A novel TOX-nanoluciferase reporter mouse for exploring modulators of T cell exhaustion

Cytotoxic T cell (CTL) exhaustion is driven by chronic T cell receptor (TCR) stimulation, leading to a dysfunctional state of cells. Exhausted CTLs exhibit diminished effector function against chronic infections and cancers. Therefore, reducing CTL exhaustion may re-establish effective adaptive immune responses. One feature of exhausted CTLs is the sustained and stable expression of transcription factor thymocyte selection-associated high mobility group box (TOX). Downregulating TOX expression in CD8+ T cells enhances their antitumor activities and improves immune checkpoint blockade (ICB) efficiency. We generated a reporter transgenic mouse to rapidly detect the expression of TOX by measuring luciferase activity. We knocked in a reporter cassette containing NanoLuc bioluminescent luciferase (Nluc) into the Tox gene locus by CRISPR/Cas9 (Tox-NLuc mice). We further generated Tox-NLuc-OT-I mice by crossing Tox-NLuc mice with OT-I mice, which allows the induction of CTL exhaustion in vitro by repeated stimulation of CD8+ T cells with OVA (257-264) peptide. Luciferase assays showed that higher luminescent signals were detected in exhausted CTLs compared to non-exhausted CTLs, which can be visualized by bioluminescence imaging. Bioluminescence changes were confirmed by measuring TOX expression by flow cytometry. The luminescence in exhausted CTLs decreased significantly when cells treated with ibrutinib and bryostatin-1, drugs that were found to directly modulate T cell exhaustion and decrease TOX expression. In summary, we have developed a novel TOX-nanoluciferase-based reporter system that can be used to monitor TOX expression and may facilitate the screening of molecules that modulate CTL exhaustion.