Development and function of specified thymic iNKT1 cells critically depend on an Ets1-Tbet transcription factor axis.

Invariant natural killer T 1 (iNKT1) cells are prototypic interferon γ-producing, innate-like T lymphocytes that play crucial roles in protection against pathogen infection and cancer. In the thymus, where these cells develop, iNKT cells contribute to the recruitment of antigen-presenting cells and promote T cell tolerance. Although the transcriptional programs guiding iNKT cell specification are well characterized, the mechanisms directing their subsequent maturation and tissue-associated effector functions remain poorly understood. Here, we reveal that the transcription factor Ets1 is essential for the maintenance of thymic iNKT1 cells and for their differentiation into a CD103+ transforming growth factor β1-dependent, tissue-resident phenotype, in addition to playing a critical role in maintaining iNKT1 effector function in the liver and spleen. Mechanistically, Ets1 limits the expression of CD8 effector T cell-associated genes and restricts the induction of the transcription factor Tbet. Elevated Tbet expression partially rescues thymic iNKT1 cell numbers in the absence of Ets1 but shifts their differentiation toward a migratory, CD103- pathway. In peripheral tissues, elevated Tbet expression failed to overcome the requirement for Ets1 in activation-induced interferon γ-production but instead drove increased cytotoxic effector protein expression. Our findings establish Ets1 as a central regulator of iNKT1 cell homeostasis, enforcing a tissue-resident identity in the thymus and supporting context-appropriate effector function.