The role of the T3/antigen receptor complex is summarized by the diagram presented in Figure 4. Signals transmitted through T3/Ti activate a phosphodiesterase. This enzyme acts on its substrate PIP2 to generate two important mediators, IP3 and diacylglycerol. IP3 mobilizes calcium from bound intracellular stones. This increase in [Ca2+]i is one intracellular signal which, in conjunction with others, induces expression of lymphokine genes by influencing pretranslational, presumably transcriptional, events. Several problems remain. Which of the five molecules in the T3/Ti complex serves as the effector molecule in the transmembrane signaling process is not known. Which molecules serve to link T3/Ti to the phosphodiesterase enzyme is under investigation. The role diacylglycerol protein kinase C and other mediators play in signalling activation is not established. Finally, for those events occurring after the early events pictured in Figure 4 that result in gene activation, the sequence is a black box. Approaches to address each of these questions are available, and answers should be forthcoming.

Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-B family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.Activation of T cells by recognition of antigens and major histocompatibility complex products on the surfaces of antigen-presenting cells initiates a series of biochemical events leading to T-cell proliferation and cytokine secretion (33). Stimulation through the T-cell receptor (TCR) alone is insufficient for T-cell proliferation or interleukin-2 (IL-2) production. In the absence of costimulation, a state of unresponsiveness, termed anergy, may develop (13, 18). The failures to produce and respond to IL-2 are the major determinants of anergy. CD28, a 44-kDa glycoprotein expressed as a homodimer on most T cells, can mediate costimulation. Induction of anergy in T-cell clones was found to be blocked by activation of CD28 by monoclonal antibodies (7).One consequence of T-cell activation is the expression of IL-2, due to both transcriptional upregulation and stabilization of IL-2 mRNA. Transcriptional upregulation is mediated by the IL-2 promoter, contained within approximately 300 bp upstream from the transcriptional start site (reviewed in references 11 and 22). Engagement of CD28 enhances activation of the IL-2 promoter resulting from either TCR stimulation or use of phorbol esters such as phorbol myristate acetate (PMA). Mutagenesis studies have demonstrated that a CD28-responsive element (CD28RE), located within the IL-2 promoter between Ϫ160 and Ϫ152 relative to the transcriptional start site, is required for CD28-induced ...