Endothelial-Leukocyte Adhesion in CAR T Cell Treatment Associated Neurotoxicity

项目总结 本项目研究嵌合抗原受体(CAR)T细胞的神经毒性机制 心理治疗。CAR T细胞是经过基因改造的患者来源的T细胞，使用CAR识别和破坏 恶性靶细胞。尽管CAR T疗法在治疗白血病和淋巴瘤方面取得了令人印象深刻的效果， 大约30%-40%的患者在接受CD19-1治疗后的第一个月内出现神经副作用。 靶向CAR T细胞。这包括认知障碍、癫痫发作，在极少数情况下还包括致命性脑水肿。 CAR T细胞输注后的全身性细胞因子释放综合征是神经毒性的公认危险因素， 但全身性炎症和大脑功能障碍之间的联系还知之甚少。 为了研究神经毒性的机制，我们建立了一个免疫活性的小鼠模型。之后 用大剂量CD19导向的小鼠CAR T细胞治疗，小鼠出现运动和平衡困难，如 以及脑部微出血。令人惊讶的是，我们发现10%的皮质毛细血管被白色遮挡 神经毒性过程中的血细胞。伴随而来的是毛细血管重塑和血管覆盖率降低。 通过周细胞。基于这些发现，我们现在提出以下实验： 目的1：是什么分子机制导致白细胞在神经毒性过程中堵塞毛细血管？我们 将在小鼠体内使用双光子成像来确定导致毛细血管堵塞的细胞类型-- 老鼠自己的T细胞还是移植来的汽车T细胞？然后我们将测量CAR T细胞治疗如何改变 黏附分子在脑毛细血管内皮细胞和白细胞的表达，并检测 阻断这些黏附相互作用可以防止毛细血管堵塞和神经毒性。 目的2：神经毒性过程中人微血管内皮细胞与白细胞的黏附增加吗？在……里面 与我们在老鼠身上所做的工作类似，我们将使用人脑毛细血管的3D体外模型来测量其溶解程度 患者血浆中的因素影响内皮细胞黏附分子的表达。然后我们将测试一下 有神经毒性的CAR T细胞患者的白细胞更倾向于堵塞合成的 模拟毛细血管的微血管，以及我们是否可以通过阻止黏附分子来防止这种堵塞。 目的3：细胞间黏附信号的强度能区分CAR T细胞的有效性和毒性吗？我们会 用定量多重免疫沉淀检测CAR T细胞中的蛋白质-蛋白质相互作用网络 对小鼠造成高或低的神经毒性，以了解什么激活状态有利于神经毒性。我们 然后将测试是否下调黏附分子的表达可以引导CAR T细胞远离毒性 表型通过削弱它们向其他细胞发出信号的能力以及附着脑微血管的能力来实现。 这项工作具有创新性，因为它结合了先进的成像、体外建模技术和蛋白质 神经科学、血管生物学和肿瘤学之间的独特合作中的网络分析。这项工作是 它具有重要意义，因为它解决了新出现的癌症免疫疗法中的关键安全问题。

PROJECT SUMMARY This project studies the mechanism of neurologic toxicity in chimeric antigen receptor (CAR) T cell therapy. CAR T cells are genetically modified, patient derived T cells that use the CAR to recognize and destroy malignant target cells. Although CAR T therapy has shown impressive results against leukemia and lymphoma, approximately 30-40% of patients experience neurologic side effects in the first month after receiving CD19- targeted CAR T cells. This includes cognitive disturbances, seizures, and in rare cases fatal cerebral edema. Systemic cytokine release syndrome after CAR T cell infusion is a well-established risk factor for neurotoxicity, but the connection between systemic inflammation and brain dysfunction is poorly understood. To study the mechanisms of neurotoxicity, we have developed an immunocompetent mouse model. After treatment with high dose CD19-directed murine CAR T cells, mice develop motor and balance difficulties, as well as brain microhemorrhages. Surprisingly, we found that >10% of cortical capillaries are obstructed by white blood cells during neurotoxicity. This was accompanied by capillary remodeling and decreased vessel coverage by pericytes. Based on these findings, we now propose the following experiments: Aim 1: What molecular mechanisms cause white blood cells to plug capillaries during neurotoxicity? We will use in vivo two-photon imaging in mice to determine which cell types cause the capillary plugging – the mouse’s own or the transferred CAR T cells? We will then measure how CAR T cell treatment changes the expression of adhesion molecules in brain capillary endothelial cells and in leukocytes, and test whether blockade of these adhesion interactions can prevent capillary plugging and neurotoxicity. Aim 2: Is neuroendothelial-leukocyte adhesion increase in human microvessels during neurotoxicity? In parallel to our work in mice, we will use a 3D in vitro model of human brain capillaries to measure how soluble factors in patient plasma affect adhesion molecule expression in the endothelium. We will then test whether white blood cells from CAR T cell patients with neurotoxicity have increased predilection for plugging synthetic microvessels that mimic capillaries, and whether we can prevent this plugging by blocking adhesion molecules. Aim 3: Can the strength of cell-cell adhesion signaling separate CAR T cell efficacy from toxicity? We will use quantitative multiplex immunoprecipitation to probe protein-protein interaction networks in CAR T cells that cause high or low neurotoxicity in mice to understand what activation states are conducive to neurotoxicity. We will then test whether knock down of adhesion molecule expression can direct CAR T cells away from a toxicity phenotype by impairing their ability to signal to other cells, and to adhere to the brain microvasculature. This work is innovative because it combines advanced imaging, in vitro modeling techniques, and protein network analysis in a unique collaboration between neuroscience, vascular biology, and oncology. The work is significant because it addresses key safety issues in emerging cancer immunotherapy modalities.