The Novel Murine CD4~+CD8~+ Thymocyte Cell Line Exhibits Lineage Commitment into Both CD4~+ and CD8~+ T Cells by Altering the Intensity and the Duration of Anti-CD3 Stimulation In Vitro

摘要

A CD4~+CD8~+ double-positive thymocyte cell line,257-20-109 was established from BALB/c mice thymocytes and used to analyze the requirements to induce CD4 or CD8 single-positive(SP)T cells.CD4SP cells were induced from 257-20-109 cells by anti-CD3 stimulation in the presence of the FcR-positive macrophage cell line,P388D1.During stimulation,maturation events,such as the down-regulation of CD24 and the up-regulation of CD69,H-2D~d,CD5,and Bcl-2,were recognized.Furthermore,these CD4SP cells appeared to be functional because the cells produced IL-2 and IL-4 when activated with phorbol ester and calcium ionophore.In contrast,CD8SP cells could be induced by stimulation with fixed anti-CD3 after removal of stimulation.To investigate the extent of signals required for CD4SP and CD8SP,the cells stimulated under either condition for 2 days were sorted and transferred to different culture conditions.These results suggested that the fate of lineage commitment was determined within 2 days,and that CD4 lineage commitment required longer activation.Furthermore,the experiments with subclones of 257-20-109 demonstrated that the lower density of CD3 did not shift the cells from CD4SP to CD8SP,but only reduced the amount of CD4SP cells.In contrast,when the 257-20-109 cells were stimulated by the combination of fixed anti-CD3 and anti-CD28,the majority of the cells shifted to CD4SP,with an enhancement of extracellular signal-regulated kinase 1 phosphorylation.Our results indicate that the signals via TCR/CD3 alone shifted the double-positive cells to CD8SP cells,but the reinforced signals via TCR/CD3 and costimulator could commit the cells to CD4SP.