CAREER: An Immunoengineering Approach to Create Purely Synthetic Microenvironments Enabling Generation of Antigen-Specific Effector B cells

一种称为B细胞的白色细胞在我们的免疫系统中起着关键作用，它可以产生抗体来对抗各种感染。然而，B细胞并不是一直处于警戒状态;它们需要在一个称为生发中心（GC）的特殊生理微环境中被选择性地激活以增殖和改变（分化）成为抗体产生细胞。由于其复杂的结构，功能和动态演变的性质，创建一个功能齐全的GC模型离体（体外）是极具挑战性的，因此需要一个跨学科和综合的工程方法。该CAREER项目旨在通过重新创建能够诱导全系列B细胞反应的GC的最关键组分来开发纯人工GC模型系统。与使用常规体内（体内）观察相比，使用对每个关键参数进行精确和独立控制的离体模型系统，将以前所未有的详细程度研究这些GC组分在产生B细胞反应中的作用。这些成果将使各个领域取得重大进展，包括疫苗接种，免疫治疗，自身免疫性疾病和癌症。作为高度跨学科项目的补充教育和推广计划，重点放在三个方面：1）开发有效的免疫工程课程，教授免疫学和工程学之间的接口主题，2）在学术界，当地研究机构和行业合作伙伴之间创造独特的教育和研究合作环境，以及3）促进北方亚拉巴马代表性不足的高中生和大学生从事免疫工程和一般STEM领域。研究者的长期研究目标是开发用于癌症，感染，和自身免疫性疾病的研究。为了实现这一目标，该CAREER项目将开发一种人工离体模型系统，该系统能够模拟双子中心（GC）最重要的功能特征：创建可以产生亲和力成熟的抗原特异性抗体的B细胞。 尽管在B细胞生物学和免疫学方面取得了巨大成就，但还没有人工模型系统能够完全再现离体GC的所有关键特征。受最先进的GC模型的研究结果的启发，该项目假设GC微环境有三个关键组成部分，如果没有正确的模拟，离体GC反应的重演将是不可能的：1）最佳的CD 40 L-CD 40信号传导，需要来自滤泡辅助T（TFH）细胞的帮助信号，2）GC的带状结构和GC B细胞的亮区（LZ）和暗区（DZ）之间的带间迁移，以及3）需要来自滤泡树突细胞（FDC）的抗原呈递的时间控制的开-关B细胞受体（BCR）信号。 该研究计划是根据三个目标，解决每个确定的关键组成部分。 每个目标都包括开发系统可控的生物材料平台，这些平台将为人工发育的GC B细胞提供关键的生物信号和微环境。 第一个目的是通过在具有可调模量和应力松弛特性的仿生/生物响应粘弹性水凝胶上提供CD 40配体（CD 40 L或CD 154）分子来控制由滤泡辅助T（TFH）细胞提供的CD 40信号传导的质量和数量。 预期的结果是通过设计生物材料平台的机械性能来将配体提呈细胞表面受体，特别是肿瘤坏死因子（TNF）超家族的各种分子，从而加深对如何控制信号事件数量和质量的理解。CD 40 L所属。 第二个目的是通过在用于人工GC B细胞的微流体装置内产生受控的趋化因子梯度来提供模拟GC（DZ和LZ）的带状结构的微环境。通过使用微流控装置提供受控的趋化因子梯度，将能够系统地研究使人工活化的B细胞迁移的条件以及在GC反应方面的带间迁移的后果。 第三个目的是通过模型抗原与基于微珠的人工FDC的可逆表面缀合来引入BCR信号传导的开和关时间调节，假设BCR信号传导需要被时间调节开和关，以便能够延长GC B细胞的增殖。预期的结果是更清楚地理解BCR信号传导在GC反应中的作用。 最后，该项目的成果有望实现功能性人工离体GC模型，该模型可以：1）通过能够识别新的B细胞表位而不依赖于T细胞，促进针对主要病原体的新疫苗的开发，对于这些病原体，还没有有效的疫苗可用，2）被开发为用于产生抗原特异性效应B细胞作为过继性细胞治疗的线性可扩展的细胞制造平台，以及3）在研究B细胞生物学和B细胞恶性肿瘤的生物医学科学中作为一个更好控制和更具成本效益的模型系统。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

A type of white blood cells called B cells play a critical role in our immune system by producing antibodies to combat various infections. However, B cells are not on alert all the time; they need to be selectively activated to proliferate and change (differentiate) to become antibody-producing cells in a special physiological microenvironment called the germinal center (GC). Due to its complicated structure, function, and dynamically evolving nature, creating a fully functional GC model ex vivo (outside the body) is extremely challenging, and thus requires an interdisciplinary and comprehensive engineering approach. This CAREER project seeks to develop a purely artificial GC model system by recreating the most critical components of the GC capable of inducing a full array of B cell reactions. Using the ex-vivo model system with precise and independent control over each critical parameter, the roles of these GC components in producing B cell reactions will be investigated in unprecedented detail compared to using conventional in-vivo (inside the body) observations. These outcomes will enable critical advancement of various fields, including vaccination, immunotherapy, autoimmune diseases, and cancers. As complementary educational and outreach plans of the highly interdisciplinary project, focus has been made on three aspects: 1) the development of effective curricula for immunoengineering that teaches the topics at the interface between immunology and engineering, 2) the creation of uniquely collaborative environment for education and research among academia, local research institutes, and industry partners, and 3) the promotion of underrepresented high school and college students in Northern Alabama to be engaged in immunoengineering and general STEM fields.The investigator’s long-term research goal is to develop translational cellular and molecular immunotherapies for cancers, infections, and autoimmune