Adaptive-like CAR-iPSC-CD4⁺ T cells outperform CD8⁺ counterparts in sustained ALL control.

BACKGROUND: Induced pluripotent stem cell (iPSC)-derived T cells offer a renewable source for off-the-shelf immunotherapy. With the advent of the artificial thymic organoid (ATO) method, the in vitro differentiation of CD4+ T cells from iPSCs has also become feasible. CD4⁺ T cells have shown superior longevity, resistance to exhaustion, and helper functions in primary settings, but whether iPSC-derived CD4⁺ T cells retain these features remains unclear. METHODS: In this study, CD4⁺ T cells were differentiated from human iPSCs using the ATO system. Primary T cells served as controls to evaluate the phenotypic and activation features of iPSC-derived CD4⁺ and CD8⁺ T cells. To assess antitumor function, we generated CD19-BBζ CAR-iPSC-T cells and employed a hematologic malignancy model using NALM6 acute lymphoblastic leukemia (ALL) cells. Both short-term and long-term cytotoxicity assays were conducted to compare iPSC-derived CD4⁺ and CD8⁺ T cells in terms of killing efficiency, cytokine secretion, persistence, exhaustion phenotype, and proliferative capacity. The helper function of iPSC-derived CD4⁺ T cells toward CD8⁺ T cells was further evaluated by Ki-67 staining and proliferation assays. Statistical analyses were performed using GraphPad Prism. RESULTS: Our study demonstrated that iPSC-derived CD4⁺ T cells exhibited both helper- and cytotoxic-like features. Compared with iPSC-derived CD8⁺ T cells or CD4⁺/CD8⁺ mixtures, iPSC-derived CD4⁺ T cells showed superior proliferation, cytokine secretion, and sustained cytotoxicity following CAR transduction. They also promoted the expansion of iPSC-derived CD8⁺ T cells and displayed helper-like functions with increased resistance to exhaustion. CONCLUSIONS: Although not identical to primary CD4⁺ T cells, iPSC-derived CD4⁺ T cells recapitulated key functional advantages, especially sustained antitumor activity, supporting their value as a renewable, off-the-shelf source for next-generation CAR-T therapies.