Functional uncoupling of T-cell receptor engagement and Lck activation in anergic human thymic CD4(+) T cells

Human thymic CD1a(-)CD4(+) T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome t oxin-1 (TSST-1). Thymic CD4(+) T-cell blasts, established by stimulating hu man thymic CD1a(-)CD4(+) T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced ad ult peripheral blood (APB) CD4(+) T-cell blasts produce high levels of inte rleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor ze ta chain induced after restimulation with TSST-1 was 2-4-fold higher in APE CD4(+) T-cell blasts than in thymic CD4(+) T-cell blasts. The tyrosine kin ase activity of Lck was low in both thymic and APE CD4(+) T-cell blasts bef ore restimulation with TSST-1, After restimulation, the Lck kinase activity increased in APE CD4(+) T-cell blasts but not in thymic CD4(+) T-cell blas ts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APE CD4(+) T-cell blasts before restimulation with TSST-1, After restimula tion, it was markedly dephosphorylated in APE CD4(+) T-cell blasts but not in thymic CD4(+) T-cell blasts. Lck fi om APE CD4(+) T-cell blasts bound th e peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505, Confocal microscopy demonstrated that colocalizat ion of Lck and CD45 was induced after restimulation with TSST-1 in APE CD4( +) T-cell blasts but not in thymic CD4(+) T-cell blasts. Further, remarkabl e accumulation of Lck in the membrane raft was observed in restimulated APE CD4(+) T-cell blasts but not in thymic CD4(+) T-cell blasts. These data in dicate that interaction between Lck and CD45 is suppressed physically in th ymic CD4(+) T-cell blasts and plays a critical role in sustaining an anergi c state.