Revealing the Biophysics of the Germinal Center Microenvironment

摘要 艾滋病毒疫苗有可能吗？疫苗是过去最成功的公共卫生干预措施之一。 世纪。几乎所有疫苗都是通过诱导保护性抗体起作用的。然而，我们对细胞的理解 疫苗免疫反应的动态，特别是围绕B细胞竞争的生物学 生发中心(GC)对复杂疫苗抗原的作用有限。对基本B细胞缺乏了解 生物因素导致无法开发出有效的艾滋病毒疫苗。有希望的是，一小部分人 HIV+个体已经发展出广泛的中和抗体(BNAbs)，这让人们重新希望艾滋病毒 疫苗是可能的。最近的研究发现，许多HIV阴性的健康人类捐赠者都有VRC01-类 BNab前体B细胞。然而，从这些研究中发现，这些潜在的bNab前体B细胞 以异常罕见的频率被发现。这表明，免疫后这些B细胞可能是 被更频繁的非中和B细胞所击败。回答围绕这一问题的免疫学问题 问题，我开发了一个利用含有人类基因的小鼠来研究生殖系恢复的VRC01的模型系统 BNab(VRC01gHL)。通过这个B细胞转移模型，我们发现抗原亲和力、亲和力和前体 在GCs中罕见的VRC01gHL B细胞的竞争成功中，频率都起着相互依赖的作用。关键的是，我们 发现稀有的具有生理亲和力的VRC01gHL B细胞可以被激活以在 GCS。然而，这些反应仅限于特定的“GC”岛，表明B细胞竞争种子。 除了地方选区内的竞争外，个别地方选区亦至为重要。综上所述，这些观察表明 B细胞在GC微环境中的免疫优势(GCME)是需要克服的主要障碍 开发一种成功的艾滋病毒疫苗。然而，与以下方面有关的知识差距很大 B细胞竞争进入GC，并在GCME内竞争的生理状态。首先，什么？ 我们是否了解GCME的生物物理和代谢特征？我们假设并发现 GCS形成低氧微环境。我推测，其他生物物理制约因素可能也在控制 GC选择事件，因为许多途径已经被证明在缺氧的肿瘤微环境中都是活跃的 (TMES)和低氧GCME。我推测，在与TME进一步相关的情况下，GCME可能包含 高乳酸水平，诱导多个代谢GPCR，降低pH，升高温度和细胞压力。 我假设GC的这些生物物理参数可以并且确实影响B细胞选择事件到复合体 抗原。在这份DP2提案中，我将通过多项研究来研究GCME细胞外环境的性质 光子定向直接测量并定义限制VRC01成功的生物物理约束- B类细胞反应。然后，我们将应用我们从GCME研究中学到的东西来操纵B细胞 GCME中的免疫优势有利于VRC01-B类细胞的竞争性选择。

Abstract Is an HIV vaccine possible? Vaccines are one of the most successful public health interventions over the past century. Nearly all vaccines work by induction of protective antibodies. However, our understanding of the cellular dynamics of immune responses to vaccines, particularly the biology surrounding B cell competition within germinal centers (GC) to complex vaccine antigens is limited. This lack of understanding of fundamental B cell biology has contributed to the inability to develop an effective HIV vaccine. Promisingly, a small population of HIV+ individuals have developed broadly neutralizing antibodies (bnAbs), giving renewed hope that an HIV vaccine is possible. Recent work has found that many HIV negative healthy human donors have VRC01-class bnAb precursor B cells. However, work from these studies revealed that these potential bnAb precursor B cells are found at an unusually rare frequency. This suggested that following immunization these B cells may be outcompeted by more frequent non-neutralizing B cells. To answer immunological questions surrounding this problem, I developed a model system utilizing mice containing human genes for the germline-reverted VRC01 bnAb (VRC01gHL). Through this B cell transfer model, we found that antigen affinity, avidity, and precursor frequency all played interdependent roles in competitive success of rare VRC01gHL B cells in GCs. Critically, we found that rare VRC01gHL B cells with physiological affinities could be primed to successfully compete within GCs. However, these responses were limited to specific “GC” islands suggesting B cell competition to seed individual GCs is critical in addition to competition within the GC. Taken together, these observations suggest that B cell immunodominance in the GC microenvironment (GCME) is a major obstacle to overcome in developing a successful HIV vaccine. However, there are significant knowledge gaps pertaining to the physiological conditions in which B cells compete to enter GCs, and compete within the GCME. To start, what do we know about the biophysical and metabolic characteristics of the GCME? We hypothesized and found that GCs form a hypoxic microenvironment. I hypothesize that other biophysical constraints may be acting to control GC selection events as many pathways have been shown to be both active in hypoxic tumor microenvironments (TMEs) and in the hypoxic GCME. I hypothesize that in further correlation with TMEs, the GCME may contain high lactate levels, induce multiple metabolic GPCRs, reduced pH, increased temperature, and cellular pressure. I posit that these biophysical parameters of the GC can and do influence B cell selection events to complex antigens. In this DP2 proposal I will investigate the nature of the extracellular milieu of the GCME through multi- photon targeted direct measurements and define the biophysical constraints that limit the success of VRC01- class B cell responses. We will then apply what we learn from studying the GCME to manipulate B cell immunodominance in the GCME to favor competitive selection of VRC01-class B cells.