Beyond subsets: single cell transcriptomics reveals multidimensional regulation of iNKT cells cross tissues and species

Abstract

Invariant natural killer T (iNKT) cells are innate-like lymphocytes that rapidly respond to lipid antigens presented by CD1d or to inflammatory cytokines and influence diverse immune responses. Much of our current understanding of iNKT cell biology derives from murine studies, which established a framework of thymic differentiation into NKT1, NKT2, and NKT17 subsets. Recent single-cell RNA sequencing (scRNAseq) studies have substantially expanded this view by revealing non-linear developmental trajectories, early epigenomic priming, and a multipotent recent thymic emigrant population that continues to differentiate after thymic egress. In peripheral tissues, iNKT cells undergo extensive remodeling driven by local environmental cues and antigen exposure, giving rise to regulatory, and effector states not observed in the thymus. At the same time, emerging human studies reveal principles that differ from those described in mice. Human iNKT cells exhibit a blended type 1/type 17 transcriptional program, limited evidence for NKT2-like populations, and functionally distinct CD4+, double-negative (DN), CD8αα+, and terminal effector-like subsets. Comparative analyses across species further suggest that differences in thymic selection, transcription factor networks, and peripheral maturation contribute to divergent patterns of iNKT cell specialization. Together, these findings support a revised view of iNKT cells as dynamic and context-dependent transcriptional states shaped by developmental history, tissue environment, antigen exposure, and species-specific regulatory programs.