Scalable expansion of human iNKT cells: single-cell profiling and in vivo control of GvHD with preserved GvL activity

Invariant natural killer T (iNKT) cells have the potential to reduce graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation (HSCT), but their limited presence in peripheral blood hinders therapeutic development. Current methods for expanding human iNKT cells, mainly using IL-2, rely on prior iNKT-cell sorting, take 6-8 weeks, and primarily expand CD4+ iNKT cells. However, CD4- iNKT cells are more effective at controlling GVHD and regulating antigen-presenting cells and T-cell activation.

Researchers have developed a scalable culture system that can directly expand human CD4- iNKT cells from total peripheral blood mononuclear cells (PBMCs) using alpha galactosylceramide (alpha-GalCer) and optimized cytokine conditions. IL-15 proved to be the most effective cytokine in this process. The 14-day protocol yielded approximately 3.8 million iNKT cells from just 2 million PBMCs, with 74% of them being CD4- iNKT cells.

Single-cell transcriptomic profiling revealed eight major iNKT subsets, various differentiation trajectories during expansion, and distinct transcriptional programs associated with IL-2 versus IL-15. iNKT cells expanded with IL-15 displayed enhanced abilities to kill monocyte-derived dendritic cells and leukemic cells in vitro. Furthermore, these iNKT cells controlled xeno-GVHD and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models.

This innovative platform enables the reproducible production of human CD4- iNKT cells at a clinically relevant scale, positioning IL-15-expanded iNKT cells as a promising immunotherapy candidate for allogeneic HSCT.