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

弥漫性大B细胞淋巴瘤（DLBCL）和滤泡性淋巴瘤（FL）是最常见的淋巴样 成人恶性肿瘤。DLBCL和FL患者中存在EZH2突变。免疫疗法、EZH 2抑制剂和CAR T细胞分别被FDA批准用于FL和DLBCL。了解患者为何有反应或耐药 对于这些方法充分发挥其潜力至关重要。但目前尚不清楚 EZH2i的作用机制是细胞自主的还是通过恢复免疫监视， EZH2i如何影响CAR T细胞杀死淋巴瘤的能力，因为还没有可视化的过程 对淋巴瘤的免疫监视。特别是，小鼠脾脏的白色髓， 淋巴瘤形成，是具有挑战性的可视化在体内由于有限的成像穿透深度， 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显微镜深两倍的成像深度，我们的目标是在体内可视化动态 淋巴瘤B细胞与它们的微环境和CAR T细胞在EZH2i处理后在脾中的相互作用。 为此，我们提出以下具体目标：（1）开发同步6色和纵向3P 小鼠脾脏中白色髓活体成像的显微镜方法和（2）进行活体3 P 用于理解EZH2抑制剂对抗淋巴瘤应答的再激活的影响及其 对增强CAR T细胞疗法的影响。在目标1中，首先，我们将确定允许的激光参数， 优化成像速度以追踪小鼠脾脏中的单个免疫细胞迁移， 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引起热和 局灶性损伤其次，我们将开发一种植入式慢性成像窗口，用于纵向成像， 小鼠脾脏数周至数月。第三，我们将开发同时6色3P成像 通过在电流中增加2个检测通道， 4-彩色3P成像系统。在目标2中，我们将同时进行6色和纵向3P显微镜 为了观察淋巴瘤B细胞与辅助性T细胞、调节性T细胞、CD8效应T细胞的动态相互作用， EZH2i后同基因淋巴瘤小鼠模型脾脏中的细胞、滤泡树突状细胞和血管 或对照载体处理。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或媒介物处理后肿瘤细胞的凋亡，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，这项研究将证明6色和纵向 3P显微镜首次用于肿瘤脾免疫系统的活体成像。成像 结果将为抗淋巴瘤免疫应答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，使我们能够产生新的假设， 联合免疫疗法的设计。

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.