CAR-NKT cells induce low cytokine release syndrome by targeting hyperinflammatory macrophages with mitigation from GM-SCF inhibition.

Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable efficacy in cancer treatment but is frequently limited by severe toxicities, including cytokine release syndrome (CRS), which is largely driven by host myeloid cell activation. CAR-engineered invariant natural killer T (CAR-NKT) cells have emerged as a promising alternative with comparable antitumor activity and reduced toxicity; however, the mechanisms underlying their improved safety profile remain poorly defined. Here, we systematically evaluate CRS induced by human CAR-NKT and CAR-T cells using a heavily tumor-bearing xenograft mouse model that recapitulates clinically relevant CRS. Compared with conventional CAR-T cells, CAR-NKT cells induce significantly lower CRS severity across multiple clinical and biochemical parameters. To elucidate the underlying mechanisms, we perform comprehensive single-cell transcriptomic profiling of mouse immune cells, including myeloid and granulocytic populations, across six anatomical compartments: tumor, blood, liver, lung, spleen, and bone marrow. This integrative analysis reveals dynamic immune remodeling during tumor progression, therapeutic intervention, and CRS development. Mechanistically, CAR-NKT cells preferentially target CD1d⁺ hyperinflammatory macrophages, a key driver of CRS, thereby limiting myeloid-mediated inflammatory responses. Furthermore, pharmacological inhibition of GM-CSF further attenuate CRS, supporting a combinatorial strategy to enhance the safety of CAR-NKT cell therapy. Collectively, these findings provide mechanistic insight into the reduced toxicity of CAR-NKT cells and support their development as a safer and effective platform for cancer immunotherapy.