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)是最常见的淋巴瘤 成人的恶性疾病。DLBCL和FL患者存在EZH2突变。免疫疗法、EZH2抑制剂和CAR T细胞分别被FDA批准用于FL和DLBCL。了解患者反应或抵抗的原因 免疫疗法是这些方法充分发挥其潜力的关键。然而，目前还不清楚 无论EZH2 i的作用机制是细胞自主的还是通过恢复免疫监视和 EZH2 i如何影响CAR T细胞杀伤淋巴瘤的能力，因为这一过程尚未可视化 在体内对淋巴瘤的免疫监测。尤其是小鼠脾的白色牙髓，关键区域 淋巴肿大，由于其成像穿透深度有限，在活体内的可视化具有挑战性。 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显微镜深两倍的成像深度，我们的目标是在体内可视化动态 EZH2治疗后淋巴瘤B细胞及其微环境与脾CAR T细胞的相互作用 为此，我们提出了以下具体目标：(1)同时开发六色和纵向3P 小鼠脾白髓活体成像的显微镜方法及(2)活体3P 了解EZH2抑制剂对抗淋巴瘤再激活的影响及其作用的显微镜观察 对加强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以 优化成像速度以跟踪单个免疫细胞在小鼠脾中的迁移，因为 深度和快速成像所需的激光激励的平均功率和脉冲能量会导致热和 局灶性损伤。第二，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 系统通过在当前增加2个检测通道来可视化6种不同细胞类型之间的交互 四色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 可视化淋巴瘤B细胞与辅助性T细胞、调节性T细胞、CD8效应器T的动态相互作用 EZH_2对同基因淋巴瘤小鼠脾细胞、滤泡树突状细胞和血管的影响 或控制车辆处理。此外，我们还将可视化淋巴瘤B细胞和CAR T细胞之间的相互作用 以及EZH2 i或赋形剂治疗后肿瘤细胞的下列凋亡情况，以了解EZH2 i如何影响 CAR T细胞杀伤肿瘤细胞的能力。总而言之，这项研究将展示六色和纵向 3P显微镜首次用于活体成像脾肿瘤免疫系统。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 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.