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 evi...