Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates.

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.

MAxSIM: multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells.

Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.

Atypical B cells in chronic infectious diseases and systemic autoimmunity: puzzles with many missing pieces.

The world's struggle to contain the SARS-CoV-2 epidemic, primarily through vaccination, has highlighted the importance of better understanding the biology of B cells that participate in defense against infectious diseases, both acute and chronic. Here, we focus on a population of human B cells, termed atypical B cells (ABCs), that comprise a distinct B-cell lineage that differentiates from naive B cells in an interferon-γ-driven process, and are infrequent in healthy individuals but significantly expanded in chronic infectious diseases, including malaria, as well as in systemic autoimmune diseases such as systemic lupus erythematosus (SLE). Recent comparisons of ABCs by single-cell RNAseq provided evidence that ABCs in diverse chronic infectious diseases and in systemic autoimmune diseases are highly related and share common drivers of differentiation and expansion. However, ABCs in different diseases are not identical and also show discrete disease-specific features. Here, we compare and contrast key features of two ABC populations, namely those that are expanded in individuals living in malaria-endemic areas of the world versus those in SLE patients. This comparison is of interest as it appears that unique features of these two diseases result in participation of autoreactive ABCs in parasite-specific responses in malaria but in pathogenic autoimmune responses in SLE. A better understanding of the commonality and differences in the ABC responses in these two diseases may provide critical insights into the development of vaccines that drive pathogen-specific antibody responses and avoid autoimmunity.

Shared transcriptional profiles of atypical B cells suggest common drivers of expansion and function in malaria, HIV, and autoimmunity.

Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD+IgMlo and IgD-IgG+ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

Analysis of B Cell Receptor-Mediated Antigen Extraction by B Lymphocytes from Plasma Membrane Sheets Using Confocal Microscopy.

High-resolution confocal imaging has provided new insights in the process of receptor-mediated endocytosis in variety of cell types. We describe here the protocol for investigating B cell receptor (BCR)-mediated internalization of membrane bound antigens using confocal microscopy. We describe the method to prepare plasma membrane sheets (PMS) in a small area, bind fluorescently tagged antigens to the PMS and activate B cells on the PMS. We also describe the method for analyzing antigen internalization using confocal microscopy and computational image analysis. This protocol is useful for the study of antigen internalization by B cells and can be applied for studying receptor-mediated endocytosis in other cells as well. The setup we describe here is especially useful for studying rare cell types when the number of cells available is limiting.

Expression of inhibitory receptors by B cells in chronic human infectious diseases restricts responses to membrane-associated antigens.

Chronic human infectious diseases, including malaria, are associated with a large expansion of a phenotypically and transcriptionally distinct subpopulation of B cells distinguished by their high expression of a variety of inhibitory receptors including FcγRIIB. Because these B cells, termed atypical memory B cells (MBCs), are unable to respond to soluble antigens, it was suggested that they contributed to the poor acquisition of immunity in chronic infections. Here, we show that the high expression of FcγRIIB restricts atypical MBC responses to membrane-associated antigens that function to actively exclude FcγRIIB from the B cell immune synapse and include the co-receptor CD19, allowing B cell antigen receptor signaling and differentiation toward plasma cells. Thus, chronic infectious diseases result in the expansion of B cells that robustly respond to antigens that associate with cell surfaces, such as antigens in immune complexes, but are unable to respond to fully soluble antigens, such as self-antigens.

Exhaustion may not be in the human B cell vocabulary, at least not in malaria.

T cells exposed to persistent antigen in the inflammatory environment of chronic infections often show progressive loss of effector functions, high expression of inhibitory receptors and distinct transcriptional programs. T cells in this functional state are termed "exhausted" and T cell exhaustion is associated with inefficient control of infections. A remarkably similar scenario has been described for B cells during chronic infections in humans, including malaria, in which case a subpopulation of atypical memory B cells (MBCs) greatly expands and these MBCs show attenuation of B cell receptor signaling, loss of the B cell effector functions of antibody and cytokine production, high expression of inhibitory receptors and distinct transcriptional profiles. The expansion of these MBCs is also associated with inefficient control of infections. Despite the similarities with exhausted T cells we speculate that at least in malaria, atypical MBCs may not be exhausted but rather may be functional, possibly even beneficial. Our recent results suggest that we simply may not have known how to ask an atypical MBC to function. Thus, exhaustion may not be in the human B cell's vocabulary, at least not in malaria.

The Differentiation in vitro of Human Tonsil B Cells With the Phenotypic and Functional Characteristics of T-bet+ Atypical Memory B Cells in Malaria.

Malaria is a deadly infectious disease associated with fundamental changes in the composition of the memory B cell (MBC) compartment, most notably a large expansion of T-bet+ MBCs, termed atypical MBCs. However, we know little about the precursors of atypical MBCs and the conditions that drive their differentiation. We compared the responses of human tonsil naïve B cells, MBCs, and germinal center B cells to a variety of stimulatory conditions. We determined that prolonged antigen presentation in the presence of CpG and IFN-γ induced maximal expression of T-bet and other phenotypic markers of malaria-associated atypical MBCs primarily in naïve B cells in vitro. Importantly T-bet+ naïve-derived B cells resembled atypical MBCs in their hypo-responsiveness to signaling through their B cell receptors. Thus, naïve B cells can be induced to differentiate into phenotypically and functionally atypical-like MBCs in vitro under conditions that may prevail in chronic infectious diseases in vivo.

Malaria-induced interferon-γ drives the expansion of Tbethi atypical memory B cells.

Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21-CD27- 'atypical' memory B cells (MBCs) that exhibit reduced B cell receptor (BCR) signaling and effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet, and that T-bet expression correlates inversely with BCR signaling and skews toward IgG3 class switching. Moreover, a longitudinal analysis of a subset of children suggested a correlation between the incidence of febrile malaria and the expansion of T-bethi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs plus BCR cross linking induced T-bet expression in naïve B cells that was abrogated by neutralizing IFN-γ or blocking the IFN-γ receptor on B cells. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsillar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data provide new insight into the mechanisms underlying atypical MBC differentiation.

Coordination of planar cell polarity pathways through Spiny-legs.

Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. We have characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, we define where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP, and provide a new understanding of the orientation of polarity and the basis of PCP phenotypes. Our results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals.

Signal transduction by the Fat cytoplasmic domain.

The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1ε encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.

Propagation of Dachsous-Fat planar cell polarity.

The Fat pathway controls both planar cell polarity (PCP) and organ growth. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP, whereas their slope can influence growth. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.