A Tonic Signaling Code Governs CAR-T Cell Efficacy in Diffuse Midline Glioma

Diffuse midline glioma (DIPG/DMG) is a uniformly fatal pediatric brain tumor. While CAR T-cell therapy holds promise, clinical responses remain inconsistent, largely due to limited CAR-T persistence and premature exhaustion. Reliable biomarkers to predict therapeutic efficacy are lacking, and proposed T-cell exhaustion or stemness markers offer limited predictive value. Here, we systematically evaluate CAR-T cells against the clinically relevant tumor antigen B7-H3 and identify tonic signaling, antigen-independent CAR activation, as a key determinant of therapeutic performance. B7-H3 CAR-T cells with minimal tonic signaling show superior tumor killing, persistence, and resistance to exhaustion in patient-derived DIPG models. Integrative single-cell and multi-omic profiling identifies a tonic signaling–associated gene signature that consistently outperforms conventional exhaustion or stemness markers in predicting CAR-T efficacy across multiple independent clinical trial datasets, including DIPG/DMG and other tumor types. Thus, our findings establish a mechanistic and predictive framework to guide CAR design and improve clinical outcomes. Overall design: Single-cell RNA sequencing was performed using the sorted 376.96, MGA271, and Hu8H9 CAR-T cells and control T cells on day 12 of culture.