Universal characterization of epitope immunodominance from a multiscale model of clonal competition in germinal centers.

We introduce a multiscale model for affinity maturation, which aims to capture the intraclonal, interclonal, and epitope-specific organization of the B-cell population in a germinal center. We describe the evolution of the B-cell population via a quasispecies dynamics, with species corresponding to unique B-cell receptors (BCRs), where the desired multiscale structure is reflected on the mutational connectivity of the accessible BCR space, and on the statistical properties of its fitness landscape. Within this mathematical framework, we study the competition among classes of BCRs targeting different antigen epitopes, and we construct an effective immunogenic space where epitope immunodominance relations can be universally characterized. We finally study how varying the relative composition of a mixture of antigens with variable and conserved domains allows for a parametric exploration of this space, and we identify general principles for the rational design of two-antigen cocktails.

Formation of the junctions between lymph follicles in the Peyer's patches even before postweaning activation.

Peyer's patches (PPs), which contain an abundance of B and T cells, play a key role in inducing pivotal immune responses in the intestinal tract. PPs are defined as aggregated lymph follicles, which consist of multiple lymph follicles (LFs) that may interact with each other in a synergistic manner. LFs are thought to be spherical in shape; however, the characteristics of their structure are not fully understood. To elucidate changes in the structure of PPs as individuals grow, we generated serial 2D sections from entire PPs harvested from mice at 2, 4, and 10 weeks of age and performed a 3D analysis using a software, Amira. Although the number of LFs in PPs was not changed throughout the experiment, the volume and surface area of LFs increased significantly, indicating that LFs in PPs develop continuously by recruiting immune cells, even after weaning. In response to the dramatic changes in the intestinal environment after weaning, the development of germinal centers (GCs) in LFs was observed at 4 and 10 weeks (but not 2 weeks) of age. In addition, GCs gradually began to form away from the center of LFs and close to the muscle layer where export lymphatic vessels develop. Importantly, each LF was joined to the adjacent LF; this feature was observed even in preweaning nonactivated PPs. These results suggest that PPs may have a unique organization and structure that enhance immune functions, allowing cells in LFs to have free access to adjacent LFs and egress smoothly from PPs to the periphery upon stimulation after weaning.

Highlight of 2023: Advances in germinal centers.

In this article for the Highlight of 2023 series, we discuss recent advances in the fundamental biology of the germinal center response. These discoveries provide important insights as to how the germinal center contributes to protection against infection, and also highlights opportunities for future vaccine development.

B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles.

B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.

B cells! Don't go the wrong way in this tumor.

The association of tertiary lymphoid structures (TLSs) with survival and immunotherapy response brought B cells to center stage. In a pan-cancer B cells atlas in Science, Ma et al. show that germinal center reaction generating anti-tumor antibody-secreting cells (ASCs) from B memory cells in mature TLSs co-exist in tumors with extra-follicular reaction generating auto-reactive ASCs from memory B cells in immature TLSs.

The Role of the Transcriptional Coactivator BOB.1/OBF.1 in Adaptive Immunity.

BOB.1/OBF.1 is a transcriptional coactivator involved in octamer-dependent transcription. Thereby, BOB.1/OBF.1 is involved in the transcriptional regulation of genes important for lymphocyte physiology. BOB.1/OBF.1-deficient mice reveal multiple B- and T-cell developmental defects. The most prominent defect of these mice is the complete absence of germinal centers (GCs) resulting in severely impaired T-cell-dependent immune responses. In humans, BOB.1/OBF.1 is associated with several autoimmune and inflammatory diseases but also linked to liquid and solid tumors. Although its role for B-cell development is relatively well understood, its exact role for the GC reaction and T-cell biology has long been unclear. Here, the contribution of BOB.1/OBF.1 for B-cell maturation is summarized, and recent findings regarding its function in GC B- as well as in various T-cell populations are discussed. Finally, a detailed perspective on how BOB.1/OBF.1 contributes to different pathologies is provided.

