A yolk sac-derived hepatic γδ T cell subpopulation develops in a hematopoietic stem cell- and thymus-independent manner.

γδ T cells are a highly heterogeneous T cell lineage with diverse immunological functions; however, the developmental origins underlying this heterogeneity, particularly the existence and physiological relevance of extrathymic pathways, remain incompletely defined. Our previously generated transcriptomic datasets indicate that Stab2 is specifically expressed in the yolk sac but not the aortic endothelial cells. Leveraging this spatially restricted expression pattern, we generated a Stab2-CrexERT2 inducible genetic lineage tracing mouse model and demonstrated its ability to label yolk sac-derived hematopoietic cells in vivo. Importantly, we detected the contribution of the yolk sac endothelial cells labeled at embryonic day 9.5 to a subset of Vγ1 γδ T cells in the adult liver but hardly to any hematopoietic progenitors in the thymus and bone marrow. Single-cell transcriptomic profiling combined with functional evaluation revealed that the Stab2-labeled yolk sac endothelial cell-derived hepatic γδ T cells preferentially enrich IFN-γ-producing γδ T1 cells and overrepresent characteristics associated with tissue homeostasis and repair. Collectively, our findings provide direct in vivo evidence for the existence of a bone marrow hematopoietic stem cell-independent and thymus-independent developmental pathway of liver γδ T cells originating from yolk sac endothelial cells, and suggest that different origins and developmental paths may confer distinct functional properties to γδ T cells.