Understanding the in vivo impact of immunotherapies in splenic lymphoma by intravital three-photon microscopy

通过活体三光子显微镜了解免疫疗法对脾淋巴瘤的体内影响

基本信息

批准号：
10576013
负责人：
CHRIS XU
金额：
$ 23.39万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-03 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10576013
关键词：
Adult Affect Aftercare Antibodies Apoptosis B-Cell Lymphomas B-Lymphocytes BCL2 gene Blood Vessels Bone Marrow Brain CAR T cell therapy CD8B1 gene Cell Communication Cell Therapy Cells Chronic Color Detection EZH2 gene FDA approved Fluorescence Follicular Dendritic Cells Follicular Lymphoma Generations Glass Goals Helper-Inducer T-Lymphocyte Hematopoietic Neoplasms Human Image Immune Immune response Immune system Immunologic Surveillance Immunotherapeutic agent Immunotherapy Implant Individual Label Lasers Lymphoma Lymphoma cell Lymphomagenesis Malignant lymphoid neoplasm Malpighian corpuscles Methods Microscopy Motion Mus Nature Optics Patients Penetration Photons Physiologic pulse Process Regulatory T-Lymphocyte Reporter Research Resistance Resolution Respiration Signal Transduction Somatic Mutation Speed Spleen System T-Lymphocyte Therapeutic Time Tissue imaging Tissues Transplantation Tumor Promotion Visualization blood vessel visualization cell motility cell type chemotherapy chimeric antigen receptor T cells data acquisition design detector effector T cell epigenetic therapy imaging capabilities imaging system immune activation in vivo in vivo imaging inhibitor intravital imaging intravital microscopy large cell Diffuse non-Hodgkin&apos s lymphoma lymph nodes meter mouse model mutant neoplastic cell recruit response restoration serial imaging side effect three photon microscopy tumor two-photon

项目摘要

Diffuse large B cell lymphoma (DLBCL) and the follicular lymphoma (FL) are the most common lymphoid malignancy in adults. EZH2 mutant occurs in DLBCL and FL patients. Immunotherapies, EZH2 inhibitor and CAR T cells are FDA approved for FL and DLBCL, respectively. Understanding why patients respond or are resistant to the immunotherapies is essential for these approaches to reach their full potential. However, it is unclear whether the mechanism of EZH2i action is cell autonomous or through restoration of immune surveillance and how EZH2i affects the ability of CAR T cells to kill lymphoma because there has not been visualized the process of immune surveillance against lymphoma in vivo. In particular, the white pulps of mouse spleen, key regions of lymphomagenesis, are challenging to visualize in vivo due to the limited imaging penetration depth of conventional two-photon (2P) microscopy. Leveraging deep tissue imaging capability of three-photon (3P) microscopy that provides twice deeper imaging depth than 2P microscopy, we aim to in vivo visualize the dynamic interplay of lymphoma B cells with their microenvironments and CAR T cells in the spleen upon EZH2i treatment. To this end, we propose the following specific aims: (1) develop simultaneous 6-color and longitudinal 3P microscopy approach for intravital imaging of the white pulps in mouse spleen and (2) perform the intravital 3P microscopy for understanding the impact of EZH2 inhibitors on reactivation of anti-lymphoma response and their impact on enhancing CAR T cell therapy. In Aim 1, first of all, we will determine permissible laser parameters to optimize the imaging speed for tracking individual immune cell migration in a mouse spleen because high average power and pulse energy of laser excitation required for deep and fast imaging can cause thermal and focal damages. Second, we will develop an implantable chronic imaging window for longitudinal imaging of mouse spleen for several weeks to several months. Third, we will develop simultaneous 6-color 3P imaging system to visualize the interaction among 6 different cell types by adding 2 more detection channels in the current 4-color 3P imaging system. In Aim 2, we will perform the simultaneous 6-color and longitudinal 3P microscopy to visualize the dynamic interaction of lymphoma B cells with helper T cells, regulatory T cells, CD8 effector T cells, follicular dendritic cells, and blood vessels in the spleen of syngeneic lymphoma mouse model upon EZH2i or control vehicle treatment. In addition, we will also visualize the interplay of lymphoma B cells with CAR T cells and the following apoptosis of the tumor cells upon EZH2i or vehicle treatment to understand how EZH2i affects the ability of CAR T cell to kill the tumor cells. Collectively, this study will demonstrate 6-color and longitudinal 3P microscopy for intravital imaging of the splenic immune system with tumors, for the first time. The imaging results will provide the first direct evidence for the nature of the anti-lymphoma immune response and the manner in which lymphomas evade host immune cells and CART cells, allowing us to generate new hypotheses for the design of combination immune therapies.

弥漫性大 B 细胞淋巴瘤 (DLBCL) 和滤泡性淋巴瘤 (FL) 是最常见的淋巴癌成人恶性肿瘤。 EZH2 突变发生在 DLBCL 和 FL 患者中。免疫疗法、EZH2抑制剂和CAR T 细胞分别获得 FDA 批准用于治疗 FL 和 DLBCL。了解患者为何有反应或耐药免疫疗法对于这些方法充分发挥其潜力至关重要。然而尚不清楚 EZH2i 的作用机制是细胞自主的还是通过免疫监视的恢复以及 EZH2i 如何影响 CAR T 细胞杀死淋巴瘤的能力，因为该过程尚未可视化体内针对淋巴瘤的免疫监视。特别是小鼠脾脏的白髓，由于淋巴瘤发生的成像穿透深度有限，在体内可视化具有挑战性传统的双光子（2P）显微镜。利用三光子 (3P) 的深层组织成像能力显微镜提供比 2P 显微镜更深两倍的成像深度，我们的目标是体内可视化动态 EZH2i 治疗后淋巴瘤 B 细胞与其微环境和脾脏中 CAR T 细胞的相互作用。为此，我们提出以下具体目标：（1）发展同步6色和纵向3P 用于小鼠脾脏白髓活体成像的显微镜方法和（2）执行活体 3P 显微镜用于了解 EZH2 抑制剂对抗淋巴瘤反应再激活的影响及其作用对增强 CAR T 细胞治疗的影响。在目标 1 中，首先，我们将确定允许的激光参数优化成像速度以跟踪小鼠脾脏中的个体免疫细胞迁移，因为高深度和快速成像所需的激光激发的平均功率和脉冲能量可能会导致热和局灶性损伤。其次，我们将开发一种可植入的慢性成像窗口，用于纵向成像小鼠脾脏持续数周至数月。第三，我们将开发同步6色3P成像系统通过在当前的系统中添加 2 个检测通道来可视化 6 种不同细胞类型之间的相互作用 4色3P成像系统。在目标 2 中，我们将进行同步 6 色和纵向 3P 显微镜检查可视化淋巴瘤 B 细胞与辅助 T 细胞、调节性 T 细胞、CD8 效应 T 细胞的动态相互作用基于 EZH2i 的同基因淋巴瘤小鼠模型脾细胞、滤泡树突状细胞和血管或控制车辆处理。此外，我们还将可视化淋巴瘤 B 细胞与 CAR T 细胞的相互作用以及 EZH2i 或媒介物治疗后肿瘤细胞的凋亡，以了解 EZH2i 如何影响 CAR T细胞杀死肿瘤细胞的能力。总的来说，这项研究将展示 6 色和纵向首次使用 3P 显微镜对肿瘤脾免疫系统进行活体成像。成像结果将为抗淋巴瘤免疫反应的性质和方式提供第一个直接证据其中淋巴瘤逃避宿主免疫细胞和 CART 细胞，这使我们能够产生新的假设联合免疫疗法的设计。