A serum- and feeder-free system to generate CD4 and regulatory T cells from human iPSCs.

iPSCs can serve as a renewable source of a consistent edited cell product, overcoming limitations of primary cells. While feeder-free generation of clinical grade iPSC-derived CD8 T cells has been achieved, differentiation of iPSC-derived CD4sp and regulatory T cells requires mouse stromal cells in an artificial thymic organoid. Here we report a serum- and feeder-free differentiation process suitable for large-scale production. Using an optimized concentration of PMA/Ionomycin, we generated iPSC-CD4sp T cells at high efficiency and converted them to Tregs using TGFβ and ATRA. Using genetic engineering, we demonstrated high, non-viral, targeted integration of an HLA-A2 CAR in iPSCs. iPSC-Tregs ± HLA-A2-targeted CAR phenotypically, transcriptionally and functionally resemble primary Tregs and suppress T-cell proliferation in vitro. Our work is the first to demonstrate an iPSC-based platform amenable to manufacturing CD4 T cells to complement iPSC-CD8 oncology products and functional iPSC-Tregs to deliver Treg cell therapies at scale.