Analyses of T-cell differentiation from hemopoietic stem cells in the G₀ phase by an in vitro method

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G₀ phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (10⁵ cells per well) in the G₀ phase were added to these thymic lobes and cocultured at 37°C in a 5% CO₂/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) β chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR β-chain variable domain (V_βS) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.