Tunable differentiation of human CD4⁺ and CD8⁺ T cells from pluripotent stem cells

Allogeneic T cell therapies are a highly desirable option to circumvent the cost and complexity of using autologous T cells to treat diseases. Allogeneic CD8⁺ T cells can be made from pluripotent stem cells (PSCs), but deriving CD4⁺ T cells from PSCs has remained a significant challenge. Using feeder- and serum-free conditions, we found that CD4⁺ vs. CD8⁺ T cell commitment from PSCs can be controlled by fine-tuning the dynamics of Notch and T cell receptor (TCR) signaling delivered to CD4⁺CD8⁺ double-positive T cells. Notch signaling negatively impacts CD4⁺ T cell commitment, and its timed removal allows generation of clonally diverse and expandable CD4⁺ T cells from PSCs. The resulting CD4⁺ T cells respond to cytokine-mediated polarization by differentiating into Th1, Th2, or Th17 cells, recapitulating canonical helper cell function. These findings represent a significant step toward using PSC-derived CD4⁺ T cells as a low-cost, off-the-shelf cell therapy.