Rapid ex vivo expansion of highly enriched human invariant natural killer T cells via single antigenic stimulation for cell therapy to prevent graft-versus-host disease.

Abstract

CD1d-restricted invariant natural killer (iNK) T cells are rare regulatory T cells that may contribute to the immune-regulation in allogeneic stem cell transplantation (ASCT). Here, we sought to develop an effective strategy to expand human iNK T cells for use in cell therapy to prevent graft-versus-host disease (GVHD) in ASCT. Human iNK T cells were first enriched from peripheral blood mononuclear cells (PBMCs) using magnetic-activated cell sorting separation, then co-cultured with dendritic cells in the presence of agonist glycolipids, alpha-galactosylceramide, for 2 weeks. The single antigenic stimulation reliably expanded iNK T cells to an average of 2.8 × 10⁷ per 5 × 10⁸ PBMCs in an average purity of 98.8% in 2 weeks (N = 24). The expanded iNK T cells contained a significantly higher level of CD4⁺ and central memory phenotype (CD45RA^-CD62L⁺) compared with freshly isolated iNK T cells, and maintained their ability to produce both Th-1 (interferon [IFN]γ and tumor necrosis factor [TNF]α) and Th-2 type cytokines (interleukin [IL]-4, IL-5 and IL-13) upon antigenic stimulation or stimulation with Phorbol 12-myristate 13-acetate/ionomycin. Interestingly, expanded iNK T cells were highly autoreactive and produced a Th-2 polarized cytokine production profile after being co-cultured with dendritic cells alone without exogenous agonist glycolipid antigen. Lastly, expanded iNK T cells suppressed conventional T-cell proliferation and ameliorated xenograft GVHD (hazard ratio, 0.1266; P < 0.0001). We have demonstrated a feasible approach for obtaining ex vivo expanded, highly enriched human iNK T cells for use in adoptive cell therapy to prevent GVHD in ASCT.