Regulation Of Germinal Center Research Articles

Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells

Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell-intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag-specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen-specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.

Novel heterozygous mutation in NFKB2 in a patient with predominantly antibody deficiency

Background: The Nuclear Factor-kappa B (NF-kB) signaling pathway plays a critical role in regulating a wide range of cellular processes such as immune function, inflammation, and tumor regulation. There are two major pathways that play a role in NF-kB activation: the canonical NF-kB1 pathway and the non-canonical NF-kB2 pathway. Abnormalities in non-canonical NF-κB2 signaling are linked with significant impairments in the immune system, mainly B cell maturation, antibody production, as well as T helper and regulatory T cell function through its effect on germinal center regulation. Methods: Our patient’s medical record was analyzed retrospectively, including her medical history, results from immune laboratory tests, and genetic analyses. Results: We present a 16-year-old female with a history of chronic cough complicated with episodes of hemoptysis and diagnosed with bronchiectasis secondary to common variable immunodeficiency. Whole exome sequencing analysis revealed a novel heterozygous variant in the NFKB2 gene (NM_001077494.3), c.931C>T resulting in p.Arg311Cys. Conclusion: The presence of NFKB2 mutations can lead to the development of early-onset common variable immunodeficiency. Statement of Novelty: We have identified a novel variant in the NFKB2 gene associated with antibody deficiency.

Germinal center B cells that acquire nuclear proteins are specifically suppressed by follicular regulatory T cells.

Follicular regulatory T cells (Tfr) restrict development of autoantibodies and autoimmunity while supporting high-affinity foreign antigen-specific humoral response. However, whether Tfr can directly repress germinal center (GC) B cells that acquire autoantigens is unclear. Moreover, TCR specificity of Tfr to self-antigens is not known. Our study suggests that nuclear proteins contain antigens specific to Tfr. Targeting of these proteins to antigen-specific B cells in mice triggers rapid accumulation of Tfr with immunosuppressive characteristics. Tfr then exert negative regulation of GC B cells with predominant inhibition of the nuclear protein-acquiring GC B cells, suggesting an important role of direct cognate Tfr-GC B cells interactions for the control of effector B cell response.

Pathologic and molecular insights in nodal T-follicular helper cell lymphomas.

T-follicular helper (TFH) cells are one of the T-cell subsets with a critical role in the regulation of germinal center (GC) reactions. TFH cells contribute to the positive selection of GC B-cells and promote plasma cell differentiation and antibody production. TFH cells express a unique phenotype characterized by PD-1hi, ICOShi, CD40Lhi, CD95hi, CTLAhi, CCR7lo, and CXCR5hi . Three main subtypes of nodal TFH lymphomas have been described: 1) angioimmunoblastic-type, 2) follicular-type, and 3) not otherwise specified (NOS). The diagnosis of these neoplasms can be challenging, and it is rendered based on a combination of clinical, laboratory, histopathologic, immunophenotypic, and molecular findings. The markers most frequently used to identify a TFH immunophenotype in paraffin-embedded tissue sections include PD-1, CXCL13, CXCR5, ICOS, BCL6, and CD10. These neoplasms feature a characteristic and similar, but not identical, mutational landscape with mutations in epigenetic modifiers (TET2, DNMT3A, IDH2), RHOA, and T-cell receptor signaling genes. Here, we briefly review the biology of TFH cells and present a summary of the current pathologic, molecular, and genetic features of nodal lymphomas. We want to highlight the importance of performing a consistent panel of TFH immunostains and mutational studies in TCLs to identify TFH lymphomas.

B cell- and T cell-intrinsic regulation of germinal centers by thymic stromal lymphopoietin signaling.

Long-lived and high-affinity antibodies are derived from germinal center (GC) activity, but the cytokines that regulate GC function are still being identified. Here, we show that thymic stromal lymphopoietin (TSLP) signaling regulates the GC and the magnitude of antigen-specific antibody responses. Both GC B cells and T follicular helper (TFH) cells up-regulate the expression of surface TSLP receptor (TSLPR), but cell-specific loss of TSLPR results in distinct effects on GC formation and antibody production. TSLPR signaling on T cells supports the retention of antigen-specific B cells and TFH differentiation, whereas TSLPR in B cells regulates the generation of antigen-specific memory B cells. TSLPR in both cell types promotes interferon regulatory factor 4 (IRF4) expression, which is important for efficient GC activity. Overall, we identified a previously unappreciated cytokine regulator of GCs and identified how this signaling pathway differentially regulates B and T cell responses in the GC.

