Single-cell transcriptomic profiling reveals diversity in human iNKT cells across hematologic tissues

Invariant natural killer T (iNKT) cells are evolutionarily conserved innate lymphocytes important for protection against pathogens, malignancies, and graft-versus-host disease, with potential for universal donor cellular therapies. While mouse studies reveal transcriptionally and functionally distinct subsets, a comprehensive understanding of human iNKT cell heterogeneity is limited. Herein, we delineate the transcriptomic diversity of human iNKT cells from multiple immunologically relevant hematologic tissues. Human iNKT cells express naive/precursor, transitional, and T helper (Th)1/17/NK-like transcriptional profiles, partially contrasting with findings in mice. Additionally, these data uncover transcription factor dynamics not previously described in mice and reveal a T effector memory RA⁺-like population. Further, two distinct expression patterns of human CD8⁺ iNKT cells are described—one resembling naive/precursor cells and another resembling Th1/17/NK-like cells, with predominant expression of CD8αα protein. These critical insights into the transcriptional heterogeneity of human iNKT cells will facilitate future functional studies and inform iNKT-based cellular therapy development.