Single-cell RNA sequencing identifies shared differentiation paths of mouse thymic innate T cells

Invariant natural killer T (iNKT), mucosal-associated invariant T (MAIT), and gd T cells are innate T cells that acquire memory phenotype in the thymus and share similar biological characteristics. However, how their effector differentiation is developmentally regulated is still unclear. Here we identify analogous effector subsets of these three innate T cell types in the thymus that share transcriptional profiles. Using single-cell RNA sequencing, we show that iNKT, MAIT and gd T cells mature via shared, branched differentiation rather than linear maturation or TCR-mediated instruction. Simultaneous TCR clonotyping analysis reveals that thymic maturation of all three type is accompanied by clonal selection and expansion. Analyses of mice deficient of TBET, GATA3 or RORgt and additional in vivo experiments corroborate the predicted differentiation paths, while human innate T cells from liver samples display similar features. Collectively, our data indicates that innate T cells share effector differentiation processes in the thymus.