The Generation and Maintenance of Human Memory B Cells

适应性免疫系统编码的能力是记住与抗原的初次接触，并在个体的一生中以快速而稳健的方式在再次接触该抗原时对该抗原做出反应。这种免疫记忆现象是适应性免疫系统的一个基本属性，也是所有疫苗开发的基础。对于大多数疫苗来说，中和抗体在保护性免疫反应中起着关键作用，因此产生和维持体液记忆的机制引起了人们的极大兴趣。长期体液免疫是在记忆B细胞(MBC)和长寿浆细胞(LLPC)中编码的，它们是初级免疫反应的一部分。LLPC是终末分化的细胞，存在于骨髓中，负责长期维持血清抗体水平，在再次感染时对病原体及其毒素的初始控制起着关键作用。MBC能够通过增殖和分化为浆细胞(PC)来建立抗原诱导的反应，从而在再次暴露于病原体时产生快速、高滴度的次级抗体反应。即使是中和抗体效价不足的个体，单核细胞集落刺激因子也可以提供保护。尽管单核细胞在对抗感染中起着核心作用，但我们对B细胞记忆产生和维持的细胞和分子机制的了解还不完全。开发新疫苗的努力将受益于对这些过程的更详细了解，特别是针对疟疾中的恶性疟原虫等病原体的疫苗，这些病原体似乎会颠覆免疫记忆。该项目是皮尔斯博士和路易斯·米勒博士以及他在疟疾疫苗发展处(MVDB)的同事之间的合作努力。在过去的一年里，我们一直致力于了解幼稚个体在接种疫苗后B细胞记忆的产生、维持和激活。在这个过程中，人们特别感兴趣的是TLR9的作用，它是一种模式识别受体，可以启动先天免疫反应。TLR9检测具有低甲基化CpG基序的微生物DNA，在人类中优先表达于浆细胞样树突状细胞(PDC)和B细胞。TLR9配体间接参与了B细胞记忆的维持，尽管目前TLR9在B细胞记忆的产生中的作用还没有得到解决。 我们利用外周血液中识别抗原特异性人类记忆B细胞的最新进展，描述了美国疟疾特异性记忆B细胞的产生和维持，以应对MVDB目前正在开发的疫苗。在过去的一年里，我们与MVDB合作，描述了在参与两项疟疾疫苗试验的志愿者的外周血液中获得抗原特异性记忆B细胞的情况，其中一项疫苗由恶性疟原虫顶膜抗原1(AMA1)和裂殖子表面蛋白1(MSP1)组成，这两种疫苗都单独或与TLR9激动剂CpG联合使用。志愿者间隔28天接种三剂疫苗，并在每次接种后3、7、14和28天采集外周血样本，并在第三次和最后一次接种后最多200天采集外周血样。这项研究的纵向设计允许详细描述一次和二次疫苗接种后MBC产生和维持的动力学特征。在对AMA1-C1疫苗试验的分析中，详细描述特征的能力最为明显，在该试验中，在每次接种疫苗后的几个时间点收集了PBMC样本。我们发现，记忆B细胞的获得是一个动态过程，在这个过程中，抗原特异性记忆B细胞池迅速扩大，然后在接种疫苗后收缩。在接种含CpG疫苗的个体中，抗原特异性记忆B细胞出现得更快、数量更多、持续时间更长。疫苗特异性记忆B细胞在再次免疫时出现的百分比预测14天后抗原特异性抗体水平，在稳定状态下，抗原特异性记忆B细胞与抗体水平呈正相关。我们还观察到，每次接种后3天，循环中总的免疫球蛋白G+记忆B细胞池中的抗原非依赖性下降，可能是佐剂诱导的记忆B细胞进入组织的结果。与这种可能性一致，我们观察到循环中浆细胞总数的大幅增加，表明被诱导离开循环的记忆B细胞产生了浆细胞。这些数据首次描述了人类记忆B细胞对疫苗接种的幼稚反应，并将作为疟疾流行地区类似分析的基线。

The adaptive immune system encodes the ability to remember an initial encounter with an antigen and to respond to that antigen upon re-exposure in a rapid and robust fashion for the life time of the individual. This phenomenon of immunological memory is a fundamental property of the adaptive immune system and is the basis for all vaccine development. For most vaccines, neutralizing antibodies plays a critical role in protective immune responses, and thus the mechanisms that underlie the generation and maintenance of humoral memory are of considerable interest. Long-term humoral immunity is encoded in memory B cells (MBC) and long-lived plasma cells (LLPC) which are generated as a part of the primary immune response. LLPC are terminally differentiated cells that reside in the bone marrow and are responsible for the long-term maintenance of serum Ab levels which play a key role in the initial control of pathogens and their toxins upon reinfection. MBC are capable of mounting an antigen-induced response by proliferating and differentiating into plasma cells (PC) resulting in rapid, high titer secondary Ab responses upon re-exposure to pathogens. MBC may provide protection even in individuals with insufficient neutralizing Ab titers. Despite the central role of MBC in combating infections, our understanding of the cellular and molecular mechanisms that underlie the generation and maintenance of B cell memory is incomplete. Efforts to develop new vaccines would benefit from a more detailed knowledge of these processes, particularly vaccines against a pathogen such as Plasmodium falciparum in malaria which appear to subvert immunological memory. This project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Branch (MVDB). Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of B cell memory in nave individuals in response to vaccination. Of particular interest in this process is the role of TLR9, a pattern recognition receptor that initiates innate immune responses. TLR9 detects microbial DNA with hypomethylated CpG motifs and in humans is preferentially expressed by plasmacytoid dendritic cells (PDC) and B cells. TLR9 ligands have been indirectly implicated in the maintenance of B cell memory although at present the role of TLR9 in the generation of B cell memory has not been addressed. We have taken advantage of recent advances in the identification of antigen-specific human memory B cells in peripheral blood to describe the generation and maintenance of malaria-specific memory B cells in the U.S. in response to vaccines currently under development in the MVDB. Over the last year, in collaboration with the MVDB, we described the acquisition of antigen-specific memory B cells in the peripheral blood of volunteers enrolled in two trials of the malaria vaccines, one composed of P. falciparum apical membrane antigen 1 (AMA1) and one of merozoite surface protein 1 (MSP1), both on alum either alone or in combination with the TLR9 agonist, CpG. Volunteers received three doses of the vaccine 28 days apart and peripheral blood samples were collected 3, 7, 14 and 28 days after each vaccination and up to 200 days following the third and last vaccination. The longitudinal design of this study permitted a detailed characterization of the kinetics of MBC generation and maintenance in response to primary and secondary vaccination. The capacity for a detailed characterization was most apparent in the analysis of the AMA1-C1 vaccine trial in which PBMC samples were collected at several time points after each vaccination. We found that the acquisition of memory B cells is a dynamic process in which the antigen-specific memory B cell pool rapidly expands and then contracts following vaccination. In individuals who received CpG-containing vaccines, antigen-specific memory B cells appeared more rapidly, in greater numbers, and persisted for longer. The percentage of vaccine-specific memory B cells present at the time of re-immunization predicted antigen-specific antibody levels 14 days later and at steady state, there was a positive correlation between antigen-specific memory B cells and antibody levels. We also observed an antigen-independent decrease in the total IgG+ memory B cell pool in circulation 3 days after each vaccination, possibly the result of adjuvant-induced trafficking of memory B cells into tissues. Consistent with this possibility we observed a large increase in the total number of plasma cells in circulation, suggesting that memory B cells induced to leave the circulation gave rise to plasma cells. These are the first data describing the naive human memory B cell response to vaccination and will serve as a baseline for similar analyses in malaria endemic areas.