All TLR-Ls induce systemic expansion of monocytes, but only R-848 and CpG-ODN induce rapid, transient expansion of CD14⁺CD16⁺ inflammatory monocytes. (A) The total monocyte population was identified within the SSC-A^hiFSC-A^hi PBMCs as the CD3⁻CD8⁻CD20⁻ HLA-DR⁺ population. Kinetics of total monocyte frequencies within PBMCs were assessed at the indicated time points after TLR-L injection. (B) Three distinct subpopulations were defined within the total monocytes by CD14 and CD16 staining: CD14⁺CD16⁻, CD14⁺CD16⁺, and CD14^dimCD16⁺⁺. The flow cytometric dot blot representation of monocyte subpopulations frequencies within the total monocytes at baseline (day −12) and at 3 and 8 hours and 1, 2, 3, 6, and 14 days after TLR-L injection are shown. The CD16⁺ cells (red) are overlaid on the total monocyte population (black). Numbers in gates represent the frequency of distinct subpopulations within the total monocytes in one representative animal from each experimental group. (C) Kinetics of frequencies of the CD14⁺CD16⁻ (gray), CD14⁺CD16⁺ (red), and CD14^dimCD16⁺⁺ (blue) subpopulations within the total monocytes were assessed at the indicated time points after TLR-L injection. Statistical analysis of cell kinetics in panels A and C indicates significant change relevant to the baseline. Data are means ± SEM. *P < .05; **P < .01; ***P < .001 by t test.