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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要朱莉安·古斯特博士提出了一项研究，以了解血脑屏障和大脑的扰动是否会影响大脑的功能。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。作为神经学、肿瘤学和免疫学交叉领域的转化临床医生科学家的独立性。在 CAR T 细胞疗法中，患者的 T 细胞用识别癌细胞表面的受体进行修饰标记物，并诱导 T 细胞杀死目标。受益于 CD19 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。对患者的神经毒性机制知之甚少。古斯特博士展示了内皮激活、神经胶质损伤、白细胞浸润和微出血的证据。为了模拟小鼠的神经毒性，Gust 博士用高剂量的 CD19-CAR T 细胞治疗野生型小鼠，其中 CD19-CAR T 细胞由与未经转导的 T 治疗的小鼠不同，同一近交系的同基因供体小鼠接受 CAR T 治疗。细胞，出现全身细胞因子释放、异常行为和广泛的脑微出血。综上所述，人类和小鼠的数据表明以下假设：血脑屏障在神经毒性（目标 1），并且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 成分（内皮细胞、紧密连接、周细胞、基底膜、星形胶质细胞末端）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她将通过细胞旁和跨细胞途径评估免疫浸润对 BBB 的贡献。通过流式细胞术和组织学进行分解，并用基质金属蛋白酶-9 进行破坏。 NVU通过结构或功能改变得到证实，我们可以得出结论，它是系统性的关键环节炎症会导致大脑功能障碍，这需要对目标 2 进行进一步详细的机制研究。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这种创新方法可以通过单个微血管实现血流动力学的可视化。通过测量血管直径和红细胞转运速度来检验 CAR T 的假设。细胞治疗导致白细胞粘附在血管壁上，从而减慢血流、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。获奖期间，Gust博士将接受动物模型开发、高级成像方面的培训技术、免疫学、血管生物学、实验设计和分析中的ligor、科学传播、在主要导师 Andy Shih 博士和她的指导下，培养人际网络和领导技能。团队中，她将使用该提案中的数据来开发 R01 应用程序，以实现向独立过渡。