Egr2 deletion prevents anti-CD3 mAb-induced anergy in vitro. (A–C) EV- or Cre-transduced T cells were left untreated or anergized with anti-CD3. The cells were washed, rested for 1–2 d, and rechallenged with immobilized anti-CD3 + anti-CD28. IL-2 production was analyzed by ELISA (A), T cell proliferation by [³H] incorporation (B), and ERK phosphorylation by immunoblot (C). (D–F) Egr2-deleted T cells were transduced with an EV or a DGK–α–myc-expressing adenovirus. The overexpression of DGK-α was confirmed by immunoblot (D). IL-2 production (E) and T cell proliferation (F) upon stimulation with immobilized 1 µg/ml anti-CD3 + 1 µg/ml anti-CD28 were analyzed. Data are presented as mean ± SD and are representative of two to three independent experiments. **, P < 0.01. In B, the difference between anergic EV and anergic Cre at 0.1 µg/ml anti-CD3 is statistically significant (P < 0.01). In C, quantitative densitometry averaged over three independent experiments showed significant difference in Erk phosphorylation between anergic EV and anergic Cre during restimulation (P < 0.05). In F, there is a significant difference between EV-EV and Cre-EV (P < 0.01) but no significant difference between EV-DGK-α and Cre-DGK-α at 0.1 µg/ml of anti-CD3.