Blood Brain Barrier Disruption during Chimeric Antigen Receptor (CAR) T cell therapy

项目总结/摘要 Juliane Gust博士提出了一项研究，以了解BBB和大脑的扰动是否 微血管灌注有助于CAR T细胞神经毒性。这项工作将为格斯特博士 作为神经学，肿瘤学和免疫学交叉点的翻译临床科学家的独立性。 在CAR T细胞疗法中，患者的T细胞被识别癌细胞表面的受体修饰 标记物，并诱导T细胞杀死靶标。数以千计的病人以前几乎没有治愈的希望， 受益于CD 19定向CAR T细胞治疗白血病和淋巴瘤。然而，约40%的人发展为神经系统疾病， 毒性，约1%死于脑水肿。神经毒性的机制知之甚少。在病人身上， 博士Gust已经显示出内皮激活、神经胶质损伤、白细胞浸润和微血管形成的证据。 为了在小鼠中建立神经毒性模型，Gust博士用高剂量的CD 19-CAR T细胞治疗野生型小鼠， 相同近交系的同基因供体小鼠。CAR T处理的小鼠，与用未转导的T 细胞，发展成全身性细胞因子释放、异常行为和广泛的脑微血管病。 总之，人和小鼠的数据表明以下假设： 神经毒性（目标1），神经毒性伴随着脑血流改变（目标2）。 对于目标1，Gust博士将使用单个NVU组分（内皮细胞，致密的）的免疫标记， 连接、周细胞、基底膜、星形胶质细胞端足）并量化细胞数量、形状和邻接性。 她将注射血管内示踪剂，然后通过荧光和电子显微镜观察示踪剂泄漏 通过细胞旁和跨细胞途径。她将评估免疫浸润对BBB的贡献 通过流式细胞术和组织学与基质金属蛋白酶-9的共标记进行分解。如果破坏 NVU通过结构或功能的改变得到证实，我们可以得出结论，它是从系统性的关键环节 炎症导致脑功能障碍，这需要进一步详细的机制研究。对于目标2，Gust博士将 通过体内双光子成像测量小鼠皮质小动脉、毛细血管和小静脉中的血流量， 一个薄的头骨窗。这种创新的方法允许可视化血流动力学与单微血管 通过测量血管直径和红细胞通过速度来确定分辨率。为了验证CAR T 细胞治疗导致白细胞粘附于血管壁，从而减慢血液流动、运输和 将定量表达GFP的CAR T细胞和罗丹明-6G标记的白细胞和血小板的滚动。 微血管血流受损的确认将指导重新考虑治疗方法。 在获奖期间，Gust博士将接受动物模型开发、高级成像 技术，免疫学，血管生物学，实验设计和分析的严谨性，科学交流， 网络和领导技能。在主要导师Andy Shih博士和她的指导下 她将使用该提案中的数据开发R 01应用程序，以过渡到独立。

PROJECT SUMMARY/ABSTRACT Dr. Juliane Gust proposes a study to understand whether perturbations of the BBB and cerebral microvascular perfusion contribute to CAR T cell neurotoxicity. This work will prepare Dr. Gust for independence as a translational clinician-scientist at the intersection of neurology, oncology, and immunology. In CAR T cell therapy, patients’ T cells are modified with a receptor that recognizes cancer cell surface markers, and induces T cell killing of the target. Thousands of patients with previously little hope of cure have benefitted from CD19-directed CAR T cells for leukemia and lymphoma. However, ~40% develop neurologic toxicity, and ~1% die from cerebral edema. The mechanism of neurotoxicity is poorly understood. In patients, Dr. Gust has shown evidence of endothelial activation, glial injury, leukocyte infiltrates, and microhemorrhages. To model neurotoxicity in mice, Dr. Gust treated wild type mice with high dose CD19-CAR T cells made from syngeneic donor mice of the same inbred strain. CAR T treated mice, unlike mice treated with untransduced T cells, develop systemic cytokine release, abnormal behavior, and widespread cerebral microhemorrhages. Taken together, the human and mouse data suggest the following hypotheses: that the BBB is disrupted during neurotoxicity (Aim 1), and that neurotoxicity is accompanied by altered cerebral blood flow (Aim 2). For Aim 1, Dr. Gust will use immunolabeling of individual NVU components (endothelial cells, tight junctions, pericytes, basement membrane, astrocyte endfeet) and quantify cell number, shape, and contiguity. She will inject intravascular tracers followed by fluorescent and electron microscopy to visualize tracer leakage via paracellular and transcellular pathways. She will assess the contribution of immune infiltrates to BBB breakdown by flow cytometry and histology with colabeling for matrix metalloprotease-9. If disruption of the NVU by structural or functional alteration is confirmed, we can conclude that it is a key link from systemic inflammation to brain dysfunction, which warrants further detailed mechanistic studies. For Aim 2, Dr. Gust will measure blood flow in mouse cortical arterioles, capillaries and venules via in-vivo two-photon imaging through a thinned skull window. This innovative approach allows visualization of hemodynamics with single microvessel resolution by measuring vessel diameter and speed of red blood cell transit. To test the hypothesis that CAR T cell treatment leads to leukocyte adherence to vessel walls and consequent slowing of blood flow, transit and rolling of GFP-expressing CAR T cells and Rhodamine-6G labeled leukocytes and platelets will be quantified. Confirmation of impaired microvascular blood flow would guide a reconsideration of therapeutic approaches. During the award period, Dr. Gust will receive training in animal model development, advanced imaging techniques, immunology, vascular biology, rigor in experimental design and analysis, science communication, networking, and leadership skills. Under the guidance of primary mentor Dr. Andy Shih and her mentoring team, she will use data from this proposal to develop an R01 application for transition to independence.