A three-stage developmental pathway for human V & gamma;9V & delta;2 T cells within the postnatal thymus

V?9Vd2 T cells are the largest population of ?d T cells in adults and can play important roles in providing effective immunity against cancer and infection. Many studies have suggested that peripheral V?9Vd2 T cells are derived from the fetal liver and thymus and that the postnatal thymus plays little role in the development of these cells. More recent evidence suggested that these cells may also develop postnatally in the thymus. Here, we used high-dimensional flow cytometry, transcriptomic analysis, functional assays, and precursor-product experiments to define the development pathway of V?9Vd2 T cells in the postnatal thymus. We identify three distinct stages of development for V?9Vd2 T cells in the postnatal thymus that are defined by the progressive acquisition of functional potential and major changes in the expression of transcription factors, chemokines, and other surface markers. Furthermore, our analysis of donor-matched thymus and blood revealed that the molecular requirements for the development of functional V?9Vd2 T cells are delivered predominantly by the postnatal thymus and not in the periphery. Tbet and Eomes, which are required for IFN-? and TNFa expression, are up-regulated as V?9Vd2 T cells mature in the thymus, and mature thymic V?9Vd2 T cells rapidly express high levels of these cytokines after stimulation. Similarly, the postnatal thymus programs V?9Vd2 T cells to express the cytolytic molecules, perforin, granzyme A, and granzyme K. This study provides a greater understanding of how V?9Vd2 T cells develop in humans and may lead to opportunities to manipulate these cells to treat human diseases.