Abstract A021: Spatiotemporal gene profiling reveals distinct <i>in vivo</i> dynamics and immune checkpoint regulations of human CAR-T and CAR-NKT cells against solid tumors

Abstract Chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies have achieved transformative success in treating hematologic malignancies. However, their efficacy in solid tumors remains modest, largely hindered by poor tumor infiltration, antigen heterogeneity, and highly immunosuppressive tumor microenvironments (TME). To overcome these challenges, invariant natural killer T (NKT) cells have recently gained attention as an alternative and potentially superior platform for CAR engineering. NKT cells possess intrinsic tissue-homing properties, innate-like cytotoxic functions, and the capacity to modulate the TME through cytokine secretion and cross-talk with other immune subsets. In this study, we performed a comprehensive preclinical comparison between CAR-T and CAR-NKT cells using multiple solid tumor models, integrated with spatiotemporal transcriptomic profiling across defined time points and tissue compartments. Our analyses revealed striking differences in their pharmacokinetic behaviors, immune landscape remodeling, and exhaustion dynamics. CAR-NKT cells demonstrated superior tumor localization, prolonged in vivo persistence, and reduced terminal exhaustion, accompanied by a distinct pattern of immune checkpoint receptor expression compared to CAR-T cells. In contrast, CAR-T cells exhibited stronger initial cytotoxic bursts but showed faster contraction and susceptibility to TME-mediated suppression. Functionally, we identified unique checkpoint dependencies between the two cell types. CAR-T cells showed enhanced antitumor activity when combined with TIGIT blockade, while CAR-NKT cells responded more robustly to CD96 blockade, indicating divergent regulatory pathways governing their persistence and effector function. Collectively, our findings highlight the complementary and distinct therapeutic dynamics of CAR-T and CAR-NKT cells in solid tumors. These insights provide a mechanistic foundation for the rational design of next-generation CAR-engineered cell therapies and tailored combinatorial strategies to improve efficacy and durability in solid tumor treatment. Citation Format: Yan-Ruide Li, Yuning Chen, Yichen Zhu, Xinyuan Shen, Lili Yang. Spatiotemporal gene profiling reveals distinct in vivo dynamics and immune checkpoint regulations of human CAR-T and CAR-NKT cells against solid tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A021.