Analysis of the In Vivo Function of Follicular Regulatory T (TFR) Cells in the Regulation of Antibody Response.

Follicular regulatory T (TFR) cells, a subset of Foxp3+ regulatory T cells (Tregs), play an essential role in the regulation of germinal center (GC) response and humoral self-tolerance. Although it is generally accepted that TFR cells suppress GC antibody responses mediated by follicular helper T (TFH) cells and B cells, the helper activity of TFR cells on GC responses has also been recently reported. Because of this, it is critical to develop specific assays that are able to precisely assess TFR cell function, particularly its in vivo activity, independent of differentiation and other Tregs. Here we describe an adoptive transfer approach in conjugation with flow cytometry and ELISA to evaluate the TFR cell function on TFH, B cells, and antibody response in vivo.

Bach2 regulates B cell survival to maintain germinal centers and promote B cell memory

The transcription factor Bach2 serves as a crucial regulator of the germinal center (GC) reaction, which is required for production of high-affinity antibodies and establishment of long-lived B cell memory. However, the stage at which Bach2 controls the GC programs and the precise mechanism underlying these processes remain poorly understood. In this study, we show that genetic ablation of Bach2 in GC B cells of mice impairs their survival and maintenance, and memory B cell formation. These defects can be rescued by enforced expression of anti-apoptotic gene Bcl2. As expected, Bach2-deficient GC B cells are defective in antibody affinity maturation, but have normal somatic hyper mutation and class switch recombination of immunoglobulin genes. Mechanistically, Bach2 controls the GC programs by directly repressing pro-apoptotic gene Bim and a set of genes involved in cell stress response and metabolic processes. Thus, our work reveals the precise roles of Bach2 in the GC biology, and demonstrates that Bach2 acts as a crucial survival regulator of GC B cells, providing a key mechanism underlying GC B maintenance and B cell memory formation.

Mettl14-Mediated m6A Modification Is Essential for Germinal Center B Cell Response.

The germinal center (GC) response is essential for generating memory B and long-lived Ab-secreting plasma cells during the T cell-dependent immune response. In the GC, signals via the BCR and CD40 collaboratively promote the proliferation and positive selection of GC B cells expressing BCRs with high affinities for specific Ags. Although a complex gene transcriptional regulatory network is known to control the GC response, it remains elusive how the positive selection of GC B cells is modulated posttranscriptionally. In this study, we show that methyltransferase like 14 (Mettl14)-mediated methylation of adenosines at the position N 6 of mRNA (N 6-methyladenosine [m6A]) is essential for the GC B cell response in mice. Ablation of Mettl14 in B cells leads to compromised GC B cell proliferation and a defective Ab response. Interestingly, we unravel that Mettl14-mediated m6A regulates the expression of genes critical for positive selection and cell cycle regulation of GC B cells in a Ythdf2-dependent but Myc-independent manner. Furthermore, our study reveals that Mettl14-mediated m6A modification promotes mRNA decay of negative immune regulators, such as Lax1 and Tipe2, to upregulate genes requisite for GC B cell positive selection and proliferation. Thus, our findings suggest that Mettl14-mediated m6A modification plays an essential role in the GC B cell response.

Dual Targeting of EZH2 and HDAC with Tazemetostat and Belinostat Promotes Immunogenicity in GC-DLBCL

