B Cell Biology

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Over the last year 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. However, small molecular weight soluble antigens (i.e. antigens that have not formed immune complexes or become complement fixed) also gain entry into SLO. We compared the responses of human tonsil nave B cells to antigen associated with membranes or in solution in vitro. 1) The role of PIEZO in B cell responses. We discovered that the early and late signaling cascades initiated by antigens associated with membranes versus in solution were different as were the outcomes of signaling in terms of cell fate decisions and differentiation. Importantly, we discovered a mechanism by which B cells sensed that they were engaging antigen on a membrane versus in solution that may account for the different outcomes of signaling. We determined that B cells express the mechano-sensitive membrane calcium channel PIEZO. When B cells interact with surfaces, such as antigen on membranes, and the B cell membrane is stretched, 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. We are pursuing these observations to identify the impact of the PIEZO-mediated calcium influx on B cell signaling both in vitro and in vivo. 2) The role of membrane antigens in triggering the metabolic clock. The metabolic clock is a term we coined to highlight the central role of metabolic fitness and in particular mitochondrial performance, in the life and death decisions of an antigen activated B cell. The metabolic clock model extends the two-signal hypothesis for B cell activation by adding a major conceptual novelty putting mitochondrial fitness as the fate-decision maker. According to this model binding of an antigen to the BCR (signal one) triggers a rapid increase in metabolic activity necessary to sustain subsequent proliferation and differentiation. However, signal one also triggers a countdown (the clock) that gradually with time dampens metabolic activity resulting in a calcium imbalance that leads to mitochondrial dysfunction and cell death. The clock ticking towards metabolic insufficiency and cell-death can be stopped by signal two coming from either a cognate B cell-T cell interaction or from toll like receptor (TLR) signaling. The requirement for antigen-specific T cells prevents activation of self-reactive B cells and TLR signaling communicates to the B cell the presence of a dangerous pathogen and the need to produce antibodies. Thus, the metabolic clock ensures that only antigen stimulated B cells that receive a second confirmatory signal undergo full-scale immune activation. We demonstrated that mitochondrial dysfunction is a direct result of gradual increases in cytoplasmic calcium levels induced by signal one and antagonized by signal two. We recently addressed the question: do both soluble and membrane-associated antigens trigger the metabolic clock in B cells? Our results show that both forms of antigen trigger the clock in mouse and human B cells although the kinetics of the clock are different. This is an important finding as it indicates that this fundamental mechanism may be insensitive to the form of the antigen. We are pursuing these observations to determine if the antigen-induced BCR activation of the metabolic clock is a feature of all B cell subpopulations and of B cells at all differentiated states. 3) 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 administered in a prime/boost series of three injections over six months have demonstrated remarkable prophylactic efficacy in clinical trials and effectiveness in national immunization programs with high rates of coverage. There is mounting evidence that the vaccines have similar efficacy and effectiveness even when administered in a single dose. 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. We have entered into a collaboration with Dr. John Schiller (NCI) who along with Dr. Doug Lowy (NCI) developed the HPV-VLP vaccines now commercially produced as Cervarix, Gardasil and Gardasil-9. HPV-VLPs are formed by the self-assembly of the HP virus major capsid protein, L1, to determine how HP-VLPs activate B cells. To do so we are creating both human B cell lines that express BCRs containing HP L1-specific mAb IgH and IgL chains as well as transgenic mice into which we have introduced the same HP L1-specific IgH and L chains. We will assess the responses to the HPV-VLP vaccines versus pentamers of the L1 protein alone. We hypothesize that HPV-VLP will function as a membrane-associated antigen as compared to the soluble HP L1 tetramer. The results of these studies could potentially reveal a property of the HPV-VLP that confers its efficacy in inducing LLPCs that could be applied to other subunit vaccines. Understanding the mechanisms underlying B cell antigen affinity maturation. As described above, current evidence nearly exclusively from mouse models, suggests that B cell memory is acquired in two phases. In phase 1 nave B cells in follicles in SLO encounter antigens presented on FDC and process and present these antigens to Tfh resulting in B cell proliferation and differentiation to germinal center GC B cells. In phase 2 newly differentiated GC B cells undergo somatic hypermutation (SHM) and affinity selection resulting in differentiation into either MBCs or LLPCs. The highest affinity GC B cells appear to differentiate into LLPCs that provide the first line of defense against reinfection by the same pathogen. In contrast, lower affinity GC B cells differentiate into MBCs that provide a second line of defense against variant pathogens that escape LLPC antibodies. Upon re-infection MBCs have three fates. IgG+ MBCs that have acquired SHMs and are of high affinity differentiate into PCs. In contrast. IgM+ MBCs with relatively low levels of SHMs re-enter GCs to undergo further SHM and affinity selection. Alternatively, MBC can undergo apoptosis. Thus, the drive to acquire high-affinity pathogen-specific antibodies is a fundamental feature of immunity to infection. We are carrying out studies to understand the cellular and molecular mechanisms underlying affinity-dependent activation and differentiation of human MBCs by investigating: 1) the antigen-affinity dependent activation and differentiation of different subpopulations of human MBCs; 2) the mechanisms by which antigen affinity modulates functional outcomes in MBCs; and 3) the influence of various infectious diseases on antigen affinity discrimination by human MBCs. Understanding affinity-dependent MBC activation has important implications for the design of vaccination strategies that can efficiently generate broadly neutralizing antibodies against infectious pathogens.