Revealing the Biophysics of the Germinal Center Microenvironment

摘要 HIV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。几乎所有的疫苗都是通过诱导保护性抗体来起作用的。然而，我们对细胞的理解 对疫苗的免疫应答动力学，特别是围绕B细胞竞争的生物学， 复杂疫苗抗原的免疫反应中心（GC）是有限的。对基本的B细胞缺乏了解 生物学上的原因导致无法开发出有效的艾滋病毒疫苗。很有希望的是， HIV+个体已经产生了广泛中和抗体（bnAbs），这给HIV感染者带来了新的希望。 疫苗是可能的。最近的工作发现，许多HIV阴性的健康人类供体具有VRC 01类 bnAb前体B细胞。然而，这些研究的工作表明，这些潜在的bnAb前体B细胞 出现的频率非常罕见这表明免疫后这些B细胞可能是 被更频繁的非中和性B细胞击败。为了回答围绕这一点的免疫学问题， 为了解决这个问题，我开发了一个模型系统，利用含有人类基因的小鼠进行生殖系回复的VRC 01 bnAb（VRC01gHL）。通过这种B细胞转移模型，我们发现抗原亲和力、亲合力和前体 频率在GC中罕见的VRC 01 gHL B细胞的竞争成功中均起相互依赖的作用。关键是，我们 发现罕见的具有生理亲和力的VRC 01 gHL B细胞可以被引发成功地竞争 GC。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GC内部的竞争之外，单个GC也至关重要。综上所述，这些观察结果表明， GC微环境（GCME）中的B细胞免疫优势是克服的主要障碍， 研制成功的艾滋病疫苗然而，在这方面存在着重大的知识差距， 在生理条件下，B细胞竞争进入GC，并在GCME内竞争。首先， 我们是否了解GCME的生物物理和代谢特征？我们假设并发现， GC形成缺氧微环境。我假设其他生物物理限制可能起作用来控制 GC选择事件，因为许多途径已被证明在缺氧肿瘤微环境中都是活跃的 （TME）和低氧GCME。我推测，在进一步与TME的相关性，GCME可能包含 高乳酸水平、诱导多种代谢GPCR、降低pH、升高温度和细胞压力。 我认为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，使其复杂化。 抗原在这个DP 2的建议，我将调查的性质的细胞外环境的GCME通过多- 光子靶向直接测量，并定义限制VRC 01成功的生物物理约束- B类细胞应答。然后我们将应用我们从研究GCME中学到的知识来操纵B细胞 GCME中的免疫优势有利于VRC 01类B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.