Epigenetic remodeling is essential for the proper differentiation and function of germinal center (GC) B cells. Competing actions of histone acetyltransferases (HATs), such as CREBBP and EP300, and inhibitory histone deacetylases (HDACs) and methyltransferases (HMTs), such as EZH2, modulate the epigenomic program of GC B cells and control key activating and immunogenic processes, such as BCR and CD40 signaling, antigen processing and presentation, and cell cycle regulation. In line with the critical importance of epigenetic regulation of GC B cell function, inactivating mutations in CREBBPor EP300and gain-of-function mutations in EZH2 have been identified in 39% and 21% of GC B-cell like diffuse large B-cell lymphoma (GC-DLBCL), respectively. Strategies to target epigenetic dysfunction in GC-DLBCL have largely focused on indirectly restoring the activity of HATs through the use of histone deacetylase (HDAC) inhibitors, such as belinostat, and by directly inhibiting EZH2 with tazemetostat. Despite showing great promise in preclinical studies, single agent therapies with HDAC or EZH2 inhibitors have shown only modest efficacy in the clinic. Recently, our group demonstrated synergistic effects of dual therapy with EZH2 and HDAC inhibitors in inducing lethality of GC-DLBCL lines. As combination therapies begin to be tested in clinical trials, whether dual inhibition of EZH2 and HDAC is able to restore immunogenic features of GC-DLBCL and lead to enhanced T cell-mediated killing in vivo remains unknown. Here, we utilize transcriptomic and flow cytometric methods to assess whether epigenetic remodeling with tazemetostat (EZH2 inhibitor) and belinostat (HDAC inhibitor) can alter the immunogenicity of GC-DLBCL. We find that combination treatment with both tazemetostat and belinostat promotes an antigen processing and presentation program in GC-DLBCL lines and results in significant increases in MHC-I and MHC-II surface expression. This effect was only modestly appreciated with single agent treatment and required at least 4-7 days post-treatment to become apparent. Importantly, the increased MHC expression in response to combination therapy was not dependent on EZH2 or CREBBPmutation status, suggesting broad applications of dual therapy in patients with diverse mutation burdens. Furthermore, we found that combination treatment also altered surface expression of costimulatory molecules, such as CD80 and CD86, suggesting that tazemetostat and belinostat can modulate interactions with T cells in a multifaceted manner. These findings thus uncover that dual targeting of EZH2 and HDAC with tazemetostat and belinostat promotes antigen presentation pathways in GC-DLBCL and suggests that dual therapies could restore immunogenicity in GC-DLBCL and enhance immune-mediated killing of tumor cells.This work was funded, in part, by the ASH HONORS Award Summer Program. [Display omitted] DisclosuresAmengual: Seagen: Consultancy; Appia Pharmaceuticals: Research Funding; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau.

Dual role of CCL3 and follicular regulatory T cells in the control of germinal center response

Abstract Follicular regulatory T cells (Tfrs) have been reported to play multiple roles in the control of B cells response. They repress foreign antigen-specific germinal center (GC) B cells at the peak of GC response. At the same time, under some conditions they promote GC B cell cycling in IL-10 dependent fashion and ensure optimal affinity maturation. Our previous studies suggested that CCL3 produced by GC centrocytes (CCs) promoted their direct contacts with Tfr cells and negative regulation of GC B cells at the peak of GC response. However, which fraction of CCs upregulates CCL3 expression, which receptors on Tfrs may respond to CCL3, and the long-term effect of CCL3 on GCs has been unclear. Based on the single cell and bulk qPCR analysis we found that CCL3 is upregulated in about 10% of CCs that express Myc and are undergoing positive selection. qPCR and transwell analysis revealed expression and synergistic involvement of CCR5 and CCR1 chemokine receptors on Tfr cells in their chemotaxis to CCL3. Interestingly, both an adoptive transfer and mixed bone marrow chimeras models suggest that after the peak of GC response B cell-intrinsic production of CCL3 promotes their prolonged participation in GCs and B cell affinity maturation, as well as better memory and plasmablast response. We also showed that both the negative and positive regulation of GC B cells by CCL3 was dependent on the presence of Tregs. To summarize, our studies suggest that in addition to the role of CCL3 and Tregs in the modest repression of foreign antigen-specific B cells at the peak of GC response, it is also important for optimal selection of B cells in GCs. Further studies are underway to determine the role of Tfr cells specifically in the negative and positive CCL3-mediated regulation of GCs.

Compromised counterselection by FAS creates an aggressive subtype of germinal center lymphoma.

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh-B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma.

Abstract PO-22: Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma

Abstract Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B-cell lymphoma (DLBCL); however, the function of Fas in the GC remains controversial. Although GC-derived DLBCL has better overall outcomes to therapy than other DLBCL types, some cases are refractory and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes B-cell death in the light zone likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. We found that FAS alterations occurred most commonly in the GC-derived genetic subtype of DLBCL, EZB. FAS alterations in EZB were associated with inferior outcomes and an enrichment of Tfh cells. Alterations in FAS co-occurred with deficiency in HVEM and PD-1 ligands that regulate the Tfh-B cell interaction and were associated with increased diversity of B-cell receptor variable genes across samples. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in a distinct subtype of GC-derived lymphoma. Citation Format: Raud Razzaghi, Shreya Agarwal, Nikita Kotlov, Olga Plotnikova, Krystle Nomie, Da Wei Huang, George W. Wright, Moyi Li, Katsuyoshi Takata, Chen Yao, John J. O'Shea, James D. Phelan, Stefania Pittaluga, David W. Scott, Jagan R. Muppidi. Compromised counterselection by FAS creates a lethal subtype of germinal center lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-22.

