2d). Higher concentrations of rapamycin (up to 100 ng/ml) did not further find more enhance T cell proliferation after TLR-7 ligation of PDC. T cells stimulated by PDC secreted proinflammatory (IFN-γ, IL-17) and anti-inflammatory (IL-10) cytokines (Fig. 2e), but no T helper type 2 (Th2) cytokines (data

not shown). Treatment of PDC with rapamycin suppressed the capacity of PDC to stimulate IFN-γ and IL-10 secretion by T cells irrespective of the mode of PDC-activation. Because rapamycin enhances the capacity of TLR-7 activated PDC to stimulate CD4+ T cells, we determined whether these CD4+ cells acquired a different cytokine production profile. CFSE-stained naive and memory T cells were stimulated by TLR-7 activated PDC that were treated or not treated with rapamycin. After 7 days these T cells were restimulated with PMA/ionomycin and intracellular IFN-γ, C59 wnt IL-17 and IL-10 accumulation was determined. Figure 2f shows that rapamycin

treatment of PDC reduced the generation of IFN-γ-producing and IL-10-producing naive Th cells, while leaving IFN-γ and IL-10 production in the memory Th cell compartment unaffected. IL-17 was not induced in naive Th cells by TLR-7 PDC (< 1%), but rapamycin treatment of PDC slightly reduced the numbers of IL-17-producing memory Th cells. To find an explanation for the observed increase in T cell proliferation induced by rapamycin-treated TLR7-activated PDC, we determined the effects

of rapamycin on the expression of major histocompatibility complex (MHC) and co-stimulatory molecules on PDC. Rapamycin did not affect expression of MHC class I and II molecules on PDC under any of the stimulation conditions (data not shown). CD40 expression on PDC was suppressed by rapamcyin in both stimulation conditions, while CD86 expression was not affected. Interestingly, rapamycin enhanced up-regulation of CD80 significantly on Non-specific serine/threonine protein kinase TLR-7-ligated PDC, but not on TLR-9-activated PDC (Fig. 3a). In the absence of rapamycin a subpopulation of TLR-7-stimulated PDC did not express CD80, while in the presence of rapamycin all PDC up-regulated CD80 expression. To determine whether the increased CD80 expression might be responsible for the increased ability of rapamycin-treated TLR-7-activated PDC to stimulate T cell proliferation, a neutralizing antibody against CD80 was added to co-cultures of TLR-7-stimulated PDC and allogeneic T cells. As rapamycin inhibits IFN-α production by TLR-7-activated PDC and IFN-α has an inhibitory effect on T cell proliferation [26, 27], we also determined the effect of a neutralizing IFN-α-R2 antibody on the T cell stimulatory capacity of TLR-7-activated PDC.