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

Abstract

Unlike conventional αβT cells, invariant natural killer T (iNKT) cells complete their terminal differentiation to functional iNKT1/2/17 cells in the thymus. However, underlying molecular programs that guide iNKT subset differentiation remain unclear. Here, we profiled the transcriptomes of over 17,000 iNKT cells and the chromatin accessibility states of over 39,000 iNKT cells across four thymic iNKT 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 iNKT precursors and different iNKT subsets and indicated that iNKT2 and iNKT17 lineage commitment may occur as early as stage 0 (ST0) by two distinct programs, while iNKT1 commitments may occur post ST0. Both iNKT1 and iNKT2 cells exhibit extensive phenotypic and functional heterogeneity, while iNKT17 cells are relatively homogenous. Furthermore, we identified that a novel transcription factor, Cbfβ, was highly expressed in iNKT progenitor commitment checkpoint, which showed a similar expression trajectory with other known transcription factors for iNKT cells development, Zbtb16 and Egr2, and could direct iNKT cells fate and drive their effector phenotype differentiation. Conditional deletion of Cbfβ blocked early iNKT cell development and led to severe impairment of iNKT1/2/17 cell differentiation. Overall, our findings uncovered distinct iNKT developmental programs as well as their cellular heterogeneity, and identified a novel transcription factor Cbfβ as a key regulator for early iNKT cell commitment.

Single cell resolution analysis of multi-tissue derived human iNKT cells reveals novel transcriptional paradigms

“…Three clusters, C0, C3, and C5, had minimal expression of TBX21 and RORC and retained expression of ZBTB16 , albeit relatively downregulated relative to clusters C1 and C4 (Fig 2A). These cells were largely thymic-derived and may represent early iNKT cells differentiating towards Th1/17-like iNKT cells, correlating with data from a recent murine study 17 (Fig 1D-E). Collectively, these results suggest that human iNKT cells can be described as Th1/17-like, Th2-like, transitional, or early differentiating cells based on their expression patterns of transcription factors canonically associated with murine iNKT cells.…”

“…Our data demonstrate that human iNKT cells form a differentiation trajectory which, when limited to post-stage 0 iNKT cells, appears to originate from a naïve/precursor stage. The reduced expression of ZBTB16 in our naïve/precursor stage relative to the Th2-like clusters corresponds with a recent report of a similar population in murine thymic iNKT cells 17 . Further, our naïve precursor stage was characterized by expression of genes known to play important roles in murine iNKT cell development.…”

“…This corresponds with previous data showing that human CB derived iNKT cells produce higher Th2 cytokines than iNKT cells derived from adult PB 34,63 . Our differentiation trajectory suggests that Th2-like iNKT cells represent a transitional phenotype between naïve/precursor iNKT cells and Th1-like iNKT cells, similar to recent findings in murine iNKT cells 16,17 . In these murine studies, iNKT2 cells (or a fraction thereof) represented a transitional population from which iNKT1 cells and iNKT17 cells independently branched off.…”

“…Interestingly, we saw that the cluster with highest RORC expression (PB-C8) also had high and unique expression of AQP3 (Fig 4D, 1.89 avg log2FC). Aqp3 expression has also been demonstrated in iNKT17 cells in mice 17 .…”

“…In these murine studies, iNKT2 cells (or a fraction thereof) represented a transitional population from which iNKT1 cells and iNKT17 cells independently branched off. Indeed, several murine studies have highlighted the distinct transcriptional clustering of iNKT1 cells and iNKT17 cells, with unique expression of Tbx21 and Rorc , respectively, which has been further demonstrated at the protein level 12,16,17,20 . In contrast, our study of human iNKT cells revealed co-expression of TBX21 and RORC in two clusters, suggesting a combined Th1/17 signature in these cells.…”