ENTRY OF CD4(-)CD8(-) IMMATURE THYMOCYTES INTO THE CD4/CD8 DEVELOPMENTAL PATHWAY IS CONTROLLED BY TYROSINE KINASE SIGNALS THAT CAN BE PROVIDED THROUGH TCR COMPONENTS

Entry of thymus-migrated precursor cells into the CD4/CD8 developmental pathway was analyzed by using the short-term organ cultures of day 14 fetal mouse thymus lobes. Organ cultures of CD4(-) CD8(-) day 14 fetal thymocytes for 1-2 days resulted in the generation of CD4(-)CD8(+) cells, which were mostly immediate precursor cells for CD4(+)CD8(+) thymocytes. This differentiation of CD4(-)CD8(-) thymocytes into CD4(-)CD8(+) cells was strongly enhanced by anti-CD3 antibodies. The anti-CD3-induced generation of CD4(-)CD8(+) cells was even found in the immunodeficient scid fetal thymus cultures, and the cell surface CD3 expression on the scid fetal thymocytes could be directly visualized, indicating that functional CD3 could be expressed on CD4(-)CD8(-) immature thymocytes without being associated with rearranged TCR components. The anti-CD3-induced generation of CD4(-)CD8(+) cells from scid and normal fetal thymus cultures was inhibited by tyrosine kinase inhibitors Herbimycin A and Tyrphostin. The generation of CD4(-)CD8(+) cells in unstimulated normal fetal thymus cultures was also markedly inhibited by the tyrosine kinase inhibitors but not by Cyclosporin A, suggesting that tyrosine kinase-dependent but calcineurin-independent signals were essential for the differentiation of CD4(-)CD8(-) thymocytes. Interestingly, the generation of CD4(-)CD8(+) cells from the normal fetal thymus cultures was modestly but consistently enhanced by anti-TCR beta antibody, suggesting that functional TCR beta in addition to CD3 was expressed on normal CD4(-)CD8(+) immature thymocytes. On the other hand, anti-TCR delta antibody did not affect this differentiation in the normal fetal thymus cultures and-the generation of CD4(-)CD8(+) cells from the fetal thymus cultures of TCR delta-deficient mice was still enhanced by anti-TCR beta or anti-CD3 antibodies, indicating that either TCR delta chains or TCR delta(+) cells were not involved in the control of the differentiation into CD4(-)CD8(+) cells. These results indicate that the entry of CD4(-)CD8(-) immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals and that these signals can be provided through the engagement of TCR-CD3 complexes with or without TCR beta chains expressed on the CD4(-)CD8(-) immature thymocytes.