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

嵌合抗原受体 (CAR) T 细胞治疗期间的血脑屏障破坏

• 批准号: 10039479
• 负责人: Juliane Gust
• 金额: $ 19.06万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10039479
• 关键词: Address; Adherence; Adhesions; Affect; Animal Model; Animals; Area; Astrocytes; Award; Basement membrane; Behavior; Biology; Blood; Blood - brain barrier anatomy; Blood Platelets; Blood Vessels; Blood capillaries; Blood flow; Brain; Brain Edema; CAR T cell therapy; CD19 gene; Caliber; Cell Adhesion; Cell Count; Cell surface; Cells; Cellular immunotherapy; Cephalic; Cerebral Edema; Cerebrovascular Circulation; Cerebrum; Chemotherapy and/or radiation; Child Health; Clinical; Communication; Control Groups; Data; Delirium; Dextrans; Dose; Edema; Electroencephalography; Electron Microscopy; Endothelial Cells; Endothelium; Erythrocytes; Experimental Designs; Extravasation; Flow Cytometry; Functional disorder; Goals; Hematologic Neoplasms; Hemorrhage; Histology; Human; Image; Imaging Techniques; Immunology; Immunotherapy; Impaired cognition; Impairment; Inbred Strain; Individual; Inflammation; Infusion procedures; Injury; Knowledge; Label; Leadership; Leukemic Cell; Leukocytes; Life; Link; Lymphoma; Malignant Neoplasms; Measures; Mechanics; Mentors; Metalloproteases; Modeling; Molecular; Mus; Neurologic; Neurologic Dysfunctions; Neurology; Oncology; Pathway interactions; Patient Care; Patients; Perfusion; Pericytes; Preparation; Program Development; Proliferating; Proteins; Refractory; Relapse; Research; Resolution; Risk; Risk Factors; Science; Scientist; Seizures; Severities; Shapes; Speed; Structure; Surface; Syndrome; T-Lymphocyte; Testing; Therapeutic; Thinness; Tight Junctions; Toxic effect; Tracer; Training; Tumor-infiltrating immune cells; Vesicle; Visualization; Wild Type Mouse; Work; animal model development; arteriole; blood-brain barrier disruption; brain dysfunction; brain health; cancer cell; cancer immunotherapy; career; career development; cell killing; cell type; cerebral microbleeds; cerebrovascular; chemotherapy; chimeric antigen receptor; chimeric antigen receptor T cells; cognitive change; common symptom; cranium; cytokine; cytokine release syndrome; experimental analysis; hemodynamics; hypoperfusion; improved; in vivo; in vivo two-photon imaging; innovation; interstitial; leukemia/lymphoma; model development; mouse model; neurotoxicity; neurovascular unit; novel; postcapillary venule; prevent; receptor; rhodamine 6G; skills; therapeutic development; translational physician; vasoconstriction; venule; young adult

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.

项目摘要/摘要 朱利安·古斯特博士提议进行一项研究，以了解血脑屏障和大脑的扰动 微血管灌流导致CAR T细胞神经毒性。这项工作将使古斯特博士为 作为一名神经学、肿瘤学和免疫学交叉学科的翻译临床医生和科学家，具有独立性。 在CAR T细胞疗法中，患者的T细胞被一种识别癌细胞表面的受体修饰 标记，并诱导T细胞对靶细胞的杀伤。数以千计以前治愈希望渺茫的患者已经 受益于CD19导向的CAR T细胞治疗白血病和淋巴瘤。然而，约40%的人会发展为神经性疾病 毒性，约1%死于脑水肿。神经毒性的机制还不是很清楚。在病人身上， 古斯特博士已经证明了血管内皮细胞激活、神经胶质损伤、白细胞渗入和微出血的证据。 为了模拟小鼠的神经毒性，古斯特博士用高剂量的CD19-CAR T细胞治疗野生型小鼠，CD19-CAR T细胞是从 同系近交系的同基因供体小鼠。CAR T处理的小鼠，不同于非转导T处理的小鼠 细胞，出现全身性细胞因子释放，行为异常，以及广泛的脑微出血。 总而言之，人类和老鼠的数据提出了以下假设：血脑屏障在 神经毒性(目标1)，神经毒性伴随脑血流改变(目标2)。 对于目标1，古斯特博士将使用免疫标记单个NVU组件(内皮细胞，紧密 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。 她将在血管内注射示踪剂，然后用荧光和电子显微镜观察示踪剂的泄漏情况 通过细胞旁和跨细胞途径。她将评估免疫渗入对血脑屏障的贡献 用流式细胞术和组织学方法检测基质金属蛋白酶-9。如果网络中断 NVU通过结构或功能的改变得到确认，我们可以从制度上得出结论，它是一个关键环节 炎症到脑功能障碍，这需要进一步的详细机制研究。对于目标2，古斯特博士将 通过体内双光子成像测量小鼠皮质小动脉、毛细血管和小静脉的血流量 一扇变薄的头盖骨窗户。这一创新的方法允许使用单个微血管进行血流动力学可视化 通过测量血管直径和红细胞转运速度来实现分辨率。来检验T车的假设 细胞治疗导致白细胞黏附于血管壁，从而减缓血液流动、转运和 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将被量化。 确认微血管血流受损将指导重新考虑治疗方法。 在获奖期间，古斯特博士将接受动物模型开发、先进成像方面的培训 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、 人际关系和领导能力。在初级导师施安迪博士和她的指导下 她将使用这个提案中的数据来开发一个R01应用程序，以过渡到独立。