Intrathymic signalling in immature CD4⁺ CD8⁺ thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain

Abstract

THYMIC selection of the developing T-cell repertoire occurs in immature CD4⁺CD8⁺ double-positive thymocytes^1–3 and is thought to be mediated by signals transduced by T-cell antigen receptor (TCR) molecules and possibly by CD4 and CD8 accessory molecules as well^{1, 4–6} It is not known, however, which signal-transduction mechanisms function in immature CD4⁺CD8⁺ thymocytes on engagement of TCR, CD4 or CD8 molecules. In mature T cells, CD4 and CD8 molecules are each associated with the src-like protein tyrosine kinase p56 lck^{7, 8} and signals transduced by TCR and CD4 activate tyrosine kinases that phosphory-late TCR-ε chains and other intracellular substrates^{9, 10}. Consequently, we examined whether tyrosine kinases could be similarly activated in immature CD4⁺CD8⁺ thymocytes. Unexpectedly, we found that TCR-εchains from CD4⁺CD8⁺ thymocytes were already phosphorylated in vivo, and that dephosphorylation of this TCR subunit occurred on removal of CD4⁺CD8⁺ cells from their intrathymic environment. Rephosphorylation of TCR-ε in cultured CD4⁺CD8⁺ thymocytes occurred rapidly in vitro, either in response to cross-linking of TCR, CD4 or CD8 by specific monoclonal antibodies, or on cell–cell contact. These observations indicate that tyrosine kinases are activated in vivo in immature CD4⁺CD8⁺ thymocytes undergoing thymic differentiation and selection. They also indicate that TCR, CD4 and CD8 molecules can function in CD4⁺CD8⁺ thymocytes as signalling molecules to activate tyrosine kinases and that phosphorylated TCR-ε serves as a marker of these signalling events.