Graph theory-based analysis of the FRC network topological robustness.

<p>(A) In silico perturbation analysis of a representative FRC network from PBS-treated control mice by random node removal for one simulation. Each image denotes the FRC network in a real coordinate system of the LN T cell zone at indicated fractions of nodes randomly removed. The number of nodes remaining and the starting number of nodes are indicated in the top right of each image. Green nodes represent the largest connected cluster, and blue nodes represent fragmented clusters. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002515#pbio.1002515.s008" target="_blank">S2 Video</a> for the full simulation. (B) Average shortest path length versus fraction of nodes removed. The dashed line represents fraction of nodes removed for the maximal value of average shortest path length, i.e., the network threshold point. (C) Relative size of the largest cluster compared to the size of the starting network at 0% versus fraction of nodes removed. The indicated value in the top right denotes estimated network robustness R. The dashed line represents the minimal damage line. Data represent mean ± SD over 1,000 simulations of random node removals for a representative FRC network (<i>n</i> = 6 mice from two independent experiments). (D) Network robustness R values for FRC networks at indicated doses of DT. Data represent mean ± SD for 3–6 mice per group from two independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 (one-way ANOVA with Tukey’s post-test). ns, not significant; na, not applicable.</p

Changes in FRC morphology following diphtheria toxin (DT)-mediated ablation.

<p>(A) Three-dimensional single-cell reconstruction of the T cell zone FRC network in <i>Ccl19</i>^{<i>eyfp/idtr</i>} mice at indicated time points after two intraperitoneal (IP) injections of 8 ng/g DT or phosphate-buffered saline (PBS)-treated controls. Scale bars represent 30 µm. (B) Global morphological analysis of the total FRC network volume from the 3-D-reconstructed EYFP channel. (C–G) Single-cell analysis of FRC surface area (C), volume (D), sphericity (E), minimal distance between FRCs (F), and connected protrusions per FRC (G). Each dot represents a measurement for a single FRC. Data represent mean ± standard deviation (SD) (B–F) and median ± interquartile range (IQR) (G) for 3–5 mice per group. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 (one-way ANOVA with Tukey’s post-test [B–F] or Kruskal-Wallis test with Dunn’s post-test [G]). ns, not significant.</p

Assessing the topology of the T cell zone FRC network.

<p>(A) Overview 2-D image of an inguinal LN section from a naive adult <i>Ccl19</i>^<i>eyfp</i> mouse stained with antibodies against the indicated markers. Rectangles indicate representative T cell zones acquired with high-resolution confocal microscopy. (B) Representative 3-D Z-stack indicating the T cell zone FRCs (left panel), merged with FRC network (middle panel) and the network representation (right panel) with nodes (FRCs) and edges (physical connections). Size of T cell zone image: 304 x 304 x 32 μm. (C) Zoom-in area of single FRCs from (B, left panel) with signals for EYFP, PDPN, merged, and network representation, respectively. (D) Representative FRC network from (B, right panel). The equivalent random network was constructed using the Erdos-Renyi model, and the regular ring lattice network was constructed with eight edges for every node (FRC network median). Lattice and random networks are shown in Kamada-Kawai representation, while the FRC network is arranged in the real coordinate system of the LN T cell zone. N denotes the number of nodes, and E denotes the number of edges for each network. Small-world parameters σ and ω are shown below. The color legend represents number of edges per node. Data are representative of six mice from two independent experiments. Scale bars represent 300 μm (A), 30 μm (B, D), and 10 μm (C).</p

Impairment of LN functionality following FRC ablation.

<p>LN cellularity as determined by flow cytometry with total numbers of CD45⁺ hematopoietic cells (A), CD8⁺ T cells (B), and CD11c⁺ DCs (C) in <i>Ccl19</i>^<i>idtr</i> mice injected twice IP with the indicated doses of DT. (D) Correlation between CD45⁺ cells and FRCs remaining in the LN for indicated doses of DT with Pearson correlation coefficient r² = 0.9448, <i>p</i> = 0.00117 (Fisher’s F test). (E–G) Two-photon microscopy analysis of adoptively transferred CD8⁺ T cells into <i>Ccl19</i>^<i>idtr</i> mice injected IP with indicated doses of DT. The migration parameters analyzed include average cell speed (E), cell arrest coefficient (F), and motility coefficient (G). (H) Total numbers of transferred TCR-transgenic Thy1.1⁺CD8⁺ T cells in <i>Ccl19</i>^<i>idtr</i> LNs at indicated doses of DT. (I) Flow cytometric analysis of CD8⁺ T cell activation in <i>Ccl19</i>^<i>idtr</i> LNs on day 3 post immunization with DC-targeting viral particles. Numbers indicate mean percentage ± standard error of the mean (SEM) of proliferating Thy1.1⁺ cells of the whole Thy1.1⁺ population. Indicated <i>p</i>-values represent comparison to the 0 ng/g group. Controls indicate PBS-treated mice without viral particles. Representative experiment for 3–6 mice per group from three independent experiments. Data represent mean ± SEM for 3–20 mice per group from three independent experiments (A–D, H). Data represent mean ± SD (E–F) or median ± range (G) for 5–10 datasets from 2–3 mice per group from two independent experiments. Plus “+” indicates mean. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 (one-way ANOVA with Tukey’s post-test [A–C, H–I] and Benferroni’s post-test [G] or Kruskal-Wallis test with Dunn’s post-test [E–F]). ns, not significant.</p