Research Article A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells In Vivo Martin Szyska 1 , Stefanie Herda 1 , Stefanie Althoff 1 , Andreas Heimann 1,2 , Josefine Russ 1 , Daniele D'Abundo 1 , Tra My Dang 1 , Isabell Durieux 1 , Bernd D€ orken 1,3,4 , Thomas Blankenstein 2,4,5 , and Il-Kang Na 1,2,3,6 Abstract Adoptive T-cell therapy (ATT) efficacy is limited when target- ing large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an in vivo monitoring tool, allowing the detection of functional para- meters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive Renilla luciferase, which supports concomitant in vivo analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8 þ T cells followed by a burst- like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) in vivo. In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-trans- genic CD4 þ T cells into the tumor, marked by transient acti- vation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expres- sion of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. Cancer Immunol Res; 6(1); 110–20. Ó2018 AACR. Introduction Adoptive T-cell transfer (ATT) has emerged as a promising option in cancer therapy, although first clinical trials have shown mixed results. With CD19-CAR T cells moving into successful clinical trials (1), the picture is changing for many types of cancer. However, ATT for treatment of large solid tumors has limited efficacy due to insufficient cell migration and infiltration into tumor tissues caused by inadequate inflammatory signals, the presence of metabolic factors like lactic acid, and T-cell inhibitory circuits being highjacked by the tumor microenvironment (2–4). Consequently, it has become evident that even an ideal T cell warrants accessory therapeutic interventions in order to overcome large solid tumors. By using antibodies targeting checkpoint inhibitory molecules, the therapeutic tools available for combi- nation with ATT have gained potency surpassing conventional approaches like chemotherapy and irradiation (5, 6). However, the exact impact and optimal therapeutic window of these approaches is difficult to determine without a bulk of experimen- tal animals or advanced noninvasive tools to monitor T-cell fate upon transfer. Bioluminescent imaging (BLI; ref. 7) offers an optimal com- bination between ease-of-use and high sensitivity unrivalled by any other in vivo method, including positron emission tomog- raphy (8). Among the few reporter systems utilizing reporter T cells, most focus on migration and proliferation (9, 10). How- ever, T-cell activation and effector function, targeted by virtu- ally all tumor defense mechanisms, remain elusive. The few existing reporter systems that allow the detection of activated T cells are either not suited for in vivo BLI (11–14) or specifically monitor granzyme expression, an effector function primarily found on cytotoxic CD8 þ T cells (15). Here, we present a transgenic T cell–reporter mouse BLITC (BLI of T cells), that allows the concomitant analysis of T-cell migration and NFAT-dependent activation using dual-lucifer- ase transgenic T cells upon transfer. We implemented this reporter for the study of two independent tumor models and gained important insights into the kinetics of T-cell activation. We propose that our reporter system enables in-depth analysis of T-cell biology in a spectrum of applications not limited to tumor immunology. 1 Experimental and Clinical Research Center (ECRC), Berlin, Germany. 2 Berlin Institute of Health (BIH), Germany. 3 Department of Hematology, Oncology and Tumor Immunology, Charit e – Universit€ atsmedizin Berlin, corporate member of Freie Universit€ at Berlin, Humboldt-Universit€ at zu Berlin, and Berlin Institute of Health, Germany. 4 Max Delbr€ uck Center (MDC) for Molecular Medicine, Berlin, Germany. 5 Institute of Immunology, Charit e, Campus Berlin Buch, Germany. 6 Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Il-Kang Na, Charit e – Campus Virchow-Klinikum, H€ amatologie, Onkologie und Tumorimmunologie, Augustenburger Platz 1, 13353 Berlin. Phone: 49-30-450-540155; Fax: 49-30-450-7540155; E-mail: il-kang.na@charite.de doi: 10.1158/2326-6066.CIR-17-0256 Ó2018 American Association for Cancer Research. Cancer Immunology Research Cancer Immunol Res; 6(1) January 2018 110 on July 13, 2020. © 2018 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst December 19, 2017; DOI: 10.1158/2326-6066.CIR-17-0256