Anergic T cells proliferate only in response to exogenous IL-2 and show markedly enhanced Fyn kinase activity. (A) Peripheral T cells were pretreated with no stimulus (resting), anti-CD3 antibody (anergic), or anti-CD3 antibody plus phorbol ester PMA (rescued) for 3 days. After 1 day of resting, the cells received either no stimulus or were restimulated with anti-CD3, anti-CD3 + anti-CD28, or anti-CD3 + IL-2. Incorporation of [³H]thymidine was measured at 72 hours and is shown as the mean value of triplicates (± SD). Data were analyzed using one-way ANOVA (∗∗, P < .01; ∗∗∗, P < .001). Fyn (B) and Lck (C) were immunoprecipitated from resting (rest.), anergic (aner.), and rescued (resc.) T cells and in vitro kinase assays (IVK) were performed. Phosphorylation was visualized by autoradiography (upper panel), the amount of kinase precipitated was determined by immunoblotting with specific antibodies (lower panel). Phosphorylation of the exogenous substrate enolase and Fyn autophosphorylation were normalized with respect to the level of kinase. These values are presented here as the fold-induction (FI). (D) Expression of Fyn and Lck from total lysate. Actin shows equal loading. (E) Resting, anergic and rescued cells were either left untreated (−) or stimulated via CD3 (MEM-92) for 2 minutes (+), lysed, and subjected to Western blotting. Changes in protein tyrosine phosphorylation are shown. Equal loading is shown with actin staining. (F) To better visualize the defects in proximal signaling, the TCR ζ chain and LAT were immunoprecipitated. Western blots of anti-phosphotyrosine staining and total protein are presented.