Institute for Experimental Immunology and Imaging, University Hospital Essen, University Duisburg-Essen, Hufelandstraße 55, D-45122, Essen, Germany.
Institute for Virology, University Hospital Essen, University Duisburg-Essen, Hufelandstraße 55, D-45122, Essen, Germany.
J Mol Med (Berl). 2018 Apr;96(3-4):349-360. doi: 10.1007/s00109-018-1628-7. Epub 2018 Feb 19.
Adoptive cell transfer approaches for antigen-specific CD8 T cells are used widely to study their effector potential during infections or cancer. However, contemporary methodological adaptations regarding transferred cell numbers, advanced imaging, and the 3R principle of animal research have been largely omitted. Here, we introduce an improved cell transfer method that reduces the number of donor animals substantially and fulfills the requirements for intravital imaging under physiological conditions. For this, we analyzed the well-established Friend retrovirus (FV) mouse model. Donor mice that expressed a FV-specific T cell receptor (TCR) and the fluorescent protein tdTomato were used as source of antigen-specific CD8 T cells. Only a few drops of peripheral blood were sufficient to isolate ~ 150,000 naive reporter cells from which 1000 were adoptively transferred into recently FV-infected recipients. The cells became activated and functional and expanded strongly in the spleen and bone marrow within 10 days post infection. Transferred CD8 T cells participated in the antiviral host response within a natural range and developed an effector phenotype indistinguishable from endogenous effector CD8 T cells. Additionally, the generated reporter cell frequency allowed single cell visualization and tracking of a physiological antiretroviral CD8 T cell response by intravital two-photon microscopy. Highly reproducible results were obtained in independent experiments by reusing the same donors repetitively for multiple transfers. Our approach allows a strong reduction of experimental animals required for studies on antigen-specific CD8 T cell function and should be applicable to other transfer models.
TCR CD8 T cells are obtained repetitively from the blood samples of single donors. One thousand transferred TCR CD8 T cells get activated, are functional, and proliferate. Several adoptive cell transfers from the same donor show reproducible results. One thousand transferred cells take part in the FV immune response without modifying it. Use of fluorescent transfer cells allows in vivo imaging and single cell tracking.
针对抗原特异性 CD8 T 细胞的过继细胞转移方法被广泛用于研究其在感染或癌症期间的效应功能。然而，关于转移细胞数量、高级成像和动物研究的 3R 原则的当代方法学适应在很大程度上被忽略了。在这里，我们介绍了一种改进的细胞转移方法，该方法大大减少了供体动物的数量，并满足了在生理条件下进行活体成像的要求。为此，我们分析了成熟的 Friend 逆转录病毒（FV）小鼠模型。表达 FV 特异性 T 细胞受体（TCR）和荧光蛋白 tdTomato 的供体小鼠被用作抗原特异性 CD8 T 细胞的来源。只需几滴外周血即可从供体小鼠中分离出约 15 万个幼稚的报告细胞，其中 1000 个被过继转移到最近感染 FV 的受体小鼠中。这些细胞在感染后 10 天内在脾脏和骨髓中被激活并发挥功能，强烈扩增。转移的 CD8 T 细胞参与了抗病毒的宿主反应，其效应表型与内源性效应 CD8 T 细胞无法区分。此外，所产生的报告细胞频率允许通过活体双光子显微镜对生理抗病毒 CD8 T 细胞反应进行单细胞可视化和跟踪。通过重复使用相同的供体进行多次转移，在独立实验中可获得高度可重复的结果。我们的方法允许大大减少研究抗原特异性 CD8 T 细胞功能所需的实验动物数量，并且应该适用于其他转移模型。
TCR CD8 T 细胞可从单个供体的血液样本中重复获得。一千个转移的 TCR CD8 T 细胞被激活、具有功能并增殖。来自同一供体的多次过继转移显示出可重复的结果。一千个转移细胞参与 FV 免疫反应而不会改变它。使用荧光转移细胞可进行体内成像和单细胞跟踪。
