Highly enriched germinal center (GC) B cell populations are essential for studying humoral immunity. Current MACS protocols that isolate untouched GC B cells require GC induction and typically require further FACS purification with direct antibody labeling to achieve sufficiently high purities. We present a MACS protocol with progressive and repeated negative selections that yields highly purified untouched GC B cells from both unimmunized and GC-induced mice and allows further FACS isolation of unlabeled GC B cells from remaining debris by scatter.

Germinal centers (GCs) are specialized structures in which B lymphocytes undergo clonal expansion, class switch recombination, somatic hypermutation, and affinity maturation. Although these structures were previously thought to contain a limited number of isolated B cell clones, recent in vivo imaging studies revealed that they are in fact dynamic and appear to be open to their environment. We demonstrate that B cells can colonize heterologous GCs. Invasion of primary GCs after subsequent immunization is most efficient when T cell help is shared by the two immune responses; however, it also occurs when the immune responses are entirely unrelated. We conclude that GCs are dynamic anatomical structures that can be reutilized by newly activated B cells during immune responses.

Higher- or lower-affinity germinal center (GC) B cells are directed either to plasma cell or GC recycling, respectively; however, how commitment to the plasma cell fate takes place is unclear. We found that a population of light zone (LZ) GC cells, Bcl6loCD69hi expressing a transcription factor IRF4 and higher-affinity B cell receptors (BCRs) or Bcl6hiCD69hi with lower-affinity BCRs, favored the plasma cell or recycling GC cell fate, respectively. Mechanistically, CD40 acted as a dose-dependent regulator for Bcl6loCD69hi cell formation. Furthermore, we found that expression of intercellular adhesion molecule 1 (ICAM-1) and signaling lymphocytic activation molecule (SLAM) in Bcl6loCD69hi cells was higher than in Bcl6hiCD69hi cells, thereby affording more stable T follicular helper (Tfh)-GC B cell contacts. These data support a model whereby commitment to the plasma cell begins in the GC and suggest that stability of Tfh-GC B cell contacts is key for plasma cell-prone GC cell formation.

This data is related to the research article entitled "Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy" (Harper et al., 2016) [2]. The data presented here focuses on the humoral autoimmune response triggered by transferred allogeneic CD4 T cells and includes details on: (a) the recipient splenic germinal center (GC) response; (b) augmentation of humoral autoimmunity and accelerated heart allograft rejection following transplantation from donors primed against recipient; (c) flow cytometric analysis of donor and recipient CD4 T cells for signature markers of T follicular helper cell differentiation; (d) in vitro donor endothelial cell migration in response to column purified autoantibody from recipient sera; (e) analysis of development of humoral responses in recipients following adoptive transfer of donor CD4 T cells and; (f) the development of humoral autoimmunity in mixed haematopoietic chimeric mice.

No abstract available

TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa) cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa) or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

CD4+ T follicular helper (TFH) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2-dependent cDC2s, but not Batf3-dependent cDC1s or Klf4-dependent cDC2s, in promoting TFH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes This effect was mediated independent of Il2ra and several Notch2-dependent genes expressed in cDC2s, including Stat4 and Havcr2 Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2-dependent cDC2 subset in supporting humoral immune responses.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.

Tingible body macrophages in lymph node are involved in cleaning up debris from apoptotic B cells. Gurwisz et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20222173) and Grootveld et al. (2023. Cell.https://doi.org/10.1016/j.cell.2023.02.004) report how tingible body macrophages, originating from tissue-resident macrophages, clear apoptotic B cells in the germinal center using a "stand-hunting" strategy.

Epigenetic heterogeneity is emerging as a feature of tumors. In diffuse large B-cell lymphoma (DLBCL), increased cytosine methylation heterogeneity is associated with poor clinical outcome, yet the underlying mechanisms remain unclear. Activation-induced cytidine deaminase (AICDA), an enzyme that mediates affinity maturation and facilitates DNA demethylation in germinal center (GC) B cells, is required for DLBCL pathogenesis and linked to inferior outcome. Here we show that AICDA overexpression causes more aggressive disease in BCL2-driven murine lymphomas. This phenotype is associated with increased cytosine methylation heterogeneity, but not with increased AICDA-mediated somatic mutation burden. Reciprocally, the cytosine methylation heterogeneity characteristic of normal GC B cells is lost upon AICDA depletion. These observations are relevant to human patients, since DLBCLs with high AICDA expression manifest increased methylation heterogeneity vs. AICDA-low DLBCLs. Our results identify AICDA as a driver of epigenetic heterogeneity in B-cell lymphomas with potential significance for other tumors with aberrant expression of cytidine deaminases.

Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4(+) follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.

T follicular regulatory cells, or Tfr cells, are a discernable population of regulatory T (Treg) cells that migrate to the B cell follicle and germinal center (GC) upon immune challenge. These cells express the transcription factor Bcl6, the master regulator required for development and differentiation of T follicular helper cells, and are among a group of previously described Treg cells that use T helper cell-associated transcription factors to adapt their regulatory function to diverse milieus for maintenance of immune homeostasis. While there is consensus that Tfr cells control B-cell autoreactivity, it has been unclear whether they regulate productive, antigen-specific GC responses. Accordingly, understanding the regulatory balancing that Tfr cells play in maintenance of B-cell tolerance while optimizing productive humoral immunity is crucial for vaccine-design strategies. To this end, we discuss recent evidence that Tfr cells promote humoral immunity and memory following viral infections, fitting with the accepted role of Treg cells in maintaining homeostasis with promotion of productive immunity, while mitigating that which is potentially pathological. We also propose models in which Tfr cells regulate antigen-specific B cell responses.

Germinal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC.

The detailed mechanism driving the germinal center (GC) reaction to B cell lymphomagenesis has not been clarified. Thioredoxin interacting protein (TXNIP), also known as vitamin D3 up-regulated protein 1 which is an important tumor repressor, is involved in stress responses, redox regulation, and cellular proliferation. Here, we report that TXNIP has a potential role in the formation of GC in peripheral lymphoid organs where B lymphocytes divide rapidly. First, we compared changes in GC from wild type mice and Txnip(-/-) mice. After immunization, Txnip(-/-) mice exhibited higher expression level of BCL-6 and larger percentage of GC B cells with the reduction in antibody production and plasma cell numbers. In addition, Txnip(-/-) spleens had a much larger population which expressed Ki-67, a marker of cell proliferation, in the red pulp border than WT spleens. Furthermore, the expression of BCL-6 was decreased in TXNIP overexpressing cells and elevated in TXNIP deficient cells. Taken together, we conclude that TXNIP may contribute to the formation of GCs after immunization. During this process, TXNIP suppresses BCL-6 expression.

Human immunodeficiency virus (HIV) infection is characterized by dysfunctional B cell responses. Here we show that chronic simian immunodeficiency virus (SIV) infection is characterized by an expansion of either lymph node germinal center (GC) B cells that co-express Bcl6, Ki-67 and IL-21R and correlate with expanded T follicular helper (Tfh) cells or B cells that lack Bcl6, Ki-67 and IL-21R but express high levels of anti-apoptotic Bcl2 that negatively correlate with Tfh cells. The lack of Tfh cells likely contributes to persistence of dysfunctional non-proliferating B cells during chronic infection. These findings have implications for protective immunity in HIV-infected individuals who harbour low frequencies of Tfh cells.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd-/-) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 103 "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2Kd MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-Kd alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2Kd-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (TFH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a-/- TCR75 CD4 T cells that were genetically incapable of providing TFH cell function. The GC response was associated with affinity maturation of the anti-Kd alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by TFH cells. Clinical strategies that target the TFH cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection, we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infections do, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin, and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID mutant mice, chronic infection selects for GC B cells with hypermutated B cell receptors (BCRs) and neutralizing antibody formation. These findings demonstrate that, unlike for CD8+ T cells, chronic viral infection drives a functional, productive, and protective GC B cell response.

The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1 In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory.

Ectopic lymphoid structures form in a wide range of inflammatory conditions, including infection, autoimmune disease, and cancer. In the context of infection, this response can be beneficial for the host: influenza A virus infection-induced pulmonary ectopic germinal centers give rise to more broadly cross-reactive antibody responses, thereby generating cross-strain protection. However, despite the ubiquity of ectopic lymphoid structures and their role in both health and disease, little is known about the mechanisms by which inflammation is able to convert a peripheral tissue into one that resembles a secondary lymphoid organ. Here, we show that type I IFN produced after viral infection can induce CXCL13 expression in a phenotypically distinct population of lung fibroblasts, driving CXCR5-dependent recruitment of B cells and initiating ectopic germinal center formation. This identifies type I IFN as a novel inducer of CXCL13, which, in combination with other stimuli, can promote lung remodeling, converting a nonlymphoid tissue into one permissive to functional tertiary lymphoid structure formation.