Spatiotemporal profiling reveals distinct dynamics and checkpoint regulations of CAR-T and CAR-NKT cells against solid tumors.

Chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies have demonstrated remarkable efficacy in hematologic malignancies; however, their clinical performance in solid tumors remains limited due to suboptimal tumor infiltration, antigen heterogeneity, and immunosuppressive tumor microenvironments (TME). Invariant natural killer T (NKT) cells have recently emerged as a promising alternative platform for CAR engineering, owing to their intrinsic tissue-homing capacity, multi-modal cytotoxicity, and ability to reshape the TME. In this study, we performed a comprehensive preclinical comparison of conventional CAR-T cells and allogeneic stem cell-derived IL-15-enhanced CAR-NKT cells in solid tumor models, integrating spatiotemporal transcriptomic profiling across multiple tissues and longitudinal time points. Our analyses revealed distinct in vivo pharmacokinetic, pharmacodynamic, and immunoregulatory profiles between the two cell therapy modalities. Compared with CAR-T cells, CAR-NKT cells demonstrated superior homing, infiltration, and localization within solid tumors, along with prolonged in vivo persistence and a unique immune checkpoint receptor expression landscape. Notably, CAR-T cells exhibited synergistic antitumor responses when combined with TIGIT blockade, whereas CAR-NKT cells showed greater sensitivity to CD96 blockade in vivo. These findings highlight the divergent therapeutic dynamics of CAR-T and CAR-NKT cells and provide mechanistic insights that inform the rational design of next-generation cell therapies and combinatorial strategies for solid tumors.