B Cell Biology

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Over the last year despite the severe disruptions caused by the COVID-19 pandemic, we continued to make important progress on our understanding of B cell signaling and memory generation. Understanding B cell responses to membrane associated antigens A fundamental feature of B cell responses to antigen in secondary lymphoid organs (SLOs) in vivo is the presentation of the antigen on the surface of FDCs. We compared the responses of human tonsil naive B cells to antigen associated with membranes, in solution or displayed as a virus-like particle in vitro. 1) The role of PIEZO in B cell responses. We discovered that human B cells express the mechano-sensitive membrane calcium channel PIEZO. Using Fluorescence Lifetime Image Microscopy we showed that when B cells interact with antigen on membranes, the B cell membrane is stretched and PIEZO opens and pumps the second messenger calcium into the B cell to modulate signaling. Specific inhibitors of PIEZO dampen B cell responses to membrane antigens but not to antigens in solution. 2) The molecular basis of the efficacy of the human papillomavirus-like particle (HPV-VLP) vaccines to induce long-lasting B cell memory. HPV-VLP vaccines when administered in a single dose demonstrated remarkable prophylactic efficacy. The unexpected potency of one dose of the HVP-VLP vaccine may largely be attributed to structural features of the particles that lead to the efficient generation of long-lived antigen-specific antibody-producing cells. To explore this possibility we established a collaboration with Dr. John Schiller (NCI) a developer of the HPV-VLP vaccines. HPV-VLPs are formed by the self-assembly of the HPV major capsid protein, L1. To determine how HP-VLPs activate B cells we are creating both human B cell lines that express BCRs containing HP L1-specific mAb IgH and IgL chains and will assess the responses to the HPV-VLP vaccines versus pentamers of the L1 protein alone. We hypothesize that HPV-VLP will function to activate the mechanosensitive membrane calcium channel PIEZO in contrast to the L1 pentamer that will not. The results of these studies have the potential to reveal a property of the HPV-VLP that confers its efficacy in inducing LLPCs that could be applied to other subunit vaccines. The biology and function of atypical B cells in chronic human infectious diseases and autoimmunity Research in my lab contributed significantly to our understanding of the differentiation and function of a novel subpopulation of B cells, that we termed atypical B cells (ABCs), that are greatly expanded in a number of diverse chronic human infectious diseases including malaria and HIV and in human systemic autoimmune diseases including SLE. 1) The progenitors of human ABCs. Based on the observation that ABCs expanded in diseases characterized by chronic inflammation and persistent antigen exposure, we investigated the nature of the antigen and inflammatory cytokines required to induce human tonsil B cell subsets to express T-bet and other ABC markers in vitro. We provided evidence that prolonged stimulation of B cells by antigen bound to membranes in the presence of the inflammatory cytokine, IFN-gamma, and the TLR9 agonist, CpG, induced the majority of naive B cells to express high levels of T-bet as well as a variety of other surface markers associated with ABCs. 2) The full impact of chronic infection on the human B cell compartment. To better understand the full impact of chronic infection on the B cell compartment we performed in-depth transcriptional analyses of B cells obtained from the peripheral blood of healthy adults living in malaria endemic Africa, HIV-infected individuals and healthy US adults. We determined unique transcriptional profiles for naive B cells, ABCs and classical and activated MBCs and validated a new set of cell surface markers, cytokines and transcription factors associated with the ABC subset. Using single cell RNA-sequencing (scRNA-seq) we discovered that ABCs differentiated from naive B cells driven by IFN-gamma signaling. Moreover, ABC clusters in malaria showed significant similarities to ABCs in HIV and in autoimmune diseases. This unexpected observation provided support for the hypothesis that ABCs represent a separate lineage with a common initiator in different chronic diseases. 3) The function of ABC. Human ABCs have been described as hypo-responsive to BCR crosslinking induced by soluble Ig-specific antibodies. For ABCs from individuals living in malaria-endemic Africa, we showed that the crosslinking of the BCRs by soluble anti-Ig failed to induce: phosphorylation of kinases in the BCR signaling pathway, proliferation, differentiation to antibody secreting cells and cytokine secretion. As described above a fundamental feature of B cell responses to antigen in SLO is their recognition of antigen on the surfaces of FDCs. We discovered that human ABCs were fully responsive to membrane-associated antigen. Moreover, we provided a mechanism underlying this observation showing that responses to membrane-bound antigen were associated with exclusion of the inhibitory receptor, FcgammaRIIB, from the BCR synapse. These results provided the first evidence that ABCs are not hypo-responsive and had the ability to respond to antigen in vivo during chronic malaria. Understanding the role of antigen-affinity thresholds in B cell memory. A defining feature of human antibody responses is the generation of immunologic memory. Primary antibody responses in naive individuals are generally short-lived and result in the production of low levels of low affinity IgM antibodies. In striking contrast, the secondary or memory response to challenge by the homologous antigen in immune individuals, results in antibody levels that are orders of magnitude higher, affinity matured, isotype switched and long-lived. At the cellular level antibody memory is dependent on the acquisition of highly antigen-selected, high affinity long-lived plasma cells (LLPCs) specific for the immunizing antigen that take up residency in the bone marrow and secrete large amounts of antibodies that persist for years. Long-lived memory B cells (MBCs) are also acquired that upon antigen challenge predominantly undergo three fates: rapid differentiation to PCs that produce antibodies specific for heterologous variant antigens; differentiation toward germinal center (GC) B cells that potentially replenish the responding MBC population or induction of apoptosis. The human MBC compartment is composed of roughly equivalent numbers of switched IgG+ and unswitched IgM+ MBCs, however how these MBC subpopulations function in response to antigen challenge is incompletely understood. We discovered that intrinsic affinity thresholds for BCR-dependent antigen-activation are set at least 100-fold higher for IgG+ as compared to IgM+ MBCs independently of a number of cell surface markers that define additional heterogeneity in MBC subsets. Although IgG+ MBCs are unable to respond to low affinity antigens, challenge in vitro with high affinity antigens induced IgG+ MBCs to differentiate almost exclusively towards PC fates. In contrast, challenge with low affinity antigens induced IgM+ MBCs to predominantly differentiate towards GC B cell fates, whereas high affinity antigen induced apoptosis. We propose that human IgG+ and IgM+ MBCs populate two functionally distinct compartments in immune individuals that are restricted to responding to antigens in distinct affinity windows that result in PC fates for high affinity antigens and GC B cell fates for low affinity antigens. These findings have important implications for the design of vaccination strategies that can efficiently generate broadly neutralizing antibodies against infectious pathogens.