CD16⁺and CD16⁻monocytes differentiate into DCs with distinct transcriptional profiles. (A) Shown is the experimental flowchart. Briefly, total monocytes were purified by negative selection using magnetic beads. Highly pure CD16⁺ and CD16⁻ monocytes were subsequently sorted by FACS on staining with CD16 Abs and a cocktail of FITC-conjugated nonmonocyte lineage-specific Abs (CD1c, CD3, CD8, CD19, and CD56; supplemental Figure 1). Immature MDDCs were generated by culturing monocyte subsets in the presence of GM-CSF and IL-4 for 6 days. Total RNA was extracted from matched CD16⁺ and CD16⁻ MDDCs (n = 5) that were exposed to media, LPS, or HIV for 24 hours. Genome-wide transcriptional profiling was performed using the Affymetrix HG Plus 2.0 microarrays. (B-C) Shown are the expressions of CD14 and CD16 on total monocytes before sort (B) and the expression of CD14, CD16, CD1c, and HLA-DR on immature CD16⁺ and CD16⁻ MDDCs on differentiation in vitro (C). Results in B-C are from 1 donor representative of results generated with cells from more than 10 donors. (D-H) Shown are Venn diagrams of differentially expressed genes in CD16⁺ vs CD16⁻ MDDCs in response to media (D), LPS (E), and HIV (G), as well as the representation of the number of commonly and differentially expressed genes on exposure to LPS (F) and HIV (H). n.s., not significant.