A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function
A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function
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DOI：
10.3389/fimmu.2018.03097
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发表时间：
2019-01-08
影响因子：
7.3
通讯作者：
Lowik, Clemens
中科院分区：
文献类型：
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作者：
Kleinovink, Jan Willem;Mezzanotte, Laura;Lowik, Clemens
用于可视化T细胞的位置和功能的非侵入性成像技术在免疫学中具有重要价值。在这里，我们描述了一种转基因小鼠的设计和产生，其中所有T细胞组成型表达绿色发光的点击甲虫荧光素酶（CBG 99），而红色发光的萤火虫荧光素酶（PpyRE 9）的表达是由活化T细胞的核因子（NFAT）诱导的，例如在T细胞活化过程中，这使得T细胞的位置和功能的生物发光成像。这种双荧光素酶小鼠，我们命名为TbiLuc，在淋巴器官如淋巴结和脾脏中显示出高组成性荧光素酶表达。离体纯化的CD 8+和CD 4 + T细胞均组成型表达荧光素酶，而B细胞未显示可检测的信号。我们将TbiLuc小鼠与T细胞受体转基因OT-I小鼠杂交以获得具有确定的抗原特异性的表达谷胱甘肽酶的幼稚CD 8 + T细胞。TbiLuc*OT-I T细胞显示T细胞活化依赖性荧光素酶的完全抗原特异性诱导。在接种疫苗的小鼠中，我们使用两种不同的荧光素酶底物D-Glucin和CycLuc 1（后者与PpyRE 9酶特异性反应）以高灵敏度观察了疫苗引流淋巴结中的T细胞定位和活化。这种双荧光素酶T细胞报告小鼠可以应用于许多研究T细胞的位置和功能状态的实验模型。
Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naive CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.