An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections
Fig 5
Differentiation of granulocyte/macrophage progenitor (GMP) cell line into PMN-DCs in vitro.
(A) ER-HoxB8 GMP cells (GFP+) were cultured for 4–5 days in the presence or absence of estrogen and then cultured for an additional 5 days with or without GM-CSF or IL-4 in the presence of bone marrow feeder cells; differentiation of GFP+ cells into PMN-DCs was tracked by CD11c and MHC class II expression. (B-C) GMP cells were matured into neutrophils (Day 0) or further differentiated into PMN-DCs (after 5 days with GM-CSF and IL-4 and feeder cells). (B) Undifferentiated (Day 0) or differentiated (Day 5) neutrophils (upper panel) were incubated overnight with DsRed A. fumigatus spores stained with Uvitex and then analyzed by flow cytometry; the association rate with live (DsRed+) or dead (DsRed-) A. fumigatus spores with each population from above plots is shown in the lower panel. (C) Undifferentiated neutrophils or differentiated PMN-DCs were incubated for 1 hour with β-glucan-coated AlexaFluor647 beads and analyzed by flow cytometry for association with cells. (D) Expression of CD11c and MHC class II on starting neutrophils (Day 0) vs. neutrophils differentiated for 5 days with or without feeder cells (MFI indicated). (E) GMP cells were matured into neutrophils (Day 0) and differentiated 5–7 days without feeder cells; representative flow plots and images of cells at day 0, 5 or 7. Mean ± SEM shown.