7a). Induction of IL12p40 expression on rhesus pDC, as observed with mAb C8·6, was confirmed by using another anti-IL-12p40/70 mAb (clone

C11·5), which gave similar percentages of positive cells for pDC as well as mDC upon TLR-7/8 stimulation (Fig. 7b). Finally, analysis of IL-12p40 mRNA in TLR-7/8 (CL097)-stimulated purified pDC, mDC and monocyte populations showed similar high expression levels in pDC relative to mDC and monocytes and no induction in pDC upon TLR-4 stimulation (Fig. 8a), thus confirming the FACS expression data. Both mDC and monocytes up-regulated TNF-α mRNA expression upon TLR-4 (LPS) as well as TLR-7/8 (CL097) stimulation, underscoring the functional capacity of these purified cell populations (Fig. 8b). In agreement with the FACS analysis, TNF-α mRNA expression in pDC was up-regulated only upon TLR-7/8 and not TLR-4 stimulation. While the mDC and pDC preparations were only 60–75% pure the other cells present were find more Dorsomorphin either granulocytes or monocytes

and this could not have affected the IL-12p40 expression data, as monocytes were observed to have only very low IL-12p40 expression and the monocyte fraction itself was >90% pure with <5% mDC and <1% pDC contamination. In this work, we adapted a whole blood stimulation assay to study functional characteristics of peripheral blood DCs and monocytes in macaques and performed a direct comparison with human blood samples. Most responses of the different subsets were very similar between macaques and humans and in agreement with previous studies, in which purified cell populations instead of whole blood stimulation had been used [2, 17, 25-28, 32]. However, we observed that, in contrast to humans, rhesus pDC expressed

IL-12p40 upon stimulation with TLR-7/8 or TLR-9. Preliminary data suggest a similar IL-12p40 expression pattern in cynomolgus macaques (V.S., to be published elsewhere). We also observed that relative to humans, mDC G protein-coupled receptor kinase and monocytes in rhesus macaques responded less well to TLR-7/8 stimulation when expressed as percentage of IL-12p40- and TNF-α-positive cells. Of note is that a similar relatively lower level of IL-12 induction has been reported previously for macaque monocyte-derived DC [23]. The capacity of rhesus pDC to produce IFN-α as well as IL-12p40 may potentially modify their response to viral infections, where pDC are known to play an important role [36]. Previous studies either did not include IL-12 in their analysis [23] or measured IL-12 cytokine production by enzyme-linked immunosorbent assay (ELISA) on either stimulated total PBMC or lineage-negative cell cultures [25-27]. Others used FACS analysis, but studied IL-12 expression only in LPS-stimulated PBMC [17], which would have given no expression in pDC. Hence, our observation was made possible by the use of FACS analysis to detect TLR-induced cytokine expression in all subsets simultaneously.