IL-15 prolongs CD154 expression via STAT5 engagement of the proximal CD154 promoter (99.48)

Abstract

Abstract CD154 expression is tightly regulated but increased expression on CD4 T cells in mouse models and humans with lupus contributes to disease. Since IL-15 is increased in lupus serum, we explored an IL-15 contribution to prolonged CD154 expression. Flow cytometry reveals increased CD154 levels on primary CD4 T cells following IL-15 plus PHA stimulation at later time points (48 hrs). Since IL-15 signals via STAT5 we explored the hCD154 promoter for possible STAT5 binding sequences (TTCNNNGAA). Gel shift assays with nuclear extracts from IL-15-activated primary human CD4 T cells reveal a proximal site (-65 bp) binds STAT5. Binding of STAT5 to the proximal CD154 promoter was confirmed in vivo by chromatin immunoprecipitation. Functionally, over-expression of wild-type STAT5 in primary CD4 T cells increases CD154 transcriptional activity in a dose-dependent fashion following T cell activation. Mutation of the STAT5 binding site eliminates this effect. Moreover, over-expression of dominant-negative STAT5 inhibits CD154 transcription. Similarly, STAT5 siRNA inhibits CD154 protein expression at late time points (48 hrs) post-T cell activation. Interestingly, STAT5 remains phosphorylated at late time points post-activation when IL-15R[alpha] expression is increased. The fact STAT5 binds the CD154 promoter in response to IL-15 provides novel approaches to CD154 inhibition via anti-IL-15 antibody, or by JAK3 inhibitors.