Nef inhibits surface expression of Fms. (A) In the left histograms, TF-1-fms-Nef-ER cells were precultured with M-CSF–containing media in the absence (upper) or presence (lower) of 0.1 mM 4-HT for 24 hours. In the right histograms, TF-1-fms-Nef-ER cells were precultured with M-CSF–free media in the absence (top) or presence (bottom) of 0.1 μM 4-HT for 12 hours. The expression of Fms on these cells was analyzed by flow cytometry with PE-labeled anti-Fms antibody. The mean fluorescence intensity (MFI) of Fms expression is indicated. (B) TF-1-Nef ER cells were precultured with GM-CSF–free media in the absence (top) or presence (bottom) of 0.1 μM 4-HT for 12 hours. The surface expression of GM-CSF receptors was analyzed with FITC-labeled anti-α chain (left) and PE-labeled anti-β chain (right) antibodies. The MFI of GM-CSF receptor expression is indicated. (C) Macrophages were nucleofected with the control CD8 plasmid or CD8-Nef plasmid and then costained with APC-labeled anti-CD8 and PE-labeled anti-Fms. Results with macrophages obtained from 2 different donors are shown as contour plots. (D) As in panel C, the nucleofected macrophages were costained with APC-labeled anti-CD8 and PE-labeled anti-Fms, or with APC-labeled anti-CD8 and PE-labeled anti-CD4. The MFI of the expression of Fms or CD4 in the populations of CD8^low/−, CD8^high, CD8-Nef^low/−, or CD8-Nef^high was analyzed. The results with macrophages obtained from 5 different donors are summarized.