Abstract
Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human 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 adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the 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 colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.
Highlights
Human thymic CD1aϪCD4ϩ T cells in the final stage of maturation [1] and cord blood CD4ϩ T cells are susceptible to anergy induction by in vitro stimulation with a superantigen, toxic shock syndrome toxin-1 (TSST-1).1 Thymic and cord blood
We addressed the question of why human thymic CD1aϪCD4ϩ T cells are induced into an anergic state after stimulation with TSST-1 by comparing signal transduction in anergic TSST-1-induced thymic CD4ϩ T-cell blasts with that in highly responsive TSST-1-induced adult peripheral blood (APB) CD4ϩ T-cell blasts
The results strongly suggest that the absence of an interaction between Lck and CD45 is responsible for maintaining the anergic state of human thymic CD1aϪCD4ϩ T cells induced by TSST-1
Summary
87.3 Ϯ 7.8 98.5 Ϯ 1.0 74.9 Ϯ 9.4 81.6 Ϯ 3.5 90.2 Ϯ 6.9 63.4 Ϯ 1.4 90.5 Ϯ 8.2 71.8 Ϯ 12.6 23.5 Ϯ 8.8. Preparation of TSST-1-induced APB and Thymic T-cell Blasts— TSST-1-induced T-cell blasts were obtained as described previously [3]. In Vitro Kinase Assay—T-cell blasts were solubilized in TNE buffer as described for immunoprecipitation, and the supernatants were subjected to immunoprecipitation for 2 h with an anti-Lck Ab. Immunoprecipitates were washed with radioimmune precipitation buffer (TNE buffer containing 0.1% sodium deoxycholate and 0.1% SDS) followed by washing buffer containing 20 mM Tris-HCl (pH 7.4) and 0.5 M LiCl. Immunoprecipitates were incubated with 30 l of kinase reaction buffer containing 40 mM HEPES (pH 7.4), 3 mM MnCl2, 10% glycerol, 10 M cold ATP, and 10 Ci of [␥-32P]ATP (PerkinElmer Life Sciences) per sample.
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