Dynamic Regulation of Tfh Selection During the Germinal Center Reaction
The germinal center is a dynamic microenvironment wherein B cells expressing high affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting numbers of T follicular helper cells 1,2 . Although much is known about the mechanisms that control B cell selection in the germinal center, far less is understood about the clonal behavior of the T follicular helper cells that regulate this process. Here we report on the dynamic behavior of T follicular helper cell clones during the germinal center reaction. We find that like germinal center B cells, T follicular helper cells undergo antigen dependent selection throughout the germinal center reaction resulting in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal center leads to increased T follicular helper cell division. Competition between T follicular helper cell clones is mediated by T cell receptor affinity for peptide-MHC ligand. T cells expanding preferentially in the germinal center show increased expression of genes downstream of the T cell receptor, genes required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to dramatic remodeling of the functional T follicular helper cell repertoire during the germinal center reaction.
Tunable dynamics of B cell selection in gut germinal centers
Germinal centers (GCs), structures normally associated with B cell immunoglobulin (Ig) hypermutation and development of high-affinity antibodies upon infection or immunization, are present in gut-associated lymphoid organs of humans and mice under steady state. Gut-associated (ga)GCs can support antibody responses to enteric infections and immunization1. However, whether B cell selection and antibody affinity maturation can take place in face of the chronic and diverse antigenic stimulation characteristic of steady-state gaGCs is less clear2–8 . Combining multicolor “Brainbow” fate-mapping and single-cell Ig sequencing, we find that 5–10% of gaGCs from specific pathogen-free (SPF) mice contained highly-dominant “winner” clones at steady state, despite rapid turnover of GC B cells. Monoclonal antibodies (mAbs) derived from these clones showed increased binding to commensal bacteria compared to their unmutated ancestors, consistent with antigen-driven selection and affinity maturation. Frequency of highly-selected gaGCs was markedly higher in germ-free (GF) than in SPF mice, and winner B cells in GF gaGCs were enriched in public IgH clonotypes found across multiple individuals, indicating strong B cell receptor (BCR)-driven selection in the absence of microbiota. Vertical colonization of GF mice with a defined microbial consortium (Oligo-MM12) did not eliminate GF-associated clonotypes, yet induced a concomitant commensal-specific, affinity-matured B cell response
Class Switch Recombination Occurs Infrequently in Germinal Centers
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 BCR 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.
Myosin IIa Promotes Antibody Responses by Regulating B Cell Activation, Acquisition of Antigen, and Proliferation
B cell responses are regulated by antigen acquisition, processing, and presentation to helper T cells. These functions are thought to depend on contractile activity of non-muscle myosin IIa. Here, we show that B cell-specific deletion of the myosin IIa heavy chain reduced the numbers of bone marrow B cell precursors and splenic marginal zone, peritoneal B1b, and germinal center B cells. In addition, myosin IIa-deficient follicular B cells acquired an activated phenotype and were less efficient in chemokinesis and extraction of membrane-presented antigens. Moreover, myosin IIa was indispensable for cytokinesis. Consequently, mice with myosin IIa-deficient B cells harbored reduced serum immunoglobulin levels and did not mount robust antibody responses when immunized. Altogether, these data indicate that myosin IIa is a negative regulator of B cell activation but a positive regulator of antigen acquisition from antigen-presenting cells and that myosin IIa is essential for B cell development, proliferation, and antibody responses.
Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses
Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.
Germinal center B cells recognize antigen through a specialized immune synapse architecture
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 show that in contrast to naive and memory B cells, which gathered antigen towards 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 (PKC-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.
WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts
The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability.