B Cell Biology

了解B细胞对膜相关抗原的反应 体内次级淋巴器官(SLO)中B细胞对抗原的反应的一个基本特征是抗原呈现在FDCs的表面。我们比较了人扁桃体幼稚B细胞对膜相关抗原、溶液中的抗原以及体外呈现为病毒样颗粒的抗原的反应。 1)压电体在B细胞反应中的作用。我们发现人类B细胞表达机械敏感的膜钙通道压电体。利用荧光寿命图像显微镜，我们发现当B细胞与膜上的抗原相互作用时，B细胞膜被拉伸，压电体打开，并允许第二信使钙流入B细胞以调节信号。特定的压电剂和击倒压电剂可抑制B细胞对膜抗原的反应，但不能抑制溶液中的抗原。 2)人乳头瘤病毒样颗粒(HPV-VLP)疫苗诱导持久B细胞记忆的分子基础。单剂接种HPV-VLP疫苗显示出显著的预防效果。一剂HVP-VLP疫苗出人意料的效力在很大程度上可能归因于颗粒的结构特征，这些颗粒导致了长寿的抗原特异性抗体产生细胞的有效产生。为了探索这种可能性，我们与HPV-VLP疫苗的开发商John Schiller博士(NCI)建立了合作关系。HPV-VLP是由HPV主要衣壳蛋白L1自组装而成。为了确定Hp-VLP如何激活B细胞，我们建立了表达含有Hp L1特异性单抗IgH和IGL链的BCR的人B细胞系，并正在评估对HPV-VLP疫苗和单独使用L1蛋白五聚体的反应。我们的初步结果表明，与单独的L1五聚体相比，HPV-VLP诱导了更强大的B细胞反应，部分原因是HPV-VLP激活机械敏感的膜钙通道压电的能力，而L1五聚体不激活。这些结果揭示了HPV-VLP的一个特性，该特性使其在诱导LLPC方面具有效力，可应用于其他亚单位疫苗。 了解抗原亲和力阈值在B细胞记忆中的作用。 在细胞水平上，抗体记忆依赖于获得高度抗原选择、高亲和力、针对免疫抗原的长寿命浆细胞(LLPC)，这些细胞占据骨髓并分泌大量持续多年的抗体。长寿记忆B细胞(MBCs)在抗原攻击时主要经历三种命运：快速分化为PC，产生针对异种变异抗原的抗体；分化为生发中心(GC)B细胞，潜在地补充相应的MBC群体或诱导细胞凋亡。人类MBC亚群由数量大致相等的转换型免疫球蛋白G+和非转换型免疫球蛋白M+单核细胞组成，然而这些单核细胞亚群如何在抗原刺激下发挥作用尚不完全清楚。我们发现，BCR依赖的抗原激活的内在亲和力阈值被设定为至少比IgM+的MBC高100倍，而不依赖于定义MBC亚群中额外异质性的一些细胞表面标志物。虽然免疫球蛋白G+巨噬细胞不能对低亲和力抗原作出反应，但在体外用高亲和力抗原攻击可诱导免疫球蛋白G+巨噬细胞几乎完全分化为PC的命运。相反，低亲和力抗原诱导IgM+单核细胞向GC-B细胞方向分化，而高亲和力抗原诱导细胞凋亡。我们认为，在免疫个体中，人的免疫球蛋白G+和免疫球蛋白M+单核细胞分布在两个功能不同的隔室中，这两个隔室仅限于对不同亲和力窗口中的抗原的反应，这导致高亲和力抗原的PC命运和低亲和力抗原的GC B细胞命运。这些发现对设计疫苗接种策略具有重要意义，该策略可以有效地产生针对传染病病原体的广谱中和抗体。 了解基于人HPV-VLP的疫苗Gardasil对LLPC和MBC隔膜的影响 Gardasil是一种高效疫苗，目前获得许可用于预防感染HPV，HPV是一种高度致癌的病毒。目前，除了最近关于冠状病毒疫苗接种的研究外，我们对目前在人类中使用的任何针对抗原特异性B细胞隔间的疫苗的结果知之甚少。与约翰·席勒(NCI)合作，我们正在对接受Gardasil疫苗的一群人的外周血液HPV特异性B细胞进行深入分析。为此，我们建立了一种高灵敏的HPV-VLP荧光探针来鉴定和分离HPV特异性的B细胞。利用这一工具，我们确定Gardasil诱导了大量的IgG+MBCs的扩增，而不是IgM+的MBCs。Ig G+MBC亚群至少由6个表型确定的亚群组成，Gardasil诱导的HPV特异性B细胞含有一定数量的ABCs和2个额外的MBC亚群。此外，我们确定含有HPV特异性B细胞群的B细胞表达固有的自身反应性VH4.34基因，这表明某种程度的自身反应性可能对加德西有好处。我们计划继续使用单细胞RNA-seq、Ig-seq和细胞表面表型对接种人群中的HPV特异性B细胞进行分析。我们相信，这些分析将为Gardasil疫苗非凡疗效的细胞学基础提供洞察。

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 nave 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. 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 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 generated human B cell lines that express BCRs containing HP L1-specific mAb IgH and IgL chains and are assessing the responses to the HPV-VLP vaccines versus pentamers of the L1 protein alone. Our preliminary results showed the HPV-VLP induced a more robust B cell response as compared to the pentamer of L1 alone, due in part to the ability of the HPV-VLP to activate the mechanosensitive membrane calcium channel PIEZO in contrast to the L1 pentamer that does not. These results reveal a property of the HPV-VLP that confers its efficacy in inducing LLPCs that could be applied to other subunit vaccines. Understanding the role of antigen-affinity thresholds in B cell memory. 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. Understanding the impact of the human HPV-VLP based vaccine, Gardasil, on the LLPC and MBC compartment Gardasil is a highly potent vaccine currently licensed for protection against infection with HPV, a highly carcinogenic virus. At present we know little about the outcome of any vaccine in use today in humans on the antigen-specific B cell compartment with the exception of recent studies on COVID vaccination. 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 Gardasil 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 Gardasil induces 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-specific B cells induced by Gardasil contain numbers of ABCs and two additional MBC subpopulations. In addition we determined that the HPV-specific B cell population contained B cells expressed the inherently autoreactive VH4.34 genes suggesting that some degree of autoreactivity may be beneficial in response to Gardasil. 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 Gardasil vaccine.