NF-κB/NFAT signaling contributes to B7-H3 TRuC-T cell cytotoxicity and supports a reporter platform for glioblastoma immunotherapy.

INTRODUCTION: Glioblastoma (GBM), the most prevalent and aggressive malignant tumor of the nervous system, presents a formidable clinical challenge due to its rapid progression and extremely poor prognosis. T cell receptor fusion constructs (TRuC) -T cells represent a highly promising engineered T-cell therapy beyond CAR-T technology. Early prediction of TRuC-T cell cytotoxicity against GBM is crucial for clinical application. METHODS: We developed B7-H3-targeting TRuC-T cells and incorporated NF-κB- or NFAT-responsive transcriptional elements into the TRuC construct. Using flow cytometry, luciferase assays, and in vitro/vivo functional studies, we evaluated whether TRuC-T cells exert cytotoxicity via NF-κB and NFAT signaling pathways. RESULTS: B7-H3 TRuC-T cells effectively suppressed GBM growth both in vitro and in vivo. Co-culture of TRuC-T cells carrying NF-κB- or NFAT-reporters with target cells induced GFP expression, indicating pathway activation. DISCUSSION: Upon engagement with B7-H3, both NFAT and NF-κB signaling pathways are activated and participate in the regulation of B7-H3-targeting TRuC-T cells. Our data demonstrate that NF-κB/NFAT transcriptional reporters integrated into these TRuC constructs can be utilized to monitor target recognition and may serve as early indicators of cytotoxic efficacy. Overall, this system suggests a potential avenue for GBM therapy and offers an initial framework for the functional assessment of TRuC-T cell therapeutic potency.