Although all NKT cells belonging to either the CD4^-CD8^- (DN) or CD4⁺ population express an invariant TCR with the same specificity, they comprise an amazing array of functionally distinct subsets classified as NKT1, NKT2, NKT17 and NKT10. Each subset has different cytokine secretion profiles and is defined by the expression of different arrays of cytokine receptors and characteristic transcription factors. In both human and mouse, the CD4^-CD8^- double-negative (DN) NKT cells, in striking contrast to CD4⁺ NKT cells, are known to elicit strong antitumor activity and Th1-type responses. These functional differences between DN and CD4⁺ NKT cells cannot, however, be adequately explained by the currently accepted “mainstream” or “CD4⁺CD8⁺ (DP) pathway” model of NKT cell development. Therefore, questions regarding the developmental origin of DN NKT cells remain unresolved.

Here we provide definitive genetic evidence by fate-mapping or conditional gene ablation of Rag2 gene approaches controlled by the DP stage-specific expression of E8_IIICre recombinase that supports the existence of an alternative developmental pathway through which a fraction of DN NKT cells develops from late DN stage thymocytes, bypassing the DP stage. Importantly, the DN pathway preferentially gives rise to Th1-NKT cells with strong cytotoxic potential and distinct distribution patterns in peripheral tissues. These observations add new insights into our understanding of the development of NKT cells, as it appears that the differentiation stage as well as the microenvironmental niche of precursor cells undergoing positive selection may play an important role in determining their further developmental programs, ultimately leading to the emergence of various functional NKT cell subsets.