Université de Nantes, INSERM, UMR1064, Centre de Recherche en Transplantation et Immunologie, ITUN, 44000, Nantes, France.
PAnTher, INRAE, École nationale vétérinaire, agro-alimentaire et de l'alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), 44307, Nantes, France.
Sci Rep. 2020 Oct 16;10(1):17546. doi: 10.1038/s41598-020-74621-9.
The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe.
创新免疫细胞疗法的发展依赖于高效的细胞跟踪策略。为此，将转染细胞转移到宿主中的多尺度荧光分析与互补技术相结合将非常有益，包括用于高通量细胞分析的流式细胞术和用于深层组织成像的双光子显微镜。理想情况下，细胞应该用单个荧光探针标记，该探针应结合这些不同技术所需的所有特性。由于大多数组织（尤其是肝脏）的固有自发荧光，远红发射也是一个重要的特性。然而，开发适合双光子显微镜且与清除方法兼容的远红发射探针，以跟踪厚样品中的标记免疫细胞仍然具有挑战性。因此，评估了一种新设计的水溶性远红发射聚合物探针 19K-6H，用于跟踪原发性免疫 CD8 T 细胞。这些细胞从小鼠脾脏中制备，可通过内吞作用有效地用 19K-6H 探针标记，该探针在高达 20 μM 的浓度下仍具有高度生物相容性。标记的原发性 CD8 T 细胞即使在 3 天的主动增殖后，也可以通过共聚焦和双光子显微镜以及流式细胞术在培养物中检测到。最后，将 19K-6H 标记的原发性 CD8 T 细胞注射到免疫介导性肝炎的经典小鼠模型中。流式细胞术（在纯化的非实质细胞上）和对 800 μm 厚清除切片的双光子显微镜对转移细胞在肝脏中的高效跟踪，证明了 19K-6H 探针的多功能性。
