Cellular Multispectral Optoacoustic Tomography (MSOT) for therapy monitoring of adoptively transferred T-Lymphocytes

过继性T细胞转移是治疗恶性肿瘤或感染性疾病的一种有前途的方法。抗原特异性细胞毒性T淋巴细胞（CTL）已被鉴定为负责消除病毒（如巨细胞病毒（CMV）或EB病毒（EBV））和介导肿瘤消退（例如，在霍奇金斯病或转移性黑色素瘤中。新的基于T细胞的疗法的出现首先需要定量和定性评估T细胞向靶标的迁移，其次是它们在生物体中的分布。用过继转移的T细胞治疗的患者的免疫监测可以通过使用主要组织相容性（MHC）/肽多聚体的流式细胞术和通过分子方法来实现，以分别检测T细胞受体和引入的标记基因。这些方法可应用于体内追踪T细胞，例如，在外周血淋巴结和肿瘤部位然而，这些技术不允许直接体内追踪CTL，而是需要血液样品或组织活检。用于体内测定T细胞运输和治疗效果的理想免疫监测方法应该是非侵入性和无毒的程序。然而，在临床上没有广泛可用的技术提供功能性分子成像数据，这对于准确跟踪和监测生物分布至关重要。这可以通过使用具有仔细选择和定制的造影剂的光学方法来实现。一种称为多光谱光声层析成像系统（MSOT）的最新进展（在活体动物中真实的时间内映射和识别不同的光吸收剂方面显示出有前途的性能，使用解混技术进行光谱识别，能够进行分子和功能成像，同时收集解剖数据，这要归功于血红蛋白、氧合血红蛋白和脂肪的特定吸收光谱。这导致了对光吸收剂的行为和生物分布的新见解，例如FDA批准的荧光团ICG，基于dPGS的荧光团或纳米颗粒如金纳米结构。本项目的目的是评估MSOT作为体内基于细胞的免疫治疗的治疗监测的成像模式。细胞毒性T淋巴细胞将用荧光团和金纳米颗粒标记，并使用MSOT在鼠肿瘤模型中可视化。此外，T细胞特异性抗体将与金纳米颗粒缀合，并且这些将在体内T细胞转移后作为造影剂进行测试。将对转移细胞的存活和这些细胞在肿瘤组织中的特异性积聚进行组织学验证，并分析不同造影剂对T淋巴细胞功能的影响。

The adoptive T-cell transfer is a promising approach for the treatment of patients with malignant tumors or infectious diseases. Antigen-specific cytotoxic T lymphocytes (CTLs) have been identified as potent effectors that are responsible for eliminating viruses, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV), and for mediating tumor regression, e.g., in Hodgkins disease or metastatic melanoma. The advent of new T cell-based therapies requires the quantitative and qualitative assessment of firstly the T-cell migration to the target and secondly their distribution in the organism. The immunological monitoring of patients treated with adoptively transferred T cells can be achieved by flow cytometry with major histocompatibility (MHC)/peptide multimers and by molecular methods in order to detect the T-cell receptors and the introduced marker genes, respectively. These methods can be applied to track T cells in vivo, e.g., in the peripheral blood, the lymph nodes, and the tumor sites. However, these techniques do not allow the direct in vivo tracking of CTLs, but require blood samples or tissue biopsies. The ideal immunomonitoring method for the in vivo determination of both T-cell trafficking and therapeutic effectiveness should be a non-invasive and non-toxic procedure. However, no technology available widely in the clinic provides functional molecular imaging data, crucial for accurate tracking and monitoring of biodistribution. This can be enabled by the use of optical methods with carefully chosen and tailored contrast agents. A recent evolution called multispectral optoacoustic tomography system (MSOT)(shows promising performance in mapping and identifying different photoabsorbers in real time in a living animal, using unmixing technique for spectral identification, enabling molecular and functional imaging while simultaneously gathering anatomical data thanks to the specific absorbance spectra of hemoglobin, oxygenated hemoglobin and fat notably. This led to new insights in the behavior and biodistribution of photoabsorbers such as the FDA approved fluorophore ICG, dPGS based fluorophores or nanoparticles like gold nanostructures. The aim of this project is the evaluation of MSOT as imaging modality for therapy monitoring of cell-based immunotherapy in vivo. Cytotoxic T-lymphocytes will be labeled with fluorophores and gold nanoparticles and visualized in a murine tumor model using MSOT. Furthermore, T-cell specific antibodies will be conjugated to gold nanoparticles, and these will be tested as contrast agent after T-cell transfer in vivo. The survival of the transferred cells and the specific accumulation of these cells in the tumor tissue will be histologically verified, and the influence of the different contrast agents to the function of T-lymphocytes will be analyzed.