Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method.
AUTOR(ES)
Toki, J
RESUMO
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 (10(5) cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37 degrees 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) beta chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR beta-chain variable domain (V beta 8) 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=52338Documentos Relacionados
- Detection and differentiation of antibodies to human T-cell lymphotropic virus types I and II by the immunofluorescence method.
- Survival of hemopoietic progenitors in the G0 period of the cell cycle does not require early hemopoietic regulators.
- Fusobacterium nucleatum inhibits human T-cell activation by arresting cells in the mid-G1 phase of the cell cycle.
- Induction of T-Cell Differentiation In Vitro by Thymin, a Purified Polypeptide Hormone of the Thymus
- Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors.