IL-4 induces monocytes to differentiate into CD14^– fibrocytes that are readily distinguished from CD14⁺ macrophages in the same culture. (A) Culture of monocyte-rich apheresis fractions from healthy donors with rhIL-4 induces differentiation into an adherent population that contains cells with a typical spindlelike appearance, consistent with a fibrocyte lineage (original magnification ×20). (B) Cell surface phenotype of IL-4–differentiated adherent cells identifies 2 subsets based on CD14 expression, which further shows differential expression of collagen and TSLPR. FMO controls on gated CD14⁺ vs CD14^– populations are shown by shaded gray histograms. This is representative of more than 5 experiments from 5 separate healthy donors. (C) Fibrocyte (defined as CD14^–CD123^–CD11c⁺) numbers in monocyte cultures incubated for 9 days with rhIL-4 alone (10 ng/mL) vs rhIL-4 plus TSLP (10 ng/mL). This is representative of 4 separate experiments. (D) CD14⁺ macrophages vs CD14^– fibrocytes generated using rhIL-4 as described in (C) were analyzed by reverse transcriptase PCR for IDO, TDO, ARG1, and INOS2. The mean ± SEM of relative increases normalized to PBMC are shown from 5 separate experiments. (E) RhIL4-induced fibrocytes suppress autologous T-cell proliferation to various concentrations of anti-CD3. Comparisons between 2 groups (with T-cell-alone group) were done using a 2-way analysis of variance for multiple comparisons, with significance determined as P < .05. This is representative of 3 experiments. (F) Using RNA expression profiles of cells generated and electronically sorted as described in (C), gene set enrichment analysis of CD14⁺ macrophages vs CD14^– fibrocytes for the Lindstedt DC maturation C gene set is shown. The most underexpressed of 49 genes in CD14^– fibrocytes vs the CD14⁺ macrophages is listed below the figure. This is representative of 3 separate expression profile-based analyses.