Faculty Bibliography

Germinal centres, the structures in which B cells evolve to produce antibodies with high affinity for various antigens, usually form transiently in lymphoid organs in response to infection or immunization. In lymphoid organs associated with the gut, however, germinal centres are chronically present. These gut-associated germinal centres can support targeted antibody responses to gut infections and immunization¹. But whether B cell selection and antibody affinity maturation take place in the face of the chronic and diverse antigenic stimulation characteristic of these structures under steady state is less clear^2-8. Here, by combining multicolour 'Brainbow' cell-fate mapping and sequencing of immunoglobulin genes from single cells, we find that 5-10% of gut-associated germinal centres from specific-pathogen-free (SPF) mice contain highly dominant 'winner' B cell clones at steady state, despite rapid turnover of germinal-centre B cells. Monoclonal antibodies derived from these clones show increased binding, compared with their unmutated precursors, to commensal bacteria, consistent with antigen-driven selection. The frequency of highly selected gut-associated germinal centres is markedly higher in germ-free than in SPF mice, and winner B cells in germ-free germinal centres are enriched in 'public' clonotypes found in multiple individuals, indicating strong selection of B cell antigen receptors even in the absence of microbiota. Colonization of germ-free mice with a defined microbial consortium (Oligo-MM¹²) does not eliminate germ-free-associated clonotypes, yet does induce a concomitant commensal-specific B cell response with the hallmarks of antigen-driven selection. Thus, positive selection of B cells can take place in steady-state gut-associated germinal centres, at a rate that is tunable over a wide range by the presence and composition of the microbiota.

B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we found that, in contrast with naive and memory B cells, which gathered antigen toward the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β-NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T cell-dependent selection of high-affinity B cells in GCs.

BACKGROUND:The gut is an environment in which the immune system closely interacts with a vast number of foreign antigens, both inert such as food and alive, from the viral, bacterial, fungal and protozoal microbiota. Within this environment, germinal centres, which are microanatomical structures where B cells affinity-mature, are chronically present and active. MAIN BODY/METHODS:The functional mechanism by which gut-associated germinal centres contribute to gut homeostasis is not well understood. Additionally, the role of T cells in class switching to immunoglobulin A and the importance of B cell affinity maturation in homeostasis remains elusive. Here, we provide a brief overview of the dynamics of gut-associated germinal centres, T cell dependency in Immunoglobulin A class switching, and the current state of research regarding the role of B cell selection in germinal centres in the gut under steady-state conditions in gnotobiotic mouse models and complex microbiota, as well as in response to immunization and microbial colonization. Furthermore, we briefly link those processes to immune system maturation and relevant diseases. CONCLUSION/CONCLUSIONS:B cell response at mucosal surfaces consists of a delicate interplay of many dynamic factors, including the microbiota and continuous B cell influx. The rapid turnover within gut-associated germinal centres and potential influences of an early-life window of immune system imprinting complicate B cell dynamics in the gut.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H₂O₂, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H₂O₂ doses. Increased survival of wild-type agr cells during H₂O₂ exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H₂O₂. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb

How B cells assess risk in the intestine.

INTRODUCTION/UNASSIGNED:Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. RESULTS/UNASSIGNED:NK cells in the bone barrow and spleen. DISCUSSION/UNASSIGNED:subunit Kir6.1 has a key role in NK-cell development.

Follicular helper T cells (T_FH) mediate B cell selection and clonal expansion in germinal centers (GCs), and follicular regulatory T cells (T_FR) prevent the emergence of self-reactive B cells and help to extinguish the reaction. Here we show that GC reactions continually recruit T cells from both the naïve conventional and naive thymic regulatory T cell (Treg) repertoires. In the early GC, newly recruited T cells develop into T_FH, whereas cells entering during the contraction phase develop into T_FR cells that contribute to GC dissolution. The T_FR fate decision is associated with decreased antigen availability and is modulated by slow antigen delivery or mRNA vaccination. Thus, invasion of ongoing GCs by newly developing T_FH and T_FR helps remodel the GC based on antigen availability.

The gut immune system is shaped by the continuous interaction with the microbiota. Here we dissect temporal, spatial and contextual layers of gut B cell responses. The microbiota impacts on the selection of the developing pool of pre-immune B cells that serves as substrate for B cell activation, expansion and differentiation. However, various aspects of the gut B cell response display unique features. In particular, occurrence of somatically mutated B cells, chronic gut germinal centers in T cell-deficient settings and polyreactive binding of gut IgA to the microbiota questioned the nature and microbiota-specificity of gut germinal centers. We propose a model to reconcile these observations incorporating recent work demonstrating microbiota-specificity of gut germinal centers. We speculate that adjuvant effects of the microbiota might modify permissiveness for B cell to enter and exit gut germinal centers. We propose that separating aspects of time, space and place facilitate the occasionally puzzling discussion of gut B cell responses to the microbiota.

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells^1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.