In Vivo CRISPR/Cas9-Mediated Gene Ablation in Murine B Cells.

CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.

Heterologous booster vaccination enhances antibody responses to SARS-CoV-2 by improving Tfh function and increasing B-cell clonotype SHM frequency.

Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2-specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV-2 vaccines that induce more robust antibody response.

Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

IL-12 induces a B cell-intrinsic IL-12/IFNγ feed-forward loop promoting extrafollicular B cell responses.

While some infections elicit germinal centers, others produce only extrafollicular responses. The mechanisms controlling these dichotomous fates are poorly understood. We identify IL-12 as a cytokine switch, acting directly on B cells to promote extrafollicular and suppress germinal center responses. IL-12 initiates a B cell-intrinsic feed-forward loop between IL-12 and IFNγ, amplifying IFNγ production, which promotes proliferation and plasmablast differentiation from mouse and human B cells, in synergy with IL-12. IL-12 sustains the expression of a portion of IFNγ-inducible genes. Together, they also induce unique gene changes, reflecting both IFNγ amplification and cooperative effects between both cytokines. In vivo, cells lacking both IL-12 and IFNγ receptors are more impaired in plasmablast production than those lacking either receptor alone. Further, B cell-derived IL-12 enhances both plasmablast responses and T helper 1 cell commitment. Thus, B cell-derived IL-12, acting on T and B cells, determines the immune response mode, with implications for vaccines, pathogen protection and autoimmunity.

Maturation of germinal center B cells after influenza virus vaccination in humans.

Germinal centers (GC) are microanatomical lymphoid structures where affinity-matured memory B cells and long-lived bone marrow plasma cells are primarily generated. It is unclear how the maturation of B cells within the GC impacts the breadth and durability of B cell responses to influenza vaccination in humans. We used fine needle aspiration of draining lymph nodes to longitudinally track antigen-specific GC B cell responses to seasonal influenza vaccination. Antigen-specific GC B cells persisted for at least 13 wk after vaccination in two out of seven individuals. Monoclonal antibodies (mAbs) derived from persisting GC B cell clones exhibit enhanced binding affinity and breadth to influenza hemagglutinin (HA) antigens compared with related GC clonotypes isolated earlier in the response. Structural studies of early and late GC-derived mAbs from one clonal lineage in complex with H1 and H5 HAs revealed an altered binding footprint. Our study shows that inducing sustained GC reactions after influenza vaccination in humans supports the maturation of responding B cells.

A branching stochastic evolutionary model of the B-cell repertoire.

We propose a stochastic framework to describe the evolution of the B-cell repertoire during germinal center (GC) reactions. Our model is formulated as a multitype age-dependent branching process with time-varying immigration. The immigration process captures the mechanism by which founder B cells initiate clones by gradually seeding GC over time, while the branching process describes the temporal evolution of the composition of these clones. The model assigns a type to each cell to represent attributes of interest. Examples of attributes include the binding affinity class of the B cells, their clonal family, or the nucleotide sequence of the heavy and light chains of their receptors. The process is generally non-Markovian. We present its properties, including as t → ∞ when the process is supercritical, the most relevant case to study expansion of GC B cells. We introduce temporal alpha and beta diversity indices for multitype branching processes. We focus on the dynamics of clonal dominance, highlighting its non-stationarity, and the accumulation of somatic hypermutations in the context of sequential immunization. We evaluate the impact of the ongoing seeding of GC by founder B cells on the dynamics of the B-cell repertoire, and quantify the effect of precursor frequency and antigen availability on the timing of GC entry. An application of the model illustrates how it may help with interpretation of BCR sequencing data.

Opposing effects of pre-existing antibody and memory T cell help on the dynamics of recall germinal centers.

