Figure 4. Inflammatory Ly6C+ monocytes provide bioactive IL-18 and IL-15 to memory CD8+ T cells.
(A–B) CD8+ T cells, cDCs, neutrophils or Ly6C+ monocytes were purified by flow-cell sorting respectively from WT B6 mice that (i) contained OT-I GFP+ memory cells or (ii) were naive or immunized with WT Lm 20 hrs earlier. Purified CD8+ T cells were incubated with each innate cell subset before staining for cell surface expression of CD8 and intracellular IFN-γ and GrB. The proportion (%) of OT-I GFP+ memory cells expressing either marker is shown. (B) CD8+ T cells were incubated with anti-IL-18R prior to adding activated Ly6C+ monocytes. (A–B) represent 3 independent replicate experiments (n=3–6) with p-values. (C) Spleen cells from WT B6 mice immunized with WT Lm (with 105 or 106 Lm) 24 hrs prior were stained with mAbs against cell surface markers discriminating conventional dendritic cells (cDC, CD11chi), neutrophils (CD11bhi, Ly6Cdim, ly6G+) and Ly6C+ monocytes (CD11bhi, Ly6Chi, T/B/NK cell exclusion), and for expression of IL-15Rα. Black gates shown on dot plots define the different cell populations and histograms overlay IL-15Rα expression levels on the distinct cell subsets in uninfected (grey) versus infected (dark) mice. (D) Relative increase of IL-15Rα expression on resting versus activated DC (red), Ly6C+ monocytes (black) and neutrophils (blue) from spleen, lung and liver of B6 WT mice immunized with WT Lm, MCMV, S. pneumoniae, P. berghei or injected with poly(I:C) at indicated times. Data are representative of 2 independent replicate experiments (n=6) with p-values.