A better understanding of the molecular basis of adjuvanticity could also promote the rational design of successful vaccines by supplementing them with the appropriate adjuvants or combinations thereof. Here, we studied the effector functions of the candidate adjuvant c-di-AMP on different immune cells in vitro as well as in vivo. We asked which cell types undergo activation and which cellular processes and molecules are involved. We previously reported that c-di-AMP promotes antigen specific T cell proliferation. C-di-AMP does not seem to directly exert this effect on T cells, thus we concluded that the observed T cell activation is mediated by other immune cell types. This was also suggested by previous in vitro studies showing that DCs which were antigen-loaded in the presence of c-di-AMP were more efficient at promoting the activation of antigen-specific T cells from TCR transgenic mice. Accordingly, we examined the response of different types of APCs to c-di-AMP. We showed that treatment with c-di-AMP induces the up-regulation of the expression of MHC class II as well as T cell co-stimulatory molecules on the surface of both murine and human DCs and MWs in vitro. These in vitro findings were further confirmed by ex vivo studies in which murine DCs and MWs were analyzed after i. n. cdi-AMP application. 24 h after i. n. administration especially DCs and not so much MWs were activated by c-di-AMP in the lymphoid compartments. Although early DC antigen presenting activity in the draining CLN was described for i. n. immunization experiments with viral antigens, our adjuvant candidate c-di-AMP alone did not notably induce T cell stimulatory molecules on APCs of the CLN after 24 h. The more pronounced DC activation was observed in the NALT, the lymphoid tissue directly associated with the site of administration, and also in the MLN. The early DC response in the MLN is in line with several studies reporting a similar observation upon i. n. immunization with model antigens and adjuvants as well as upon i. n. infection with influenza virus. We also demonstrated that murine DCs and MWs/monocytes/granulocytes respond to in vivo administration of c-di-AMP by the upregulation of IFN-b expression. Taken together, we observed the effects of c-di-AMP on its target cells on two different aspects of immune regulation. First, we found up-regulation of CD80 and CD86 on the surface of mouse and human DC subsets treated with c-di-AMP. These molecules are known to interact with CD28 on the T cell surface to deliver co-stimulatory signals after TCR engagement with the antigen presenting MHC molecule. This costimulation represents an important regulator of T cell immune tolerance toward activation in response, for example by promoting T cell proliferation and differentiation. Nevertheless, both CD80 and CD86 are also ligands for the T cell surface molecule CTLA-4 which facilitates negative T cell activation signaling.