Integrative scATAC-seq and scRNA-seq analyses map thymic i NKT cell development and identify Cbfβ for its commitment

Unlike conventional αβT cells, invariant natural killer T ( i NKT) cells complete their terminal differentiation to functional i NKT1/2/17 cells in the thymus. However, underlying molecular programs that guide i NKT subset differentiation remain unclear. Here, we profiled the transcriptomes of over 17,000 i NKT cells and the chromatin accessibility states of over 39,000 i NKT cells across four thymic i NKT developmental stages using single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to define their developmental trajectories. Our study discovered novel features for i NKT precursors and different i NKT subsets and indicated that i NKT2 and i NKT17 lineage commitment may occur as early as stage 0 (ST0) by two distinct programs, while i NKT1 commitments may occur post ST0. Both i NKT1 and i NKT2 cells exhibit extensive phenotypic and functional heterogeneity, while i NKT17 cells are relatively homogenous. Furthermore, we identified that a novel transcription factor, Cbfβ , was highly expressed in i NKT progenitor commitment checkpoint, which showed a similar expression trajectory with other known transcription factors for i NKT cells development, Zbtb16 and Egr2 , and could direct i NKT cells fate and drive their effector phenotype differentiation. Conditional deletion of Cbfβ blocked early i NKT cell development and led to severe impairment of i NKT1/2/17 cell differentiation. Overall, our findings uncovered distinct i NKT developmental programs as well as their cellular heterogeneity, and identified a novel transcription factor Cbfβ as a key regulator for early i NKT cell commitment.