Re-exposure to an antigen generates abundant antibody responses and drives the formation of secondary germinal centers (GCs). Recall GCs in mice consist almost entirely of naïve B cells, whereas recall antibodies derive overwhelmingly from memory B cells. Here, we examine this division between cellular and serum compartments. After repeated immunization with the same antigen, tetramer analyses of recall GCs revealed a marked decrease in the ability of B cells in these structures to bind the antigen. Boosting with viral variant proteins restored antigen binding in recall GCs, as did genetic ablation of primary-derived antibody-secreting cells through conditional deletion of Prdm1, demonstrating suppression of GC recall responses by pre-existing antibodies. In hapten-carrier experiments in which B and T cell specificities were uncoupled, memory T cell help allowed B cells with undetectable antigen binding to access GCs. Thus, antibody-mediated feedback steers recall GC B cells away from previously targeted epitopes and enables specific targeting of variant epitopes, with implications for vaccination protocols.

Activated PI3Kδ Specifically Perturbs Mouse Regulatory T Cell Homeostasis and Function Leading to Immune Dysregulation.

FOXP3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3Kδ is required for normal Treg development and function. However, the impacts of dysregulated PI3Kδ signaling on Treg function remain incompletely understood. In this study, we used a conditional mouse model of activated PI3Kδ syndrome to investigate the role of altered PI3Kδ signaling specifically within the Treg compartment. Activated mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation, as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-γ secretion, spontaneous germinal center responses, and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype, including increased PD-1 expression and reduced competitive fitness. Consistent with these findings, Treg-specific aPIK3CD mice mounted an elevated humoral response following immunization with a T cell-dependent Ag, which correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3Kδ activity is critical for Treg homeostasis and function, suggesting that PI3Kδ signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.

A multiscale spatial modeling framework for the germinal center response.

The germinal center response or reaction (GCR) is a hallmark event of adaptive humoral immunity. Unfolding in the B cell follicles of the secondary lymphoid organs, a GC culminates in the production of high-affinity antibody-secreting plasma cells along with memory B cells. By interacting with follicular dendritic cells (FDC) and T follicular helper (Tfh) cells, GC B cells exhibit complex spatiotemporal dynamics. Driving the B cell dynamics are the intracellular signal transduction and gene regulatory network that responds to cell surface signaling molecules, cytokines, and chemokines. As our knowledge of the GC continues to expand in depth and in scope, mathematical modeling has become an important tool to help disentangle the intricacy of the GCR and inform novel mechanistic and clinical insights. While the GC has been modeled at different granularities, a multiscale spatial simulation framework - integrating molecular, cellular, and tissue-level responses - is still rare. Here, we report our recent progress toward this end with a hybrid stochastic GC framework developed on the Cellular Potts Model-based CompuCell3D platform. Tellurium is used to simulate the B cell intracellular molecular network comprising NF-κB, FOXO1, MYC, AP4, CXCR4, and BLIMP1 that responds to B cell receptor (BCR) and CD40-mediated signaling. The molecular outputs of the network drive the spatiotemporal behaviors of B cells, including cyclic migration between the dark zone (DZ) and light zone (LZ) via chemotaxis; clonal proliferative bursts, somatic hypermutation, and DNA damage-induced apoptosis in the DZ; and positive selection, apoptosis via a death timer, and emergence of plasma cells in the LZ. Our simulations are able to recapitulate key molecular, cellular, and morphological GC events, including B cell population growth, affinity maturation, and clonal dominance. This novel modeling framework provides an open-source, customizable, and multiscale virtual GC simulation platform that enables qualitative and quantitative in silico investigations of a range of mechanistic and applied research questions on the adaptive humoral immune response in the future.

Germinal center versus extrafollicular responses in systemic autoimmunity: Who turns the blade on self?

