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The CD28 costimulatory signal enhances antigen-mediated induction of interleukin-2 (IL-2) gene transcription through activation of an enhancer termed the CD28-responsive element (CD28RE). Although various nuclear proteins have been shown to bind to CD28RE, their in vivo functions in the regulation of this enhancer remain elusive. In this report, we show that CD28RE binds distinct transcription factors in cells treated with different mitogenic stimuli. Stimulation of the T-cell receptor (TCR) complex in the absence of a CD28 costimulatory signal induces a member of the nuclear factor of the activated T cells, NF-ATp; however, this treatment fails to activate the CD28RE enhancer activity. Significant activation of CD28RE was detected when the cells were treated with both the TCR stimulators and an anti-CD28 monoclonal antibody (anti-CD28), which induces the NF-kappaB/Rel enhancer binding proteins in addition to NF-ATp. The costimulatory activity of anti-CD28 can be further enhanced by a phorbol ester. Kinetic analyses demonstrate that activation of endogenous IL-2 gene transcription is correlated with the binding of CD28RE by NF-ATp and different NF-kappaB/Rel species. Transient-transfection studies reveal that expression of either NF-ATp or the p50-RelA NF-kappaB heterodimer leads to the potent transactivation of both the CD28RE enhancer and the intact IL-2 promoter in mitogen-stimulated cells. Remarkably, coexpression of these two families of enhancer-binding proteins in Jurkat T cells results in the transactivation of the CD28RE enhancer even in the absence of any cellular stimuli. Together, these results suggest that activation of IL-2 gene transcription by the TCR- and CD28-mediated signals involves the interaction of CD28RE with NF-ATp and various NF-kappaB/Rel transcription factors.