Functional disparity of distinct CD28 response elements toward mitogenic responses

Activation of T cells through the antigen-specific T-cell receptor in combination with a costimulatory signal results in efficient cytokine gene transcription. The CD28-induced signal represents a major costimulatory signal for T cells. A CD28 response element, named CD28RE, was first identified in the interleukin-2 (IL-2) promoter region, Here we demonstrate that the NF-kappa B sequence in the IL-6 promoter functions as a CD28 response element. Mutations in this sequence rendered the IL-6 promoter unresponsive to CD28 costimulation, Moreover, this element could replace the IL-2 CD28RE in conferring CD28 responsiveness to the IL-2 promoter. In analogy to the known CD28 response elements IL-2 CD28RE, IL-8 CD28RE, and the human immunodeficiency virus-1 (HIV-1) NF-kappa B motif, the IL-6 NF-kappa B motif efficiently bound c-Rel, c-Rel/NFKB1, and the recently identified inducible T-cell factor NF-MATp35, However, the IL-6 NF-kappa B sequence together with the IL-8 CD28RE and HIV-1 NF-kappa B sequence differed from the IL-2 CD28RE in the binding of NF-kappa B/Rel family proteins. Although the IL-2 CD28RE exerted selective binding with c-Rel and c-Rel/NFKB1, the other CD28REs allowed efficient binding of a wide range of NF-kappa B/Rel family proteins. The difference in binding specificity correlated with the capacity of the distinct CD28 response elements to function in the context of the IL-6 promoter in response to T-cell activation. Domain swapping experiments revealed that the IL-8 CD28RE and HIV-1 NF-kappa B motif conferred similar responsiveness as the genuine IL-6 NF-kappa B motif in the transcriptional activation of the IL-6 promoter upon CD28 costimulation, In contrast, replacement of the IL-6 NF-kappa B sequence by the IL-2 CD28RE motif strongly reduced the responsiveness of the IL-6 promoter. These data indicate that despite the sequence similarity, two different classes of CD28 responsive elements exist that differ in their NF-kappa B binding capacity and the ability to confer CD28 costimulatory responsiveness toward a heterologous promoter.