Spontaneously formed germinal centers (GCs) have been reported in most mouse models of human autoimmune disease and autoimmune patients, and have long been considered a source of somatically-mutated and thus high affinity autoantibodies, but their role in autoimmunity is becoming increasingly controversial, particularly in the context of systemic autoimmune diseases like lupus. On the one hand, there is good evidence that some pathogenic lupus antibodies have acquired somatic mutations that increase affinity for self-antigens. On the other hand, recent studies that have genetically prevented GC formation, suggest that GCs are dispensable for systemic autoimmunity, pointing instead to pathogenic extrafollicular (EF) B-cell responses. Furthermore, several lines of evidence suggest germinal centers may in fact be somewhat protective in the context of autoimmunity. Here we review how some of the conflicting evidence arose, and current views on the role of GCs in autoimmunity, outlining mechanisms by which GC may eliminate self-reactivity. We also discuss recent advances in understanding extrafollicular B cell subsets that participate in autoimmunity.

Transcriptional regulation of Tfh dynamics and the formation of immunological synapses.

Inside germinal centers (GCs), antigen-specific B cells rely on precise interactions with immune cells and strategic localization between the dark and light zones to clonally expand, undergo affinity maturation, and differentiate into long-lived plasma cells or memory B cells. Follicular helper T (Tfh) cells, the key gatekeepers of GC-dependent humoral immunity, exhibit remarkable dynamic positioning within secondary lymphoid tissues and rely on intercellular interactions with antigen-presenting cells (APCs) during their differentiation and execution of B-cell-facilitating functions within GCs. In this review, we briefly cover the transcriptional regulation of Tfh cell differentiation and function and explore the molecular mechanisms governing Tfh cell motility, their interactions with B cells within GCs, and the impact of their dynamic behavior on humoral responses.

The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6.

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.

In Situ Characterization of Human Follicular Helper CD4 T Cells.

The development of an effective humoral response to pathogens and immunogens is a multiphase biological process, which is mediated by the coordinated function of specialized immune cell types in secondary lymphoid organs and particularly in T cell and follicular areas. More specifically, within the follicular/germinal center area, the orchestrated interplay between B cells, follicular helper CD4 T cells (Tfh), and stromal cells triggers a cascade of immune reactions leading to the development of memory B cells and plasma cells able to generate effective, antigen-specific antibodies. The role of Tfh cells in this process is critical. Given the need for vaccines capable to induce antibodies of high affinity, neutralizing activity, and durability, understanding the cellular and molecular mechanisms regulating Tfh cell development is of great importance. Here, we describe novel approaches for the comprehensive understanding of these cells and possible implications for future studies in vaccine development and the understanding of the pathogenesis of relevant diseases.

Interleukin-1 regulates follicular T cells during the germinal center reaction.

Introduction: Antibody production and the generation of memory B cells are regulated by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in germinal centers. However, the precise role of Tfr cells in controlling antibody production is still unclear. We have previously shown that both Tfh and Tfr cells express the IL-1R1 agonist receptor, whereas only Tfr cells express the IL-1R2 decoy and IL-1Ra antagonist receptors. We aimed to investigate the role of IL-1 receptors in the regulation of B cell responses by Tfh and Tfr. Methods: We generated mice with IL-1 receptors inactivated in Tfh or Tfr and measured antibody production and cell activation after immunisation. Results: While IL-1ß levels are increased in the draining lymph node after immunisation, antigen-specific antibody levels and cell phenotypes indicated that IL-1ß can activate both Tfh and Tfr cells through IL-1R1 stimulation. Surprisingly, expression of IL-1R2 and IL-1Ra on Tfr cells does not block IL-1 activation of Tfh cells, but rather prevents IL-1/IL-1R1-mediated early activation of Tfr cells. IL-1Rs also regulate the antibody response to autoantigens and its associated pathophysiology in an experimental lupus model. Discussion: Collectively, our results show that IL-1 inhibitory receptors expressed by Tfr cells prevent their own activation and suppressive function, thus licensing IL-1-mediated activation of Tfh cells after immunisation. Further mechanistic studies should unravel these complex interactions between IL-1ß and follicular helper and regulatory T cells and provide new avenues for therapeutic intervention.