Morphology and phenotype of thymic DC precursors freshly isolated and after in vitro stimulation with IL-3. For confocal laser scanning microscope analysis, CD1a⁻CD3⁻CD4⁺CD8⁻thymocytes were sorted and either directly cytocentrifuge preparations were made or cells were cultured for 5 days in the presence of IL-3, resuspended and layered on precoated glass slides. Both fresh and cultured cells were stained for HLA-DR. For FACS analysis, CD1a⁻CD3⁻CD4⁺CD8⁻thymocytes were cultured for 5 days in the presence of IL-3 and CD40L⁺ mouse fibroblasts. (A) Freshly isolated DC precursors show a rounded morphology and weak staining for HLA-DR. (B) Activated DCs upon culture in IL-3 acquire a dendritic morphology and more intense staining for HLA-DR (bar, 5 μm). (C) IL-3– and CD40L-activated DCs display a phenotype specific for mature DC. (D) The expression of IL-3R and IL-7R on thymic DC precursors compared with CD4 ISP pre-T cells. Solid lines represent staining with specific antibodies as compared with staining with isotype-matched controls indicated with dotted lines.