IFN production in pDCs is the hallmark response of TLR7 activation [10] and was elicited by R848 as expected. technology was used to quantify signaling pathway activation and cytokine production across all major immune cell populations in human whole-blood stimulated with SV with the small SC79 molecule agonist resiquimod (R848), which is specific for TLRs 7 & 8. Influenza RNA has been shown to activate human TLRs 7 and 8 [20]. Unexpectedly, the SV rapidly activated multiple signaling pathways across various cell populations but overall yielded a proteomic signature distinct from TLR7/8 stimulation (Figures 1A and 1B). In myeloid cells SV induced phosphorylation of ERK, the S6 ribosomal protein (S6), CREB, and Histone H3molecules involved in MEK, PI3K, and mTOR signaling [21]. SV did not however stimulate stress kinases such as p38 and MAPKAPK2 or the NFB pathway as indicated by a lack of total IB degradation. In contrast, R848 activated almost all signaling pathways in the myeloid lineage. Importantly, p38 phosphorylation is a hallmark of almost all TLR responses and was thus a key difference between these signaling profiles. Open in a separate window Figure 1 Comparison of signaling network activation induced by either SV or a TLR7/8 agonist. (A and B) Freshly isolated human whole-blood was stimulated with PBS, SV (15ug/ml), or R848 (10ug/ml) for 30mins prior to RBC lysis and fixation. Cells were then SC79 stained with isotope labeled mAbs against surface proteins and signaling proteins and prepared for 31-parameter mass cytometric analysis. Cell populations were identified as SC79 CD11c+ CD33+ HLADR+ CD14hi Monocytes, CD11c+ CD33+ HLADR+ CD16hi Monocytes, CD66+ Granulocytes, HLADR+ CD123+ pDCs, HLADR+ CD1c+ cDCs, CD3? CD7+ NK cells, CD19+ CD20+ B cells, and CD3+ T cells. See Figure S1 for detailed gating strategy. Signaling induction was calculated as the difference of arcsinh median intensity compared with PBS control. A representative experiment is shown from 4 independent mass cytometry experiments conducted on 7 adult donors. SV induced activity also differed from TLR7/8 stimulation in lymphocytes. NK cells were activated by the vaccine but did not respond to R848, which is consistent with a lack of TLR7/8 expression in these cells [22]. B-cells were activated by R848 but only weakly affected by SV. T-cells were not activated by either stimulus. Both SV and R848 also induced shedding of CD16 (FcRIII) in monocytes and NK cells (data not shown). SV responses in monocytes were observed in all donors sampled while NK cell and dendritic cell responses were observed stably but only in particular donors. No early activity was observed in the STAT pathways in any cell type at 30mins (data not shown). Next we sought to compare the sensitivity of human immune cells to SV and R848 in order to ensure that SV was active at low concentrations. Similar to R848, SV demonstrates potent activity at nanogram per milliliter concentrations C representing 1/500th of a single vaccine dose (Figure 2). To ensure SV activity was not due to non-influenza related components in the vaccine, we stimulated whole-blood with gelatin, octylphenol ethoxylate, and thimerosal and found no detectable activity (data not shown). Open in a separate window Figure 2 Dose response dynamics of SV versus TLR7/8 stimulation. Whole-blood was stimulated at varying concentrations of either SV or R848 and monitored for S6 phosphorylation using 10-parameter Rabbit polyclonal to SORL1 flow cytometry. Cell populations were defined as CD33+ HLADR+ CD14hi Monocytes, CD33+ HLADR+ CD16hi Monocytes, CD66+ Granulocytes, CD33+ HLADR+ CD14? CD16? cDCs, CD56+ NK cells, CD20+ B cells, and CD3+ T cells. Signaling induction was calculated as the % of cells showing greater than basal S6 SC79 phosphorylation. Mean data points of replicates from 1 donor are shown. Comparison of cytokine production between the SV vaccine and a TLR7/8 agonist Signaling pathway activation in immune cells frequently causes the production of cytokines that mediate intercellular communication. A mass cytometry based staining panel capable of measuring pan-immune cytokine production was used to compare SV to TLR7/8 stimulation. This intracellular cytokine staining (ICS) panel consists of the surface markers previously mentioned as well as mAbs against IL-1, IL-1RA, IL-2, SC79 IL-4, IL-6, IL-12 (p40), IL-17A, MCP1 (CCL2), TNF, IFN, IFN, Perforin, and GM-CSF. Whole-blood.