A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells In Vivo.

Martin Szyska,Isabell Durieux,Il-Kang Na,Andreas Heimann,Tra My Dang,Bernd Dörken,Josefine Russ,Thomas Blankenstein,Stefanie Herda,Stefanie Althoff,Daniele D'Abundo

doi:10.1158/2326-6066.cir-17-0256

Abstract

Adoptive T-cell therapy (ATT) efficacy is limited when targeting 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 parameters 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+ 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-transgenic CD4+ T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression 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. ©2018 AACR.

Highlights

Adoptive T-cell transfer (ATT) has emerged as a promising option in cancer therapy, first clinical trials have shown mixed results
bioluminescence imaging of T cells (BLITC) reporter: in vitro and in vivo features After generating the NFAT-click beetle luciferase (CBR) transgenic mouse (Fig. 1A) and successfully testing its suitability for detecting in vivo T-cell activation in a classical (C57BL/6 to Balb/c) major mismatch setting (Supplementary Fig. S1), we proceeded by crossing NFAT-CBR mice with ChRluc mice [10] that constitutively express Renilla luciferase (Rluc) to enable detection of bulk T-cell accumulation and migration
Antigen-specific stimulation, using dendritic cells (DCs) pulsed with a dilution series of H-Y peptide, revealed high specificity for the cognate antigen and sensitivity comparable to standard activation markers detected via flow cytometry but surpassed by ELISA detection of IL2 and IFNy, as shown in Supplementary Fig. S2A and S2B for MH-BLITC

Summary

Introduction

Adoptive T-cell transfer (ATT) has emerged as a promising option in cancer therapy, first clinical trials have shown mixed results. 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 [2,3,4]. By using antibodies targeting checkpoint inhibitory molecules, the therapeutic tools available for combination with ATT have gained potency surpassing conventional approaches like chemotherapy and irradiation [5, 6]. The exact impact and optimal therapeutic window of these approaches is difficult to determine without a bulk of experimental animals or advanced noninvasive tools to monitor T-cell fate upon transfer

Methods

Results

Conclusion