Title: γδ-Thymocyte Maturation and Emigration Running title: γδ-Thymocyte Maturation and Emigration Authors: K. Joannou,

The thymus is the site of both αβ and γδ-T cell development. After several unique waves of γδ-T cells are generated in, and exported from, the fetal/neonatal thymus, the adult thymus continues to produce a stream of γδ-T cells throughout life. One intriguing feature of γδ T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus, however, it is less clear whether this paradigm holds true in adult animals. To investigate the relationship between maturation and time since V(D)J recombination in adult-derived γδ-thymocytes, we used the Rag2pGFP model. Immature (CD24) γδ-thymocytes expressed high levels of GFP while only a small minority of mature (CD24) γδ-thymocytes were GFP. Similarly, most GFP γδ-splenocytes were immature, while some were mature. Analysis of γδ-recent thymic emigrants (RTEs) indicated that most γδ-T cell RTEs were CD24 and GFP and adoptive transfer experiments showed that immature γδ-thymocytes could be maintained in the periphery for at least 3 days over which time they matured. With respect to the mature γδ-thymocytes that were GFP, parabiosis experiments demonstrated that mature γδ-T cells did not recirculate from the periphery. Instead, a population of mature γδ-thymocytes remained resident in the thymus for at least 60 days while mature γδ-thymocytes derived solely from adult hematopoiesis were mostly lost from the thymus within 60 days. Collectively, these data demonstrate two streams of actively developing γδ-T cells in adult mice: an immature subset that quickly leaves the thymus and matures in the periphery, and one that completes maturation within the thymus over a longer period of time. Furthermore, there is a fetal-derived and heterogeneous population of resident γδ-thymocytes of unknown functional importance.