Infusion of ex‐vivo expanded human TCR‐αβ + double‐negative regulatory T cells delays onset of xenogeneic graft‐ versus ‐host disease

Summary Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)‐αβ + double‐negative regulatory T cells (DN T regs ), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ∼3500‐fold ex‐vivo expansion of human DN T regs within 3 weeks with > 97% purity. Ex‐vivo ‐expanded DN T regs suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell‐to‐cell contact. In vivo , we demonstrate for the first time that infusion of human DN T regs delayed an onset of xenogeneic graft‐ versus ‐host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex‐vivo ‐expanded DN T regs with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN T regs can be expanded ex vivo to therapeutic numbers. The expanded DN T regs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN T regs to mitigate GVHD.