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.
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.
Nishida
Takashi
T
Department of Parasitology and Immunology, National Defense Medical College, Tokorozawa, Saitama, Japan.
Matsuki
Yasunori
Y
Ono
Takeshi
T
Oguma
Takemi
T
Tsujimoto
Kyoko
K
Sato
Masaki
M
Tadakuma
Takushi
T
D000818:Animals; D018106:CD28 Antigens / Q000502:physiology; D017252:CD3 Complex / Q000032:analysis / Q000276:immunology*; D015704:CD4 Antigens / Q000032:analysis*; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology*; D016827:CD8 Antigens / Q000032:analysis*; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D002460:Cell Line; D019070:Cell Lineage; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D013997:Time Factors
D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D020928:Mitogen-Activated Protein Kinases