Brief homogeneous TCR signals instruct common iNKT progenitors whose effector diversification is characterized by subsequent cytokine signaling

Innate-like T cell populations expressing conserved TCRs play critical roles in immunity through diverse developmentally acquired effector functions. Focusing on the prototypical lineage of invariant natural killer T (iNKT) cells, we sought to dissect the mechanisms and timing of fate decisions and functional effector differentiation. Utilizing induced expression of the semi-invariant NKT cell TCR on double positive thymocytes, an initially highly synchronous wave of iNKT cell development was triggered by brief homogeneous TCR signaling. After reaching a uniform progenitor state characterized by IL-4 production potential and proliferation, effector subsets emerged simultaneously, but then diverged toward different fates. While NKT17 specification was quickly completed, NKT1 cells slowly differentiated and expanded. NKT2 cells resembled maturing progenitors, which gradually diminished in numbers. Thus, iNKT subset diversification occurs in dividing progenitor cells without acute TCR input but utilizes multiple active cytokine signaling pathways. These data imply a two-step model of iNKT effector differentiation. [Display omitted] •Precise timing of steps and processes driving iNKT cell development•Homogeneous TCR signals instruct iNKT progenitor, but not effector differentiation•iNKT subsets emerge simultaneously from the proliferating common progenitor state•NKT17 identity is acquired in 3–5 days, while NKT1 cells fully mature in 14–20 days Innate-like T cells undergo effector differentiation in absence of infection. Leveraging a genetically induced wave of iNKT cell development, Bortoluzzi et al. reveal that early TCR signaling instructs a common progenitor state, while effector subsets emerge later without further TCR input. The precise timing of these events suggests a distinct two-step process for iNKT cell differentiation.