T Cell Receptor (TCR) Engagement in Apoptosis-defective, but Interleukin 2 (IL-2)–producing, T Cells Results in Impaired ZAP70/CD3-ζ Association

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.