Differentiation of band cells into neutrophil-DC hybrids in culture. (A-B) Gr-1^high/CD48⁻ band cells purified from C57BL/6 mice were cultured with GM-CSF in the presence of crude BM feeder cells from B6 SJL mice. (A) CD45.2⁺/CD45.1⁻ populations purified at the indicated time points were analyzed for nuclear shape (top panels) and morphology (bottom panels; bar represents 20 μm). (B) Changes in nuclear shape were examined by analyzing >2000 cells/time point after nuclear staining. Recovery rates of viable CD45.2⁺/CD45.1⁻ cells relative to the originally plated cell numbers are shown on the right. (C-E) Crude BM cells from CD45.2 mice were cultured for 2 d with GM-CSF. Gr-1^high/CD48⁻ band cells were purified from these precultures and then co-cultured with BM feeder cells from CD45.1 mice in the presence of GM-CSF for an additional 6 d. (C) The starting population (left) and the CD45.2⁺/CD45.1⁻ population purified from the co-culture (right) were analyzed for nuclear shape and morphology. The latter population was also examined for surface phenotype (D) and APC function to present OVA peptides to OT-II CD4 or OT-I CD8 T cells (means ± SD from triplicate cultures) (E). (F) CD15⁺/CD10⁻/CD64⁻/CD14⁻ band cells purified from human BM samples were examined for the surface phenotype before (top) and after culturing for 7 d in the presence of GM-CSF, TNFα, and IL-4 (bottom). (G) The above-band cell population (top) and the CD14⁺ monocyte population purified from the same human BM samples (bottom) were cultured for 7 d in parallel and then examined for surface phenotype. Data are representative of 3 independent experiments.