Identification of a CD28 response element in the CD40 ligand promoter

Ligation of the T cell coreceptor CD28 or CD2 by its cognate ligands B7-1 o r LFA-3, respectively, greatly aids the Ag-induced up-regulation of several genes, including IL-2 and CD40 ligand (CD40L), Using luciferase reporter c onstructs under the control of the 1.2 kb of 5 ' noncoding region of the hu man CD40L gene, we have found that stimulation through CD28 was required fo r a strong transcriptional activity of the CD40L promoter in response to TC R ligation, while the activity induced by CD2 was slightly lower than CD28, Deletion analysis demonstrated that the transcriptional elements mediating this effect were located within a 300-bp region upstream of the start site . Further dissection of this region and gel shift analyses demonstrated the presence of a CD28 response element in a region located between nucleotide s -170 to -164 relative to the start site. Transcriptional studies with a C D40L enhancer-promoter carrying a mutation in this putative CD28 response e lement revealed that the activity was reduced by 80 and 70% after B7-1 and LFA-3 costimulation, respectively. The transcription factor complex bound t o this site contained at least JunD, c-Fos, p50, p65, and c-Rel, but not c- Jun. Mutations introduced into the CD28RE also blocked the binding of this complex. These observations identify an important role for the CD28 signali ng pathway in the regulation of CD40L promoter transcriptional activity. Th e Journal of Immunology, 2001, 166: 2437-2443.