Human NKT10 cells are enriched in cord-derived invariant natural killer T cells and mediate immune-regulation in a xenogeneic graft-versus-host disease model

CD1d-restricted invariant natural killer (iNK) T cells are innate T cells known for their ability to shape adaptive immunity toward inflammation or immune-suppression via the rapid production of Th1-, Th2-, and Th17-type cytokines from corresponding iNKT subsets such as NKT1, NKT2, and NKT17. IL-10-producing invariant NKT cells, termed NKT10 cells, are thought to play an immunoregulatory role, but their potential clinical use remains underexplored. We characterized human NKT10 cells from cord-derived iNKT cells and investigated their therapeutic utility in allogeneic stem cell transplantation. Cord and cord-derived iNKT cells contained a high frequency of CD4 + CD25 + CD161 low FoxP3 + iNKT cells and showed Th2/Th10-biased cytokine production upon antigenic stimulation. Accordingly, cord-derived iNKT cells displayed a distinct gene expression profile with upregulated genes related to NKT2, NKT10, and regulatory T cells compared with adult donor-derived iNKT cells. Furthermore, single-cell RNA sequencing analysis of cord-derived iNKT cells confirmed the presence of NKT10-like subset that was enriched with multiple immunoregulatory pathways and genes related to immune-checkpoints ( NRP1 , PD1 , CLTA-4 , and GITR ) and NKT10 ( MAF , HIF1A , and FoxP3 ), whereas the NKT1/17-like subset present in adult donor-derived iNKT cells showed upregulation of genes related to cytotoxicity ( GZMA/B , KLRD1 , and PRF1 ), NKR ( KLRK1 , KLRB1 , KLRG1 , and NKG7 ), NKT1 ( EOMES and TBX21 ), and NKT17 ( RORC ). Lastly, cord-derived iNKT cells suppressed alloreactive T cell proliferation in vitro and ameliorated xenogeneic graft-versus-host disease where the immunodeficient NSG mice received human peripheral blood mononuclear cells supplemented with cord-derived iNKT cells. Thus, NKT10-enriched, cord-derived iNKT cells are candidate cell therapeutics for immune-modulation in allogeneic stem cell transplantation and other autoimmune diseases.