Feeder-free generation of CD4 single-positive helper T cells from human iPSCs via stage-specific modulation of Notch and TCR Signaling

Induced pluripotent stem cell (iPSC)-derived T cells (iPS-T cells) represent a promising platform for adoptive immunotherapy, offering an unlimited source of T cells amenable to gene editing and rigorous quality control. While feeder-free in vitro redifferentiation systems have been successfully developed for CD8+ cytotoxic T cells, the generation of CD4+ helper T cells under similar conditions has remained elusive—despite their essential role in sustaining long-term immune responses. In this study, we established a feeder-free culture system for the generation of CD4 single-positive (SP) helper T cells from iPSCs, under conditions that exclude TCR signaling, which otherwise biases differentiation toward the cytotoxic lineage. By precisely modulating Notch and integrin signaling in a stage-specific manner, we induced the differentiation of CD4 SP iPS-T cells expressing key helper-associated molecules, including CD40L and ThPOK, capable of promoting dendritic cell maturation. Notably, these cells also acquired cytotoxic activity upon repeated expansion while retaining robust proliferative capacity. Our findings provide a foundation for the scalable and consistent production of both CD4 SP helper and killer T cells from pluripotent stem cells, advancing the potential of iPSC-based immunotherapies.