Figure 1.
C-di-AMP does not directly affect murine T cell proliferation in vitro.
Murine splenocytes were sorted for CD4+ and CD8+ T cells (CD62Lhigh, CD44low, CD25−) and treated with either 5 µg/ml c-di-AMP, 5 µg/ml concanavalin A (ConA; positive control), or left untreated (control) in the presence of 3H-thymidine. As a measure for proliferation, 3H-thymidine incorporation in T cells was determined by scintillation. The error bars show SEM for n = 3. Differences were statistically significant at p<0.001 (***), p<0.05 (*) or non-significant (n. s.) with respect to the control.
Figure 2.
C-di-AMP activates murine immune cells in vitro.
Murine bone marrow derived model dendritic cells (DCs, CD11c+) and model macrophages (MΦs, CD11b+) were incubated for 24 h with 5 µg/ml c-di-AMP or medium without additives (control). Cells were decorated with fluorescently labeled antibodies against MHC class II and the co-stimulatory molecules CD80 and CD86. The diagram shows the normalized median fluorescence intensity (fold increase as compared to the control) analyzed by flow cytometry. Error bars are SEM for n = 3.
Figure 3.
C-di-AMP up-regulates T cell co-stimulatory molecules preferentially on conventional murine dendritic cells (DCs).
(A) In vivo effects of c-di-AMP on mouse APCs. Flow cytometric analysis of DCs (CD11c+) and MΦs (CD11c−, CD11b+) from the nose-associated lymphoid tissue (NALT), the cervical lymph nodes (CLN) or the mediastinal lymph nodes (MLN) 24 h after i. n. administration of c-di-AMP in mice. (B) In vitro effects of c-di-AMP on mouse DCs. Murine bone marrow derived model conventional (cDCs) and plasmacytoid DCs (pDCs) were generated by culturing in the presence of Flt3l and incubated for 24 h with 5 µg/ml c-di-AMP or without additives (control). Cells were decorated with fluorescently labeled antibodies against identification markers CD11c (DCs), CD11b (cDCs), B220 (pDCs), and MHC class II, CD80 and CD86. The diagrams show the normalized median fluorescence intensity (fold increase as compared to the control) analyzed by flow cytometry. Error bars are SEM for n = 3. Differences are statistically significant at p<0.01 (**) or p<0.05 (*).
Figure 4.
C-di-AMP induces IFN-β production in murine dendritic cells (DCs).
(A) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo. Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm2/steridian). Error bars are SEM. (B) C-di-AMP induces IFN-β production in DCs in vitro. BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.
Figure 5.
C-di-AMP preferentially activates human myeloid dendritic cells (DCs) in vitro.
PBMC-derived human plasmacytoid DCs (pDC) or myeloid (conventional) DCs (mDC) were incubated for 24 h in the presence of 60 µg/ml c-di-AMP or without additive (control). (A) Cells were decorated with fluorochrome-conjugated antibodies specific for the identification markers CD11c (mDC), CD303 (pDC) CD80, CD83 and CD86, and analyzed by flow cytometry. Normalized median fluorescence intensity (MDFI) is shown as fold increase compared to the control. Error bars show SEM for n = 3. (B) The medium from the cultured mDC or pDC was analyzed for IFN-β secretion by ELISA. Error bars show SEM for n = 3. Differences are statistically significant at p<0.001 (***) or p<0.05 (*).