Serum- and Feeder-Free Generation of CD4 Single Positive T Cells and Regulatory T Cells from Human Induced Pluripotent Stem Cells

Abstract Text: Increased interest in developing regulatory T cell (Treg) therapies is driven by the medical need to treat autoimmune and inflammatory diseases caused by loss or dysfunction of Tregs. However, primary Treg cell products are limited in cell numbers, subject to donor variability and difficult to multiplex edit. Human induced pluripotent stem cell (iPSC)-derived Tregs (iPSC-Tregs) could solve these issues by serving as a renewable source of a consistent edited cell product. While generation of iPSC-derived CD4 single-positive (CD4sp) T cells and CD4sp FOXP3+ Tregs using organoids was reported, here we show, for the first time, a serum- and cell feeder-free differentiation process suitable for largescale production. Using PMA and ionomycin, we generated CD4sp T cells from iPSCs at high efficiency (~3700 CD4sp/iPSC) and converted them to Tregs using TGFb and ATRA. We also demonstrated 80% non-viral, targeted integration of an HLA-A2 CAR in iPSCs by electroporating zinc finger nuclease mRNA and donor plasmid. The iPSC-Tregs +/- HLA-A2 CAR phenotypically resemble primary Tregs, are demethylated (>85%) at FOXP3 TSDR and secrete IL-10 but no proinflammatory cytokines (IL-2, IFN-g, TNFa). Comparing a validated Treg gene signature among iPSC-Tregs, primary Tregs, induced Tregs, and conventional CD4 T cells by single-cell RNA sequencing, iPSC-Tregs and primary Tregs clustered more closely together. Importantly, the iPSC-Tregs suppress T cell proliferation via HLA-A2 CAR and TCR stimulation in vitro. Our work is the first to demonstrate an iPSC-based platform for generating functional Tregs under serum- and feeder-free conditions amenable to manufacturing for delivering Treg therapies at scale.