Table 1.
Anti-human antibodies used in this study.
Fig 1.
Young and elderly mDC1s increase CD40, CD80, IFN-γ, TNF-α and IL-6 after LPS/IFN-γ.
Young and elderly PBMCs were left unstimulated or stimulated with LPS/IFN-γ for 24 hours, then stained with a lineage cocktail (containing CD3, CD14, CD16, CD19, CD20 and CD56), markers of blood DC subsets (CD1c, CD141, CD123 and CD303), and activation markers (CD40, CD80, and intracellular IFN-γ, TNF-α, IL-6 and IL-12) for flow cytometric analysis. Within PBMC (A), single cells (B) and viable lineage negative cells (C) gates, blood DC subsets were gated as: mDC1s (CD1c+CD123-CD303-; D), mDC2s (CD141+CD123-CD303-; E) and pDCs (CD123+CD303+CD1c-; D or CD123+CD303+CD141-; E). Marker expression on blood DCs was measured using percentage of cells positive (representative graph shown in F) and geometric mean fluorescence intensity (MFI) expression levels (representative graph shown in G). In graph (H), each line represents an individual volunteer, and compares the percentage of mDC1s positive for each activation marker in their LPS/IFN-γ-stimulated sample to their unstimulated control. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 10 young volunteers, n = 10 elderly volunteers, * = p<0.05, ** = p<0.005, *** = p<0.0005 comparing LPS/IFN-γ-mDC1s to unstimulated mDC1s from the same volunteer.
Fig 2.
Elderly mDC1s increase TGF-β and elderly pDCs increase PD-L1 after LPS/IFN-γ.
Young and elderly PBMCs were left unstimulated or stimulated with LPS/IFN-γ for 24 hours, and analysed via flow cytometry for CD1c+ mDC1s and CD123+CD303+ pDCs, and expression of regulatory markers (CD39, CD73, A2AR, A2BR, PD-L1, GAL-9, and intracellular IL-10 and latent TGF-β), as per Fig 1A–1G. Percentages of mDC1s positive for regulatory markers (A), TGF-β (B) and TGF-β expression levels (C), and percentages of PD-L1+ pDCs (D) and PD-L1 expression levels (E) were measured. Each line in (A-E) represents an individual volunteer, and compares their LPS/IFN-γ-stimulated sample to their unstimulated control. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 10 young volunteers, n = 10 elderly volunteers, * = p<0.05, ** = p<0.005, **** = p<0.0001 comparing LPS/IFN-γ-DCs to unstimulated DCs from the same volunteer.
Fig 3.
Young and elderly LPS/IFN-γ-MoDCs up-regulate CD40, CD80, CD86, IL-6, CD39 and PD-L1.
Young and elderly monocytes differentiated into immature CD11c+CD14- MoDCs using GM-CSF and IL-4 for seven days, were left unstimulated or stimulated with LPS/IFN-γ for a further two days, and analysed via flow cytometry for antigen-presenting and co-stimulatory markers (MHC-I, CD1a, CD40, CD80 and CD86), intracellular pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6 and IL-12), and regulatory markers (CD39, CD73, A2AR, A2BR, PD-L1 and GAL-9, and intracellular IL-10 and latent TGF-β). Percentages of CD11c+CD14- MoDCs positive for antigen-presenting and co-stimulatory molecules (A), pro-inflammatory cytokines (B), and regulatory markers (C) were measured; each line in A-C represents an individual volunteer, and compares their LPS/IFN-γ-stimulated sample to their unstimulated control. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 7–23 young volunteers, n = 7–34 elderly volunteers, * = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.0001 comparing LPS/IFN-γ-MoDCs to unstimulated MoDCs from the same volunteer.
Fig 4.
Elderly LPS/IFN-γ-MoDCs have variable changes in MCP-1, IL-6 and IL-8 secretion and partially down-regulate antigen-processing capacity.
Concentrations of MCP-1 (A), IL-6 (B), and IL-8 (C) were measured in culture supernatants from unstimulated/immature and LPS/IFN-γ-stimulated young and elderly MoDCs via cytokine bead array. Antigen processing ability of young and elderly MoDCs was assessed by incubating MoDCs with a fluorescent ovalbumin conjugate (DQ-OVA), and measuring the percentage of DQ-OVA+ cells via flow cytometry (D). Each line in (A-D) represents an individual volunteer, and compares their LPS/IFN-γ-stimulated sample to their unstimulated control. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 10–12 young volunteers, n = 10–25 elderly volunteers, * = p<0.05, **** = p<0.0001 comparing LPS/IFN-γ-MoDCs to unstimulated MoDCs from the same volunteer.
Fig 5.
Elderly LPS/IFN-γ-MoDCs induce greater T cell proliferation at lower DC: T cell ratios.
Young and elderly immature/unstimulated and LPS/IFN-γ-stimulated MoDCs were co-cultured with allogeneic, CFSE-labelled young T cells at DC: T cell ratios of 1:5, 1:20 and 1:50 for 5–8 days, and T cell proliferation analysed via flow cytometry as per S2A–S2E Fig. Percentages of young CD4+ (A) and CD8+ T cell (B) proliferation induced by LPS/IFN-γ-MoDCs were compared to those induced by age-matched unstimulated MoDCs. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 4–16 young volunteers’ MoDCs, n = 9–22 elderly volunteers’ MoDCs, * = p<0.05 comparing LPS/IFN-γ-MoDCs to age-matched unstimulated MoDCs.
Fig 6.
Young and elderly LPS/IFN-γ-MoDCs induce up-regulation of CD25, IL-12, CD39, A2AR, IL-10 and TGF-β on daughter T cells.
Young CD4+ and CD8+ T cells were stimulated by young and elderly LPS/IFN-γ-MoDCs in an MLR assay, at a ratio of 1 MoDC: 5 T cells, as described in Fig 5. Percentages of parent and daughter CD4+ (A) and CD8+ T cells (B) positive for activation markers (CD25 and intracellular IFN-γ and IL-12), and regulatory markers (CD39, CD73, A2AR, and intracellular IL-10 and latent TGF-β) were measured using flow cytometry. Changes in marker expression from parent to daughter CD4+ (A) and CD8+ T cells (B) were compared for samples stimulated by young versus elderly LPS/IFN-γ-MoDCs, where each line represents the change in marker expression from parent to daughter T cells induced by one volunteer’s MoDCs. Statistical comparisons were also performed between young and elderly volunteers within each condition. Data shown as individual values, n = 8–10 young volunteers’ MoDCs, n = 8–10 elderly volunteers’ MoDCs, * = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.0001, comparing parent to daughter T cells.