Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p < or = 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-gamma, TNF, and IL-17, and transiently expanded FOXP3(+) regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.