%A Kleinovink,Jan Willem %A Mezzanotte,Laura %A Zambito,Giorgia %A Fransen,Marieke F. %A Cruz,Luis J. %A Verbeek,J. Sjef %A Chan,Alan %A Ossendorp,Ferry %A Löwik,Clemens %D 2019 %J Frontiers in Immunology %C %F %G English %K T cell,activation,Mouse,Dual-color,luciferase,bioluminescence,imaging,transgenic,reporter,NFAT %Q %R 10.3389/fimmu.2018.03097 %W %L %M %P %7 %8 2019-January-08 %9 Original Research %# %! Dual-color T cell imaging %* %< %T A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function %U https://www.frontiersin.org/articles/10.3389/fimmu.2018.03097 %V 9 %0 JOURNAL ARTICLE %@ 1664-3224 %X 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 naïve 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.