SOSTDC1 for germinal center regulation

SOSTDC1 for germinal center regulation

In vivo Screen Identifies Zdhhc2 as a Critical Regulator of Germinal Center B Cell Differentiation.

Germinal center (GC) B cell differentiation is critical for the production of affinity-matured pathogen-specific antibodies, the dysregulation of which may lead to humoral immunodeficiency or autoimmunity. The development of an in vivo screening system for factors regulating GC B cell differentiation has been a challenge. Here we describe a small-scale in vivo screening system with NP-specific B1-8hi cells and a retroviral shRNA library targeting 78 candidate genes to search for B cell-intrinsic factors that specifically regulate GC B cell differentiation. Zdhhc2, a gene encoding palmitoyltransferase ZDHHC2 and highly expressed in GC B cells, is identified as a strong positive regulator of GC B cell differentiation. B1-8hi cells transduced with Zdhhc2-shRNA are severely compromised in differentiating into GC B cells. A further analysis of in vitro differentiated B cells transduced with Zdhhc2-shRNA shows that Zdhhc2 is critical for the proliferation and the survival of B cells stimulated by CD40L, BAFF, and IL-21 and consequently impacts on their differentiation into GC B cells and post-GC B cells. These studies not only identify Zdhhc2 as a novel regulator of GC B cell differentiation but also represent a proof of concept of in vivo screen for regulators of GC B cell differentiation.

Regulation of germinal center response by proinflammatory chemokine CCL3 and follicular regulatory T cells

Abstract Follicular regulatory T cells (Tfrs) have been reported to play multiple roles in the control of B cell responses. Tfr repress foreign antigen-specific B cells and plasma cells at the peak of germinal center (GC) response. At the same time they promote GC B cell cycling in IL10-dependent fashion and support optimal affinity maturation. Ex vivo Tfrs can act directly on both Tfh and GC B cells. However, relative contribution of direct Tfr-mediated control of Tfh cells vs GC B cells in vivo has been unclear. Our previous work suggested that GC B cell-intrinsic production of proinflammatory chemokine CCL3 promoted direct GC B cell contacts with Tfr cells and was important for modest negative regulation of B cells at the peak of GC response. In this follow up study we have addressed the role of CCL3 in the long-term regulation of GCs. We have also assessed, which subset of GC B cells upregulates production of CCL3 and which receptors on Tfr cells may respond to this chemokine. We found that in addition to the role of CCL3 in the modest repression of foreign antigen-specific B cells at the peak of GC response, it was also important for longer-term participation of B cells in GCs. Both the negative and positive regulation of GC B cells by CCL3 was dependent on the presence of Tregs. We also found that only about 10% of GC centrocytes that were Myc-positive upregulated expression of CCL3 and that Tfr chemotaxis to CCL3 chemokine ex vivo was dependent on both CCR5 and CCR1 chemokine receptors. Further studies are underway to determine the role of Tfr cells in the negative and positive CCL3-mediated regulation of GCs.

Metabolic Reprogramming Induces Germinal Center B Cell Differentiation Through Epigenetic Control of <i>Bcl6</i> Expression

The germinal center (GC) reaction is essential for long-lived humoral immunity. However, molecular requirements for the induction of Bcl6, the master regulator for GC B cell differentiation, remain unclear. Through screening for cytokines and other stimuli that regulate Bcl6 expression, we identified IL-4 as the strongest inducer. IL-4-signaling altered the metabolomic profile in activated B cells and induced accumulation of the TCA cycle intermediate α-ketoglutarate (αKG), which was required for epigenetic activation of the Bcl6 gene locus. Mechanistically, after IL-4-treatment, STAT6 bound to the known enhancers in the Bcl6 locus and recruited UTX, a demethylase for the repressive histone mark H3K27me3 that requires αKG as a cofactor. In turn, the H3K27me3 demethylation activated the enhancers and transcription of the Bcl6 gene. We propose that IL-4-medicated metabolic reprogramming in B cells is the key factor that regulates epigenetic activation of the master regulator of GC B cell differentiation.

