Only classical Ly-6C^highmonocytes acquire suppressor function and NO production independent of proliferation. Fresh murine BM cells were labeled with the cell proliferation dye eFluor670 and grown under the influence of either GM-CSF, M-CSF, G-CSF, or Flt3L at 50 U/mL each for 3 days. (A) Then, cells were harvested and counterstained for Ly-6G or for Ly-6C. Dotted line separates Ly-6C^high from Ly-6C^low monocytes (1 representative of 4 experiments shown). (B) Murine BM cells cultured in GM-CSF within the gates as indicated in panel A were separated by cell-sorting and individual cytospin preparations were stained with hematoxylin and eosin (n = 2). The sorted cell populations show morphologies of differentiated neutrophils with segmented nuclei (1) that correspond to nondividing cells, dividing preneutrophils with band or ring-shaped nuclei (2) and dividing (3) and nondividing monocytes (5). Population 4 showed a mix composed of preneutrophilic and monocytic cells, indicating that preneutrophilic cells (2) that downregulated the Ly-6G marker during proliferation merge with proliferating monocytic cells. Scale bars (images 1-5), 10 μm. (C) T-cell suppressor assay testing the sorted cell populations as shown in panel A. Populations 1 and 2 were pooled. One representative experiment of 3 is shown. For statistics, granulocytes were compared with L-Mono from pooled data from 3 independent experiments. (D) BM cells of wild-type mice were labeled with eFluor670 and cultured with the indicated doses of GM-CSF for 3 days. Then eFluor670 dilution as a measure for proliferation was tested by FACS analysis (n = 2). (E) Identification of the monocyte subset that acquires T-cell suppressive activity following a 10-day treatment with GM-CSF. (Left) Gating strategy for separating total CD11b⁺ Ly-6G⁻ myelomonocytic cells (excluding polymorphonuclear granulocytes [PMNs]) into Ly-6C^high CD43⁺ classical monocytes, Ly-6C^int CD43⁺ intermediate monocytes, Ly-6C⁻ CD43⁺ nonclassical monocytes, and Ly-6C⁻ CD43⁻ double negative cells. (Right) T-cell suppression assay with the sorted cell subsets. One representative experiment is shown. Statistics compare double negative vs classical monocytes from pooled data of 2 independent experiments. (F) Intracellular IL-6 and iNOS expression induced by overnight stimulation with LPS/IFN-γ in Ly-6C^high classical monocytes freshly isolated from SP, BM, or L-Mono. Values correspond to the median fluorescence intensity (MFI) ± SD of 2 independent experiments. For all panels, unpaired Student t test was used. *P < .05, **P < .01, ***P < .001. Error bars show SD.