diseases through the unconventional amalgamation of biomaterials engineering and immunology, namely immunoengineering. Towards this goal, this CAREER project will develop an artificial ex-vivo model system that enables the mimicry of the most important functional feature of the geminal center (GC): to create B cells that can produce affinity-matured antigen-specific antibodies. Despite tremendous achievements in B-cell biology and immunology, no artificial model system has yet been fully capable of recapitulating all the critical features of the GCs ex vivo. Motivated by findings from state-of-the-art GC models, this project hypothesizes that there are three critical components of the GC microenvironments, without the correct mimicry of which, the recapitulation of ex-vivo GC reactions would be impossible: 1) the optimal CD40L-CD40 signaling that requires help signals from T-follicular helper (TFH) cells, 2) the zonal structure of the GC and interzonal migration between the light zone (LZ) and the dark (DZ) of the GC B cells, and 3) the temporally controlled on-and-off B cell receptor (BCR) signals that require antigen presentations from follicular dendric cells (FDCs). The Research Plan is organized under three objectives that address each of the identified critical components. Each objective includes development of systematically controllable biomaterial platforms that will provide critical biological signals and microenvironments to artificially developing GC B cells. The FIRST Objective is to control the quality and quantity of the CD40 signaling provided by follicular helper T (TFH) cells, by providing CD40 ligand (CD40L or CD154) molecules on bio-mimetic/bio-responsive viscoelastic hydrogels that have tunable modulus and stress relaxation characteristics. The expected outcome is enhanced understanding of how to control the quantity and quality of signaling events by designing mechanical properties of biomaterials platforms for the presentation of ligands to cell-surface receptors, especially to various molecules of the tumor necrosis factor (TNF) superfamily to which CD40L belongs. The SECOND Objective is to provide a microenvironment mimicking the zonal structures of GC (DZ and LZ) by creating controlled chemokine gradients within a microfluidic device for the artificial GC B Cells. By providing a controlled chemokine gradient using microfluidic devices, a systematic study about the conditions that enable artificially activated B cells to migrate and the consequences of the interzonal migrations in terms of GC reactions will be enabled. The THIRD Objective is to introduce On and Off temporal regulation of BCR signaling via reversible surface conjugation of model antigens to microbead-based artificial FDCs, hypothesizing that the BCR signaling needs to be temporally regulated on-and-off in order to enable an extended proliferation of GC B cells The expected outcome is a clearer understanding of the role of BCR signaling in GC reactions. Finally, the outcomes of this project are expected to enable realization of a functional artificial ex-vivo GC model that could: 1) facilitate the development of novel vaccines against major pathogens for which no effective vaccines are yet available by enabling recognition of novel B-cell epitopes without T-cell dependency, 2) be developed as a linearly scalable cell-manufacturing platform for generation of antigen-specific effector B cells as adoptive cell therapy, and 3) serve as a better-controlled and more cost-effective model system in biomedical sciences that studies B-cell biology and B-cell malignancies.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.