Dissecting the role of proinflammatory chemokine CCL3 in the selection of germinal center B cells and in secondary antibody responses

Abstract Expression of CCL3 and CCL4 chemokines is upregulated in B cells upon crosslinking of B cell receptors and in germinal center (GC) centrocytes. However, the role of these proinflammatory chemokines in B cell responses is still unclear. Single cell qPCR analysis suggests that only a small fraction of GC centrocytes upregulates expression of CCL3/4. While B cell-intrinsic production of CCL3 promotes B cell interactions and negative control by follicular regulatory T cells at the peak of GC response, at the later stages of GC response it promotes B cells selection. Consistent with that, secondary antibody responses in CCL3 knockout mice are reduced. Our findings suggest that CCL3 is involved into multifaceted regulation of GC responses. Analyses of the mechanisms promoting both the negative and positive regulation of GC B cells in CCL3-dependent fashion are underway. These studies should be important for dissecting the role of CCL3 in normal immune responses and in B cell-originated lymphomas, where CCL3/4 are often elevated.

The role of ERK2 in germinal center B cell selection

Abstract The regulation of B cell activation and survival is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the coordination of several intracellular signaling pathways. Dysregulation of these pathways can lead to the production and survival of autoreactive memory B cells and plasma cells (PCs), resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is involved in the transduction of stimulating and apoptotic signals such as those received through the B cell receptor (BCR) and the Fas receptor (CD95), respectively. Extracellular signal regulated kinases 1 and 2 (ERK1/2), important effectors of this pathway, play a major role in B cell activation and survival. Though ERK2 specifically regulates the pro-apoptotic protein, Bmf, its role in regulating GC B cell selection is unknown. Our lab has developed a novel mouse model in which the deletion of Erk2 is accompanied by the expression of the fluorescent reporter protein, eYFP, allowing us to identify and isolate viable Erk2Δ B cells using flow cytometry. Preliminary data using this model has shown that peripheral Erk2Δ B cells express several surface markers associated with follicular T helper cell interactions and immunoglobulin class switching, such as DEC205 and CD80, respectively. Thus we hypothesize that ERK2 is a critical regulator of GC B cell selection. We are currently investigating the ability of Erk2Δ B cells to respond to various activating stimuli in vitro as well as to differentiate into antigen-specific PCs. Our results will contribute to a better understanding of the molecular programs involved in B cell selection, potentially offering new approaches to treating B cell-driven autoimmune diseases.

Regulation of the Germinal Center Response.

The germinal center (GC) is a specialized microstructure that forms in secondary lymphoid tissues, producing long-lived antibody secreting plasma cells and memory B cells, which can provide protection against reinfection. Within the GC, B cells undergo somatic mutation of the genes encoding their B cell receptors which, following successful selection, can lead to the emergence of B cell clones that bind antigen with high affinity. However, this mutation process can also be dangerous, as it can create autoreactive clones that can cause autoimmunity. Because of this, regulation of GC reactions is critical to ensure high affinity antibody production and to enforce self-tolerance by avoiding emergence of autoreactive B cell clones. A productive GC response requires the collaboration of multiple cell types. The stromal cell network orchestrates GC cell dynamics by controlling antigen delivery and cell trafficking. T follicular helper (Tfh) cells provide specialized help to GC B cells through cognate T-B cell interactions while Foxp3+ T follicular regulatory (Tfr) cells are key mediators of GC regulation. However, regulation of GC responses is not a simple outcome of Tfh/Tfr balance, but also involves the contribution of other cell types to modulate the GC microenvironment and to avoid autoimmunity. Thus, the regulation of the GC is complex, and occurs at multiple levels. In this review we outline recent developments in the biology of cell subsets involved in the regulation of GC reactions, in both secondary lymphoid tissues, and Peyer's patches (PPs). We discuss the mechanisms which enable the generation of potent protective humoral immunity whilst GC-derived autoimmunity is avoided.

Regulation of Subunit-Specific Germinal Center B Cell Responses to the HIV-1 Envelope Glycoproteins by Antibody-Mediated Feedback.

The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1.