Neuroprotective B Cell Immunotherapy for Contusion Traumatic Brain Injury

摘要：B淋巴细胞除了产生抗体外，还是免疫系统的有效调节者 无论是通过细胞间的直接相互作用，还是通过分泌可溶性分子。最近的调查显示 强调抗炎(调节)B细胞在中枢神经系统(CNS)中的有益作用， 研究表明，B细胞枯竭会加重神经退行性疾病的症状。我们有 首次证明外源性B细胞可用于治疗以恢复多种疾病的功能 损伤模型，包括心肌梗死、急性和慢性伤口的愈合以及受控皮质 撞击(CCI)创伤性脑损伤(TBI)。在我们的小鼠CCI模型中，一次向脑内注射B细胞 损伤时的实质显著减少学习和记忆障碍，通过 40-60%，损伤后35天可见胶质细胞增生和小胶质细胞活化。初步研究表明，B细胞 直到CCI后6h给药，在减少运动学习障碍方面仍然同样有效。鲜为人知 关于B细胞在脑外伤中神经保护作用的机制。在这里，我们建议调查 CCI后B细胞介导的神经保护的细胞和分子机制，最终定位 基于细胞的新疗法在脑挫伤中的临床应用。在目标1中，我们将多次评估 指出B细胞实质内注射对神经元存活和轴突变性的影响(1a)， 浸润性免疫细胞(1b)和驻留小胶质细胞(1c)的调节以及细胞的增殖和分化 损伤部位(1D)。基于B细胞在伤口愈合和CCI中的初步机制研究， 我们假设放置在CCI部位的幼稚B细胞感知局部炎症信号和损伤- Toll样受体(TLR)和B细胞受体(BCR)依赖的相关分子模式 途径，并采用一种调节表型。这种细胞状态与抗炎物质的产生有关 细胞因子(包括IL-10，但也包括IL-4、IL-35和转化生长因子β)，作用于邻近浸润性和滞留性(小胶质细胞) 免疫细胞并使其表型偏向抗炎、神经保护状态。在《目标2》中我们将 在CCI模型中询问介导B细胞神经保护效应的传感器和效应器通路。 靶向基因敲除小鼠模型将被用来确定关键分子通路的参与 (依赖TLR与依赖CD19/BCR)在损伤部位(2a)的B细胞环境感知中，并确定 调节B细胞对损伤反应所需的关键效应分子(2b)。调查……的作用 小胶质细胞作为IL-10调节炎症的下游介质，我们将调节IL-10R的表达 并测试在没有反应性小胶质细胞伙伴的情况下B细胞治疗的效果(2c)。 拟议研究的结果将为临床开发一种新的、安全的和成本低廉的- 有效的免疫细胞疗法，以解决脑损伤，这是一个主要的未得到满足的医疗需求。

ABSTRACT: In addition to antibody production, B lymphocytes are efficient regulators of the immune system both through direct cell-cell interactions and through secretion of soluble molecules. Recent investigations have underscored the beneficial role of anti-inflammatory (regulatory) B cells in the central nervous system (CNS), and shown that B cell depletion can worsen the symptoms of neurodegenerative diseases. We have demonstrated for the first time that exogenous B cells can be applied therapeutically to restore function in diverse injury models, including myocardial infarction, healing of acute and chronic wounds, and controlled cortical impact (CCI) traumatic brain injury (TBI). In our mouse CCI model a single injection of B cells to the brain parenchyma at the time of injury significantly reduced learning and memory deficits, reduced lesion volume by 40-60%, as well as gliosis and microglial activation at 35 days post-injury. Preliminary studies show that B cells administered as late as 6h after CCI remain equally effective in reducing motor learning deficits. Little is known about the mechanisms underlying the neuroprotective effects of B cells in TBI. Here, we propose to investigate the cellular and molecular mechanisms of B cell-mediated neuroprotection after CCI, ultimately positioning this novel cell-based therapy for clinical use in the context of contusion TBI. In Aim 1 we will assess at multiple time points the effect of intraparenchymal B cell administration on neuronal survival and axonal degeneration (1a), regulation of infiltrating immune cells (1b) and resident microglia (1c), and cell proliferation and differentiation at the injury site (1d). Based on preliminary mechanistic investigations of B cells in both wound healing and CCI, we hypothesize that naïve B cells placed at the site of CCI sense local inflammatory signals and damage- associated molecular patterns (DAMPs) via Toll-like receptor (TLR)- and B cell receptor (BCR)-dependent pathways, and adopt a regulatory phenotype. This cell state is associated with production of anti-inflammatory cytokines (including IL-10, but also IL-4, IL-35, and TGFβ), that act on adjacent infiltrating and resident (microglia) immune cells and bias their phenotype towards an anti-inflammatory, neuroprotective state. In Aim 2 we will interrogate the sensor and effector pathways that mediate the neuroprotective effects of B cells in a CCI model. Targeted gene knockout mouse models will be used to determine the involvement of key molecular pathways (TLR-dependent vs. CD19/BCR-dependent) in B cell environmental sensing at the injury site (2a) and to define key effector molecules required for mediating the B cell response to injury (2b). To investigate the role of microglia as down-stream mediators of inflammatory regulation via IL-10, we will modulate IL-10R expression on local microglia and test the efficacy of B cell treatment in the absence of responsive microglial partners (2c). Findings from the proposed studies will establish a foundation for clinical development of a novel, safe and cost- effective immune cell therapy to address TBI, a major unmet medical need.