Single-cell analysis maps the dynamic landscape of mouse iNKT cell development and identifies Cbfb as a key regulator for early commitment

Abstract

Abstract Invariant natural killer T (iNKT) cells are innate-like T cell that share the characteristics of T cell and NK cell. iNKT cell acquire an effector function and differentiate into iNKT1, iNKT2 and iNKT17 sub-lineages prior to the thymic export. Although remarkable advancements in iNKT cell development have been made, the underlying molecular programs that guide their differentiation and heterogeneity remain unclear. Here, we profiled the transcriptomes of over 17,000 iNKT cells and the chromatin accessibility states of over 39,000 iNKT cells from the mouse thymus across stage 0 to stage 3 using scRNA-seq and scATAC-seq. By integrating iNKT cells transcriptome and chromatin accessibility profiles, we found that both iNKT2 and iNKT17 lineage commitment occur at stage 0, while iNKT1 cells initiate their differentiation as early as stage 1. Furthermore, iNKT1 and iNKT2 cells exhibit extensive phenotypic and functional heterogeneity, while iNKT17 cells are relative identical. Reconstruction of iNKT developmental trajectories revealed two possible pathways stemming from iNKT progenitors. More importantly, we identified Cbfβ (core-binding factor subunit beta) and known Egr2 and Slamf6 exhibit similar expression pattern in iNKT precursors. Deletion of Cbfβ in the mice blocked iNKT cell development at stage 0 and stage 1, and Cbfβ-deficient iNKT cells were failed to be differentiated into effector iNKT subsets. Bone marrow transfer experiments further indicated that the defective iNKT cell development in Cbfβ KO mice was cell-intrinsic. Overall, our findings revealed the cellular heterogeneity of iNKT cells, mapped the potential developmental trajectories, and identified Cbfβ as a key regulator for early iNKT cell commitment.