The effects of cytotoxic lymphocyte antigen 4 (CTLA-4) on CD3/CD28 monoclonal antibody (mAb) activation of CD4 ؉ /CTLA-4 ؉ blastoid T cells were studied in an in vitro model system. As previously reported, coligation of CTLA-4 mAb results in suppression of T cell proliferation and cytokine production. The proliferation but not the interleukin 2 (IL-2) production could be restored by addition of exogenous IL-2, suggesting that the inhibitory effect occurred at the level of IL-2 production rather than at the regulation of the IL-2 receptor pathway. To study the effects on nuclear factors critical for T cell activation, we analyzed the levels of the transcription factors NF-B and AP-1. These were potently induced in CD3/CD28 mAb-restimulated T cells. In contrast, CTLA-4 ligation strongly suppressed the induction of both transcription factors. The compositions of NF-B and AP-1 family members were similar, irrespective of stimulation conditions. Analyses of the NF-B regulator IB-␣ revealed similar levels of IB-␣ protein in the preparations. However, a reduced phosphorylation of IB-␣ in CTLA-4 coengaged T cell blasts compared with T cells ligated with CD3/CD28 was found. Previous studies have concluded that CTLA-4 ligation regulates T cell activation by inhibiting the T cell receptor-mediated signals. However, the present findings propose that the major impact of CTLA-4 ligation is inhibition of signals mediated by CD28.The T cell surface receptors CD28 and CTLA-4 (CD152), and their ligands CD80 and CD86 on professional antigen-presenting cells, regulate the activation of T cell receptor (TCR) 1 -triggered T cells. CD28 is expressed on both resting and activated cells, whereas CTLA-4 is only detectable on activated cells (1, 2). The CD28/B7 pathway is known to be essential for the development and maintenance of T cell responses (3). CTLA-4 was originally thought to have a similar function as CD28. In agreement with this original thought, B7-dependent costimulation of CD28-deficient T cells has been demonstrated, which suggests that CTLA-4 ligation results in costimulation (4). However, there is accumulating data showing that CTLA-4 coligation has a negative regulatory effect on T cells, such as down-regulation of interleukin 2 (IL-2) production and cell cycle progression (5, 6). This negative regulatory effect is supported not only by the phenotype of CTLA-4-deficient mice (7,8) but also by the effects observed after administration of CTLA-4 monoclonal antibody (mAb) or Fab in animal models of autoimmune diseases and infection (6, 9 -11).The IL-2 gene is regulated by signals mediated by TCR as well as CD28 signaling that ultimately leads to DNA binding of nuclear factors such as the prominent NF-B and AP-1 transcription factors (12). After TCR stimulation, the immunoreceptor tyrosine-based activation motifs within the TCR/CD3 chain interact with intracellular signaling molecules. The tyrosine residues within immunoreceptor tyrosine-based activation motifs become phosphorylated and create binding sites for Src homolo...