table_1_Photoconversion of Alloreactive T Cells in Murine Peyer’s Patches During Acute Graft-Versus-Host Disease: Tracking the Homing Route of Highly Proliferative Cells In Vivo.docx

The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer’s patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer’s patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions.

免疫细胞在全身范围内的迁移调控对于保障免疫监视和维持免疫稳态至关重要。对这些细胞的标记与追踪已被证明对于研究免疫细胞递送机制及体内细胞间相互作用具有重要意义。光转换技术因其能够在不手术干预相关细胞微环境的前提下，实现对组织内细胞的无接触、时间和空间特异性标记，而成为体内应用的理想技术。然而，在分裂细胞中，转化的荧光蛋白可能会迅速衰减。在此，我们详细阐述了光转换技术的原理及其在追踪高度增殖的T细胞从T细胞激活的启动位点至效应功能的外周靶器官的体内应用。在急性移植物抗宿主病（GvHD）的启动阶段，对Dendra2+ T细胞在派伊尔斑进行光转换，并通过流式细胞术和活体双光子显微镜追踪其通过肠系膜淋巴结和周围血液至小肠。光转换的移植物抗宿主T细胞在肠黏膜固有层中保留了完整的增殖能力、归巢和迁移特性。我们最终证实，派伊尔斑中高度增殖的移植物抗宿主T细胞的光转换是研究移植物抗宿主T细胞在生理条件下的递送以及至GvHD靶组织的有效工具。此技术亦适用于研究炎症和非炎症条件下免疫细胞的追踪。