γδ T cells expressing CD8 or CD4^low appear early in murine foetal thymus development

Three-colour flow cytometry was used to study the distribution of TCRγδ⁺ cells among CD4⁺CD8^-, CD4^-CD8⁺, CD4⁺CD4⁺ CD8⁺, and CD4^-CD8^- cell populations during thymic development. Thymocytes were obtained either directly from embryos at different stages of gestation (ex vivo) or from organ cultures maintained in vitro. In both cases, TCRγδ⁺ cells were found predominantly among the double negative (CD4^-CD8^-) and CD8 single positive subsets. These cells were actively dividing as demonstrated by 7 amino actinomycin D (7AAD) labelling. A small population of TCRγδ⁺ cells expressing low levels of CD4 was identified early and transiently (days 15-18) during development, but this subset was rare in the adult thymus. In newborn mice, adult mice, and late during organ culture, TCRγδ⁺ cells were found mainly within the CD4^-CD8^- compartment of thymocytes, although a minor population of CD8⁺ cells (5-10%) bearing γδ receptor was routinely observed. In contrast, few γδ cells were contained among the CD4⁺CD8⁺ subset at any timepoint studied. These data highlight differences between the ontogeny of αβ and γδ cells in the thymus, and suggest that a CD4⁺CD8⁺ intermediate may not be a requisite for the intrathymic differentiation of murine γδ T cells.