理由：神经胶质瘤等内在脑肿瘤在很大程度上对包括免疫检查点阻断在内的免疫疗法具有抵抗力。过继细胞疗法（ACT）包括针对神经胶质瘤相关抗原的嵌合抗原受体（CAR）或T细胞受体（TCR）转基因T细胞疗法是神经胶质瘤免疫治疗的一个新兴领域。然而，目前缺乏对归巢到神经胶质瘤微环境的过继转移 T 细胞进行非侵入性监测的成像技术。方法：超小的氧化铁纳米粒子 (NP) 可以通过磁共振成像 (MRI) 和专用 MRI 序列（例如 T2* 映射）进行非侵入性可视化。在这里，我们开发了一种用氧化铁纳米颗粒对小鼠和人类 TCR 转基因细胞和 CAR T 细胞进行有效离体标记的方案。我们通过流式细胞术和透射电子显微镜 (TEM) 评估标记效率和 T 细胞功能。过继 T 细胞转移后，通过体内 9.4 T 的 MRI 使 NP 标记的 T 细胞可视化，并与通过光片显微镜 (LSM) 获得的透明大脑 3D 模型相关联。结果：NP 以高标记效率整合到亚细胞胞质囊泡中的 T 细胞中，不会干扰 T 细胞活力、增殖和效应功能（通过细胞因子分泌和体外抗原特异性杀伤测定进行评估）。我们进一步证明，在小鼠神经胶质瘤模型中，过继转移的 T 细胞可以通过 9.4 特斯拉的高场 MRI 进行纵向肿瘤内监测，具有高灵敏度。我们发现，通过 T2* 成像评估，T 细胞流入和 TME 内 T 细胞的均匀空间分布可预测肿瘤对 ACT 的反应，而不完整的 T 细胞覆盖会导致治疗耐药。结论：本研究展示了通过氧化铁纳米颗粒对胶质瘤中的过继性 T 细胞疗法进行非侵入性监测的合理性，以跟踪肿瘤内 T 细胞流入并最终预测治疗结果。© 作者。

Rationale: Intrinsic brain tumors, such as gliomas are largely resistant to immunotherapies including immune checkpoint blockade. Adoptive cell therapies (ACT) including chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cell therapy targeting glioma-associated antigens are an emerging field in glioma immunotherapy. However, imaging techniques for non-invasive monitoring of adoptively transferred T cells homing to the glioma microenvironment are currently lacking. Methods: Ultrasmall iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Here, we develop a protocol for efficient ex vivo labeling of murine and human TCR-transgenic and CAR T cells with iron oxide NPs. We assess labeling efficiency and T cell functionality by flow cytometry and transmission electron microscopy (TEM). NP labeled T cells are visualized by MRI at 9.4 T in vivo after adoptive T cell transfer and correlated with 3D models of cleared brains obtained by light sheet microscopy (LSM). Results: NP are incorporated into T cells in subcellular cytoplasmic vesicles with high labeling efficiency without interfering with T cell viability, proliferation and effector function as assessed by cytokine secretion and antigen-specific killing assays in vitro. We further demonstrate that adoptively transferred T cells can be longitudinally monitored intratumorally by high field MRI at 9.4 Tesla in a murine glioma model with high sensitivity. We find that T cell influx and homogenous spatial distribution of T cells within the TME as assessed by T2* imaging predicts tumor response to ACT whereas incomplete T cell coverage results in treatment resistance. Conclusion: This study showcases a rational for monitoring adoptive T cell therapies non-invasively by iron oxide NP in gliomas to track intratumoral T cell influx and ultimately predict treatment outcome.© The author(s).