To determine if the stimuli enhanced buy BYL719 the S6 phosphorylation, PDC were stimulated with CpGA or loxoribine in the presence of IL-3 and intracellular p-S6 expression was determined with flow cytometric staining (Fig. 1b). CpGA stimulation resulted in the same fluorescence intensity as IL-3 treatment alone, while loxoribine stimulation slightly increased the p-S6 expression. CpG-A was a more effective stimulus than loxoribine to induce IFN-α secretion (Fig. 1c). While 20 ng/ml rapamycin inhibited loxoribine-induced IFN-α secretion by 64%, it inhibited CpG-A-induced IFN-α secretion by only 20%, despite almost complete suppression of mTOR-signalling. In contrast, secretion of the proinflammatory cytokines IL-6 and TNF-α was inhibited

by rapamycin with similar efficacy in both stimulation conditions (Fig. 1d). The observed inhibitory effects of rapamycin were not due to

general impairment of PDC function, because no inhibition of CXCL-10 secretion was observed (Fig. 1d) and rapamycin did not induce apoptosis, as demonstrated by the absence of active caspase-3 (data not shown). As mTOR inhibition decreased cytokine secretion by PDC, we reasoned that mTOR stimulation might increase cytokine production. Therefore we added 10 nM VO-OHpic trihydrate, a specific inhibitor of PTEN, during PDC activation. The upstream signalling pathway that activates mTOR is initiated by phosphatidylinositol 3-kinase (PI3K), which generates 3-phosphorylated inositol lipids (PIP3) [23]. PTEN is a negative regulator of PIP3K-signalling

because it dephosphorylates PIP3 [24], and therefore inhibition of PTEN can abrogate negative regulation of mTOR phosphorylation. find more Buspirone HCl The addition of VO-OHpic trihydrate to TLR-activated PDC in a concentration that increased generation of PDC from human CD34+ progenitor cells [25] did not, however, affect p-S6 expression and cytokine production by PDC (data not shown), suggesting that PI3K-mTOR signalling is not limited by PTEN in human PDC. Together, these data show that a clinically relevant concentration of rapamycin inhibits proinflammatory cytokine production by TLR-7-activated PDC and TLR-9-activated PDC, while it suppresses IFN-α secretion in TLR-7-activated PDC but almost not in TLR-9-engaged PDC. To study the effects of mTOR inhibition on the T cell stimulatory capacity of PDC, we activated PDC with TLR ligands for 18 h and then added allogeneic CD3+ T cells. After activation in the presence or absence of rapamycin, PDC were washed carefully to remove rapamycin before T cells were added. Activation of PDC via TLR-7 in the presence of rapamycin increased their capacity to stimulate T cell proliferation, while the addition of rapamycin during TLR-9 activation did not (Fig. 2a). The increased proliferation of T cells upon mTOR inhibition in TLR-7-activated PDC was confined to enhanced expansion of the CD4 compartment (Fig. 2b), and was observed in both memory (CD45RO+) and naive (CD45RA+) T cells (Fig. 2c).