A committed postselection precursor to natural TCRαβ⁺ intraepithelial lymphocytes

The intestine is a major immune organ with several specialized lymphoid structures and immune cells. Among these are thymus-derived natural intraepithelial lymphocytes (IELs) that lack expression of the classical co-receptors CD4 or CD8αβ (double negative (DN)). Natural IELs are both αβ⁺ and γδ⁺ T cells that play important roles in the maintenance of the epithelial barrier at steady state and during inflammation. The transcription factor T-bet is essential for the peripheral development of natural IELs, but its role during thymic development has remained less clear. Here we show that a T-bet gradient in DN TCRαβ⁺NK1.1^- thymocytes (IEL precursors (IELPs)) determines IEL fate in natural TCRαβ⁺ IELs. Employing T-bet ZsGreen reporter mice in in vitro cultures and in vivo transfer experiments, we demonstrate that with increasing expression of T-bet, DN TCRαβ⁺NK1.1^- thymocytes are gradually restricted to a DN IEL fate. Furthermore, we show that the natural TCRαβ⁺ IELs seed the intestine within the first month of life. This in turn is preceded by the appearance of T-bet^- and T-bet⁺ IELPs that egress from the thymus in a sphingosine-1-phosphate (S1P)-dependent manner. In summary, the use of T-bet reporter mice has enabled us to identify and refine an immediate and clearly committed postselection precursor of natural TCRαβ⁺ IELs.