechanistically, we found that transcripts of IRF4 and XBP1 were methylated and bound by YTHDF2, which led to these mRNAs degradation. This mechanism suppresses the expression of these genes in pre-GC B cells and is critical for promoting GC seeding and for an effiecient immune response.
Aim 3. Define how post-translational modification and protein degradation control B cell selection in GCs.
Using transgenic mouse models and proteomic screens, we revealed that the kinase DYRK1A and its target MSH6, a DNA mismatch repair protein, play a critical role in antibody-mediated protection from viral infection.
Specifically, we show that:
1. Dyrk1a-deficient B cells are unable to switch their antibody isotype in vitro, whereas cell activation and proliferation remain intact.
2. Proteomic and phospho-proteomic screens revealed a strong reduction in MSH6 phosphorylation in Dyrk1a-deficient B cells, whereas the total protein levels remained unchanged.
3. Kinase activity assay using recombinant proteins demonstrated that DYRK1A directly phosphorylates MSH6. Furthermore, deletion of a single phosphorylation site on MSH6 impaired class switch recombination.
4. B cell-specific deletion of Dyrk1a prevented class switch recombination in immunized mice without affecting germinal center formation.
5. Mice that lack expression of DYRK1A in their B cells do not survive a viral infection that depends on class-switched antibodies for effective protection.
Our study introduces a novel molecular mechanism critical for the antibody response against viral infection.
The first part focuses on the crucial process of B cell selection in germinal centers, where the generation of high-affinity antibodies is essential for effective protection against infections. While previous transcriptomic analyses and experiments in transgenic mice have revealed key genes involved in the germinal center response, they lack the temporal and spatial information required to understand gene expression dynamics in situ. To address this limitation, we developed a single-molecule fluorescence in situ hybridization (smFISH) approach that enabled us to precisely quantify changes in gene transcript levels within individual B cells during their interaction with T cells. This study is almost ready for publication.
Specifically, we report the following key findings:
Delivery of T cell help increases the number of Myc-positive cells over time, rather than the total amount of Myc transcripts per cell.
Furthermore, visualization of transcription start sites revealed that T cell help enhances the rate of Myc mRNA production through an increase in the frequency of transcriptional activity. Thus, the continuous burst activity of the Myc gene in response to T cell help controls B cell selection, rather than the total amount of Myc transcript at a given time point.
Combination of imaging and in situ gene expression analyses revealed that active Myc transcription primarily occurs at the edge of the light zone, a novel niche that we term the "light zone rim”.
Aim 2. Determine how post-transcriptional modifications through m6A machineries control GC B cell functions.
Our results establish the core principles of the m6A mechanism and functions in differentiated cells of the adaptive immune response under physiological conditions. (Two studies were published in the Journal of Experimental Medicine and Cell Reports)
1. Using three mouse strains in which the methyltransferase, Mettl3, was deleted at different time points during the immune response, we show that m6A is required for both early B cell activation and for a sustained germinal center reaction..
2. METTL3 functions are essential for expression of cell cycle-related genes, including Myc and MYC-related gene programs.
3. Myc mRNA is highly methylated, and METTL3 activity stabilizes Myc transcripts in GC B cells.
4. The typical m6A “reader” YTHDF2, which enhances degradation of methylated mRNAs, does not play a role in expression of Myc and MYC-related genes in the antibody immune response.
5. The atypical m6A binder IGF2BP3 was found to functionally phenocopy Mettl3 in the germinal center by maintaining effective levels of Myc transcripts and activating the Myc pathway.
Collectively, we conclude that Myc and Myc-related genes are controlled by specific m6A molecular modifications in the adaptive immune response.
In the second study, we show that:
1. Single-cell RNA-seq analyses of antigen-specific B cells revealed a pre-germinal center (GC) subpopulation that expresses high levels of RNA binding proteins, including YTHDF2, which degrades methylated mRNA.
2. YTHDF2-deficient B cells differentiate into pre-GC B cells in response to vaccination or viral infection but cannot differentiate into GC B cells.
3. RNA-seq and protein expression analyses revealed that YTHDF2-deficient pre-GC B cells express high levels of plasma cells genes, including IRF4 and XBP1, as well as high levels of secreted immunoglobulin transcripts.
4. Mechanistically, we found that transcripts of IRF4 and XBP1 were methylated and bound by YTHDF2, which led to these mRNAs degradation. This mechanism suppresses the expression of these genes in pre-GC B cells and is critical for promoting GC seeding and for an effiecient immune response.
Aim 3. Define how post-translational modification and protein degradation control B cell selection in GCs.
Using transgenic mouse models and proteomic screens, we revealed that the kinase DYRK1A and its target MSH6, a DNA mismatch repair protein, play a critical role in antibody-mediated protection from viral infection.
Specifically, we show that:
1. Dyrk1a-deficient B cells are unable to switch their antibody isotype in vitro, whereas cell activation and proliferation remain intact.
2. Proteomic and phospho-proteomic screens revealed a strong reduction in MSH6 phosphorylation in Dyrk1a-deficient B cells, whereas the total protein levels remained unchanged.
3. Kinase activity assay using recombinant proteins demonstrated that DYRK1A directly phosphorylates MSH6. Furthermore, deletion of a single phosphorylation site on MSH6 impaired class switch recombination.
4. B cell-specific deletion of Dyrk1a prevented class switch recombination in immunized mice without affecting germinal center formation.
5. Mice that lack expression of DYRK1A in their B cells do not survive a viral infection that depends on class-switched antibodies for effective protection.
Our study introduces a novel molecular mechanism critical for the antibody response against viral infection.
Currently, we are actively working on writing and submitting aim 1 findings for publication. Our efforts involve expanding the in situ analysis technique to investigate gene expression in B cells across various organs in vaccinated mouse models.
Simultaneously, we have initiated the exploration of other facets of aim 3 in our study. This involves delving into the analysis of Fbxw7 genes and scrutinizing Myc regulation at the protein level. Our upcoming investigations will include the examination of B cell immune responses in Fbxw7-deficient mice and animals characterized by overexpression of Myc, as outlined in the grant proposal.
Simultaneously, we have initiated the exploration of other facets of aim 3 in our study. This involves delving into the analysis of Fbxw7 genes and scrutinizing Myc regulation at the protein level. Our upcoming investigations will include the examination of B cell immune responses in Fbxw7-deficient mice and animals characterized by overexpression of Myc, as outlined in the grant proposal.