Research Project
10249232
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.
