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.

B cell responses to membrane-presented antigens require the function of the mechanosensitive cation channel Piezo1.

The demand for a vaccine for coronavirus disease 2019 (COVID-19) highlighted gaps in our understanding of the requirements for B cell responses to antigens, particularly to membrane-presented antigens, as occurs in vivo. We found that human B cell responses to membrane-presented antigens required the function of Piezo1, a plasma membrane mechanosensitive cation channel. Simply making contact with a glass probe induced calcium (Ca2+) fluxes in B cells that were blocked by the Piezo1 inhibitor GsMTx4. When placed on glass surfaces, the plasma membrane tension of B cells increased, which stimulated Ca2+ influx and spreading of B cells over the glass surface, which was blocked by the Piezo1 inhibitor OB-1. B cell responses to membrane-presented antigens but not to soluble antigens were inhibited both by Piezo1 inhibitors and by siRNA-mediated knockdown of Piezo1. Thus, the activation of Piezo1 defines an essential event in B cell activation to membrane-presented antigens that may be exploited to improve the efficacy of vaccines.

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.

Intrinsic properties of human germinal center B cells set antigen affinity thresholds.

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

IgG3 regulates tissue-like memory B cells in HIV-infected individuals.

Immunoglobulin G3 (IgG3) has an uncertain role in the response to infection with and vaccination against human immunodeficiency virus (HIV). Here we describe a regulatory role for IgG3 in dampening the immune system-activating effects of chronic HIV viremia on B cells. Secreted IgG3 was bound to IgM-expressing B cells in vivo in HIV-infected chronically viremic individuals but not in early-viremic or aviremic individuals. Tissue-like memory (TLM) B cells, a population expanded by persistent HIV viremia, bound large amounts of IgG3. IgG3 induced clustering of B cell antigen receptors (BCRs) on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR (IgM-BCR). The inhibitory IgG receptor CD32b (FcγRIIb), complement component C1q and inflammatory biomarker CRP contributed to the binding of secreted IgG3 onto IgM-expressing B cells of HIV-infected individuals. Notably, IgG3-bound TLM B cells were refractory to IgM-BCR stimulation, thus demonstrating that IgG3 can regulate B cells during chronic activation of the immune system.

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.

Toll-like receptor 9 antagonizes antibody affinity maturation.

Key events in T cell-dependent antibody responses, including affinity maturation, are dependent on the B cell's presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells' capture and presentation of antigen.

FcRL4 Expression Identifies a Pro-inflammatory B Cell Subset in Viremic HIV-Infected Subjects.

In autoimmune diseases, toll-like receptor (TLR)-stimulated pro-inflammatory IL-6-secreting B cells exert pathogenic roles. Similarly, B cell Fc receptor-like 4 (FcRL4) expression amplifies TLR stimulation, and in rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B cell subset. B cells from HIV-infected subjects also express heightened levels of FcRL4 and secrete high levels of IL-6: a critical mediator of HIV disease progression. In this study, we sought to determine if FcRL4 identifies a pro-inflammatory B cell subset in HIV-infected subjects and further elucidate the mechanisms underlying FcRL4 amplification of TLR stimulation. We determine that tissue-like memory B cells express the highest endogenous levels of FcRL4 positively correlating with IL-6 expression (p = 0.0022, r = 0.8667), but activated memory B cells exhibit the highest frequency of FcRL4hiIL-6hi cells. FcRL4hi B cells exhibit an activated TLR-signaling pathway identified by elevated phosphorylation levels of: pERK (p = 0.0373), p38 (p = 0.0337), p65 (p = 0.1097), and cJUN (p = 0.0239), concomitant with significantly elevated expression of the TLR-signaling modulator hematopoietic cell kinase (HcK, p = 0.0414). Compared to FcRL4neg B cells from healthy controls, TLR9-stimulated FcRL4pos B cells express significantly higher levels of lL-6 (p = 0.0179). Further, TLR9-stimulated B cells also upregulate the expression of FcRL4 (p = 0.0415) and HcK (p = 0.0386). In B-cell lines, siRNA-mediated HcK knockdown downmodulates TLR9-induced FcRL4-mediated activation quantified by CD23 upregulation (p = 0.0553). We present data suggesting that, in viremic HIV-infected individuals, FcRL4 expression identifies unique IL-6 producing pro-inflammatory B-cell subsets. Further, TLR stimulation likely modulates FcRL4 expression and FcRL4 expression is associated with Hck, potentially enhancing the activation of TLR-signaling associated transcription factors. Pathogenic B-cells have been identified in other disease settings, and this study represents a novel report describing a pro-inflammatory B cell subset in HIV-infected patients.

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.

Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function.

Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.

The autoinhibitory C-terminal SH2 domain of phospholipase C-γ2 stabilizes B cell receptor signalosome assembly.

The binding of antigen to the B cell receptor (BCR) stimulates the assembly of a signaling complex (signalosome) composed initially of the kinases Lyn, spleen tyrosine kinase (Syk), and Bruton's tyrosine kinase (Btk), as well as the adaptor protein B cell linker (BLNK). Together, these proteins recruit and activate phospholipase C-γ2 (PLC-γ2), a critical effector that stimulates increases in intracellular Ca(2+) and activates various signaling pathways downstream of the BCR. Individuals with one copy of a mutant PLCG2 gene, which encodes a variant PLC-γ2 that lacks the autoinhibitory C-terminal Src homology 2 (cSH2) domain, exhibit PLC-γ2-associated antibody deficiency and immune dysregulation (PLAID). Paradoxically, although COS-7 cells expressing the variant PLC-γ2 show enhanced basal and stimulated PLC-γ2 activity, B cells from PLAID patients show defective intracellular Ca(2+) responses upon cross-linking of the BCR. We found that the cSH2 domain of PLC-γ2 played a critical role in stabilizing the early signaling complex that is stimulated by BCR cross-linking. In the presence of the variant PLC-γ2, Syk, Btk, and BLNK were only weakly phosphorylated and failed to stably associate with the BCR. Thus, BCRs could not form stable clusters, resulting in dysregulation of downstream signaling and trafficking of the BCR. Thus, the cSH2 domain functions not only to inhibit the active site of PLC-γ2 but also to directly or indirectly stabilize the early BCR signaling complex.