Fig. 3: Identification of thymic TE-DCs.

a Representative MP-IVM micrograph of a thymus graft in a CD11c-YFP BM chimeric mouse (left panel). In all, 2 hours after adoptive transfer of labeled DCs, both homed DCs (blue) and endogenous DCs (green) are readily detectable in proximity to microvessels, which were visualized after injection of TRITC-labeled 2 MDa dextran (red). Circles identify typical DCs that are closely associated with microvessels. The box highlights one perivascular cell, which is shown in the right panel after the digital surface rendering of the DC and microvascular lumen (Supplementary Movie 3). Scale bars represent 50 μm (left panel) and 10 μm (middle panel), respectively. b The frequency of parenchymal and perivascular DCs in transplanted thymi was evaluated in confocal micrographs. Cells were considered as perivascular when the shortest distance between the margin of the cell and the lumen of the blood vessel was <1 μm. n = 5; ***P = 0.0003 (Student’s two-tailed, paired t test). c Confocal micrograph of a frozen thymic (ET) section showing CD11c⁺ DCs (red) extending protrusions through CD31⁺ vascular endothelium (white). The upper panels show merged two-color overlays at low (left) and high magnification (middle) and a schematic illustration of the latter (right). The lower panels show single channels for CD31 (left) and CD11c staining (right) of the same high-power field. Scale bars reflect 10 μm. d 3D reconstruction and surface rendering from a confocal z-stack of a frozen thymic section similar to that described in c, showing the vasculature in blue (CD31 staining), perivascular CD11c⁺ DCs in green (GFP signal), and their intravascular protrusions pseudocolored in red. To facilitate visualization of the transendothelial protrusions, only the perivascular GFP⁺ DCs are shown in the analysis. (Supplementary Movie 4). e Zoomed-in detail showing the constriction of the cell body of a perivascular GFP⁺ DC presumably protruding across the endothelial junction (arrow). DCs dendritic cells, Par-DCs parenchymal dendritic cells. See also Supplementary Movie 5.