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（Juliane 微血管灌注有助于CAR T细胞神经毒性。这项工作将为Gust博士做好准备 独立在神经学，肿瘤学和免疫学的交集中是翻译的临床科学家。 在CAR T细胞疗法中，患者的T细胞通过识别癌细胞表面的接收器修饰 标记，并诱导靶标的T细胞杀死。数千名以前没有治愈希望的患者有 受益于CD19指导的CAR T细胞，用于白血病和淋巴瘤。但是，大约40％的神经系统 毒性，〜1％死于脑水肿。神经毒性的机制知之甚少。在患者中 Gust博士表明了内皮激活，神经胶质损伤，白细胞浸润和微毛的证据。 为了模拟小鼠的神经毒性，阵风博士用由高剂量CD19卡尔T细胞处理的野生型小鼠 同一近交菌株的合成供体小鼠。与未转导的T治疗的小鼠不同 细胞，发展全身细胞因子释放，异常行为和宽度脑微视力。 综上所述，人类和鼠标的数据提出了以下假设： 神经毒性（AIM 1），并且神经毒性伴随着脑血流的改变（AIM 2）。 对于AIM 1，Gust博士将使用单个NVU组件的免疫标记（内皮细胞，紧密 连接，周细胞，地下膜，星形胶质细胞端毛）和量化细胞数，形状和连续性。 她将注入血管内示踪剂，然后进行荧光和电子显微镜，以可视化示踪剂泄漏 通过细胞细胞和跨细胞途径。她将评估免疫浸润对BBB的贡献 流式细胞术和组织学因基质金属蛋白酶9的分解。如果破坏了 NVU通过结构或功能变化得到了确认，我们可以包括它是系统性的关键链接 对脑功能障碍的炎症，需要进一步详细的机理研究。对于AIM 2，Gust博士将 通过体内两光子成像，测量小鼠皮质艺术，毛细血管和静脉的血液流动 一个稀薄的头骨窗。这种创新的方法允许使用单微血管可视化血液动力学 通过测量血管直径和红细胞运输速度的分辨率。测试汽车T的假设 细胞处理导致白细胞遵守血管壁，因此血液流动，过境和 将量化表达GFP的CAR T细胞和标记为白细胞和血小板的Rhodamine-6G的滚动。 确认微血管血流受损将指导治疗方法的重新考虑。 在奖励期内，Gust博士将接受动物模型开发，高级成像的培训 技术，免疫学，血管生物学，实验设计和分析中的严格，科学沟通， 网络和领导技能。在主要导师安迪·希赫（Andy Shih）和她的指导下的指导下 团队，她将使用此提案中的数据来开发R01申请，以过渡到独立性。