B Cell Biology

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

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 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, PIEZO opens and allows the second messenger calcium to flow into the B cell to modulate signaling. Specific inhibitors of PIEZO and knock-down of PIEZO dampen B cell responses to membrane antigens but not to antigens in solution. We concluded from these results that the activation of Piezo1 defines an essential event in B cell activation to membrane-presented antigens and provides a stimulus for B cell activation that is absent in soluble antigens. 2) The molecular basis of the efficacy of the human papillomavirus-virus like particle (HPV-VLP) vaccines to induce long-lasting B cell memory. HPV-VLP vaccines even when administered in a single dose demonstrated remarkable prophylactic efficacy. The unexpected potency 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 plasma cells. To explore this possibility we established a collaboration with Dr. John Schiller (NCI) a developer of the HPV-VLP vaccines. To better understand the efficacy of HPV-VLP vaccines, we compared the activation of HPV-specific human B cells in vitro by an HPV-VLP vaccine, composed of 72 pentamers of the HPV L1 protein versus single L1 pentamers, termed capsomeres. HPV-VLPs and capsomeres bound exclusively to HPV-specific B cells and HPV-VLP binding induced calcium responses that greatly exceeded the levels and duration of calcium responses induced by capsomere binding over a wide range of HPV-VLP/capsomere concentrations. B cell responses to HPV-VLPs, but not to capsomeres, required the function of the mechanosensitive plasma membrane ion channel, Piezo1, that we showed was necessary for human B cell responses to membrane-associated antigens but not to soluble antigens, The enhanced response to HPV-VLP correlated with increased phosphorylation of PLCgamma2 but not with increased phosphorylation of the B cell receptor (BCR) itself, suggesting activation of Piezo1 may function to increase PLCgamma2 phosphorylation to regulate calcium responses. We speculate that these unique features of HPV-VLPs evolved to mimic membrane presentation of antigens to B cells that efficiently drive antibody responses. Understanding the role of antigen-affinity thresholds in B cell memory At the cellular level antibody memory is dependent on the acquisition of long-lived plasma cells (LLPCs) specific for the immunizing antigen that secrete large amounts of antibodies that persist for years. Antibody memory is also dependent on the acquisition memory B cells (MBCs) 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 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 differentiate towards GC B cell fates, whereas high affinity antigen induced apoptosis. We proposed 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. Understanding the impact of the human HPV-VLP vaccine on the human naive B cell compartment In an era of predicted recurring pandemics the ability to produce highly-effective, long-lived pathogen-specific vaccines will require understanding the characteristics of naive B cells in nonimmune individuals and the requirements for vaccines to activate these. We characterized the human naive B cell peripheral blood (PB) compartment (CD19+CD27-CD45RBMEM-CD38) that represents approximately 60% of all PB B cells and were separable into two subsets based on expression of CD73. The vast majority of naive B cells (80-90%) were CD73+ and of these the majority (60-70%) showed an anergic phenotype (IgMlowIgD+). In addition, the CD73+ naive B cell compartment contained the majority of all PB B cells expressing the inherently autoreactive VH4-34 heavy chain. Plasma blasts from younger individuals (18-30 years old) that received a flu vaccine seven days earlier showed that nearly half of IgM+ germline VH4-34 sequences had accumulated no or only a small number of mutations (<.05%) suggesting that the plasma blast expressing these sequences were from recently activated anergic naive B cell that were diversifying the vaccine-specific repertoire. In contrast, in older individuals (70-100 years old) nearly all IgM+ VH4-34 sequences (>85%) had accumulated a large number of mutations (0.5-.16%) suggesting that these plasma blasts were recruited from an antigen-experienced population of B cells such as IgM+ memory B cells. These findings suggest that effective vaccines in nonimmune individuals must have the capacity to activate anergic naive B cells to somatically mutate and expand the diversity of the naive B cell repertoire. Having described the anergic characteristics of naive human B cells in nonimmune individuals and the outcome of flu vaccination on these anergic cells we are investigating the outcome of an HPV-VLP vaccine in use today in humans for the protection from HPV infections and cancer on the HPV-specific B cell compartment. In collaboration with John Schiller (NCI) we are carrying out an in-depth analysis of peripheral blood HPV-specific B cells from a cohort of individuals who received the HPV-VLP vaccine. To do so we established a highly sensitive fluorescent HPV-VLP probe to identify and isolate HPV-specific B cells. Using this tool we determined that HPV-VLP induced a large expansion of IgG+ MBCs but not of IgM+ MBCs. The IgG+ MBC compartment is composed of at least six phenotypically defined subpopulations and unexpectedly the HPV-VLP vaccine failed to induce ABCs and two additional MBC subpopulations. We plan to continue our analyses of the HPV-specific B cells in vaccinated individuals using single cell RNA-seq, Ig-seq and cell surface phenotyping. We believe those analyses will provide insight into the cellular bases of the extraordinary efficacy of the HPV-VLP vaccine.