Fig 1.
Detailed 3D images of the conduit network in a whole LN.
EVIS imaging of an entire popliteal LN generated a 3D volume image of which interior slices can be viewed individually (A). Three-dimensional image reconstruction of the entire LN shows (B) lymphatic channels filled with the tracer molecule WGA (red glow) together with dextran-labeled blood vessels (green), or the blood vasculature alone (C). An interior view of 20-μm thick optical sections (D—I) and a 1 μm slice (J) permits detailed insights into the LN architecture. A cross-section of the LN displays the location of cell-specific zones (D), and close-ups reveal anatomical details, including the arrangement of long conduits descending from the SCS at the edges of a B cell follicle (arrowheads, E), an MC with a central blood vessel situated amongst the WGA-filled sinuses of the medulla (F), and the transition from dense to sparse conduit networks in the superficial to the deep TCZ (G). The conduit network forms a highly organized grid within the TCZ (white, H and I; red, J) interspersed with CS (arrowhead, panel J) and blood vessels (green), including HEVs, which are closely surrounded by cells displaying a cobblestone-like morphology (arrowhead, panels H and J). Besides larger blood vessels, small blood vessels are frequently enclosed by conduits (asterisks, G and J). Image rendering was performed in Voxx (A—I) and ImageJ (J). See also S1 Fig, S1 and S2 Videos. CS, cortical sinus; EVIS, extended-volume imaging system; HEV, high endothelial venule; LN, lymph node; MC, medullary cord; SCS, subcapsular sinus; TCZ, T-cell zone; WGA, wheat germ agglutinin.
Fig 2.
Network topology of the LN conduits in the TCZ.
Deploying custom-developed image-processing tools, a description of the conduit network in terms of nodes and links was generated from the 3D image data and used to estimate network parameters. Three-dimensional projections of the blood vasculature (red) and the conduit network in the TCZ (blue) as a whole (A) and magnified views of the TCZ (B, C) expose the complexity and the high level of detail in this data set. The full conduit data include large segments with diameters over 5 μm (D), but these represent blood vessels as the overlay with the blood vessel model (yellow) indicates (E). A blood vessel-free conduit network (F) was obtained by removing the majority of blood vessels from the 3D image prior to the network extraction in a semi-automated process. This TCZ conduit network excluding blood vessels was employed to calculate the distribution of segment diameters (G), lengths (H), the branching pattern (J), branching angles (K), and segment orientation (L), and the full data set including blood vessels was used to calculate the minimum distance to the nearest conduit (I). Simulation of T-cell motility utilizing these conduit data provides the cell displacement at a mean speed of 13 μm min−1 (M), motility coefficients for different speeds (N), and a spider plot representation of migration paths in a network with and without blood vessels (O). Values for each data point can be found in S1 Data. See also S2 and S3 Figs, and S3 Video. LN, lymph node; TCZ, T-cell zone.
Fig 3.
Comparison of conduit network parameters in the deep and superficial TCZ.
Differences in conduit density between the deep and superficial TCZ can be visualized by averaging and color-coding pixel densities over small image volumes in a “moving average” display, shown as a rainbow spectrum (A, B). A cross-section of the moving average display exposes how dense regions in the periphery of the LN (blue) surround an inner region of lower conduit density (green), directly representing dense or sparse occurrence of conduit segments in the corresponding section of the conduit network image (dark blue, left panel), respectively (A). Volume rendering (B) of the entire TCZ using this approach shows the dense superficial zone (blue) enclosing a central region of sparse conduits (green). Alternatively, the TCZ conduit map was employed for calculating the distances to the nearest conduit, and voids with a distance of over 8 μm were displayed in red, indicating larger distances within the deep TCZ as opposed to outer regions (C). From these 2 zones, 10 subregions were selected for comparative analysis (D), including the number of vertices (E), the number of conduit segments (F), the conduit volume (G), conduit segment diameters (H), conduit segment lengths (I), and the combined conduit length (J). The distributions of the minimum distances to the nearest conduit (K) and the average minimum distance (L) further exemplify the larger spacing within the deep TCZ. Simulation of T-cell motility predicts similar motility coefficients within the deep TCZ and the surrounding superficial zone (M). Data are from 1 experiment (each point represents one 100 μm subregion, N = 10) and plots show means ± SD. ****p < 0.0001, ***p < 0.001, ns = not significant, Student t test. Values for each data point can be found in S1 Data. See also S4 Fig. LN, lymph node; TCZ, T-cell zone.
Fig 4.
Three-dimensional EVIS images of a popliteal LN with WGA-labeled conduit paths contain brightly labeled multilobular spaces (red arrowheads) within the otherwise unstained B cell Fo underneath the SCS that can be repeatedly seen in 3D projections of 20 μm thickness (A) and 2D image slices (B). In immuno-labeled tissue sections of WGA-perfused inguinal LNs, WGA (green) is found in follicular conduits descending from the SCS (white arrowheads) connecting to cellular clusters expressing the FDC marker CD21/CD35 (C, D). The morphology and location of WGA-labeled cell clusters within B cell follicles (D) are generally consistent with the anatomy of FDCs in these regions (E, F) as co-staining with markers for B cells (CD45R/B220), T cells (CD3), and collagen I confirms. High-resolution confocal images (with a voxel resolution of 0.36 × 0.36 × 1 μm) of a WGA-perfused popliteal LN provide insights into the staining pattern within and around the WGA accumulations (G) and show a particularly bright cluster in several z steps (H) directly neighboring spaces with weaker labeling (red arrowheads). Images are representative of at least 6 LNs (from N = 5 mice) in which multilobular cell clusters could be observed. See also S4 and S5 Videos. CD,; EVIS, extended-volume imaging system; FDC, follicular dendritic cell; Fo, follicle; LN, lymph node; SCS, subcapsular sinus; WGA, wheat germ agglutinin.