1. Volume 45, Issue 4, Pages 877-888 (October 2016)
Clonal Proliferation and Stochastic Pruning Orchestrate Lymph Node Vasculature Remodeling 
Isabelle Mondor, Audrey Jorquera, Cynthia Sene, Sahil Adriouch, Ralf Heinrich Adams, Bin Zhou, Stephan Wienert, Frederick Klauschen, Marc Bajénoff 
Immunity 
Volume 45, Issue 4, Pages (October 2016)
DOI: /j.immuni
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2. Immunity 2016 45, 877-888DOI: (10.1016/j.immuni.2016.09.017)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Three-Dimensional Imaging of the LN Vascular Tree at High Resolution
(A) Flow cytometric analysis of LN CD45-depleted stromal cells and CD45+ hematopoietic cells harvested from Cdh5RFP mice treated or not treated with tamoxifen for 1 week. Values indicate the percentage of RFP+ cells within the indicated cell populations (n = 4 mice). Abbreviations are as follows: HEV, high endothelial venule cell (pink); cBEC, capillary blood endothelial cell (blue); and LEC, lymphatic endothelial cell (orange).
(B) LNs from tamoxifen-treated Cdh5RFP mice were sectioned either with a cryostat (20 μm thickness, left panel) or with a vibratome (200 μm thickness, right panel), stained for CD146 (white) and PNAd (green) expression, and clarified. Confocal images acquired in small fields of view were tiled to reconstruct a 3D portion of the LN vascular network (n = 4 mice). Scale bar represents 200 μm. See also Movie S1.
(C) For localization of functional PNAd+ HEVs in situ, total LN cells were labeled with the fluorescent dye DDAO-SE and injected i.v. 10 min before the harvest of the LN (n = 3 mice). Scale bar represents 100 μm.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Expansion of the LN Vasculature Relies on the Sequential Assembly of EPUs
(A) Cdh5Ubow mice were treated with tamoxifen, and their peripheral LN cells were analyzed 1 week later by flow cytometry (n = 4 mice).
(B) Predicted, non-mutually exclusive models of LN EC dynamics revealed by the Cdh5Ubow mice. In model 1, all labeled ECs divided and gave rise to small clusters of ECs sharing a similar color. In model 2, a few ECs (∗) massively divided to expand the vascular tree, forming large monocolored clusters of ECs. In model 3, sprouting angiogenesis generated neo-vessels.
(C) Cdh5Ubow mice were treated with tamoxifen and immunized 1 week later with CFA in the ears and rear footpads. 3 weeks later, total LN cells were labeled with DDAO-SE and injected i.v. into mice for 10 min to label functional HEV cells. LNs were sliced (200 μm thickness), clarified, and imaged by confocal microscopy. Pictures show the appearance of contiguous and large monocolored segments of HEV cells only in inflamed LNs. Inserts display high-magnification views of HEV cells. Images are representative of four experiments (n = 3 mice per group). Scale bars represent 300 μm. See also Figure S1 and Movies S2, S3, and S4.
(D) All confocal datasets similar to those shown in (C) were processed for the generation of Voronoi tessellations (see Experimental Procedures) for quantifying clusters and computationally evaluating the significance of cluster formation by Monte Carlo simulations with the ClusterQuant software.
(E) The experimentally observed (original) cluster characteristics (numbers of CFP2+, YFP2+, and CFP+YFP+ HEV cells per cluster) were compared to the results of the Monte Carlo simulations, in which the cells were randomly distributed (scrambled) on the basis of the observed microanatomy (displayed are 3 of 10,000 simulations). Images are representative of four experiments (n = 3 mice). ∗∗∗p < 10−4; bars represent medians.
Immunity  , DOI: ( /j.immuni )
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5. Figure 3
Expansion of the LN Vascular Tree Relies Exclusively on LN-Resident ECs
(A) UbcTOM mice were sub-lethally irradiated and reconstituted with the BM of UbcGFP mice. Chimeric mice were immunized with CFA in the ears and rear footpads and the EC fraction of their draining LNs was analyzed 3 weeks later by flow cytometry (n = 6 mice).
(B) UbcTOM mice were surgically joined to WT mice for the creation of parabiotic pairs of mice sharing common blood circulation. 12 weeks later, the WT partner was immunized with CFA in the rear footpad. The contribution of the UbcTOM partner to the EC fraction of the draining LN of the WT partner was analyzed 3 weeks later by flow cytometry (n = 5 pairs of parabiotic mice).
Immunity  , DOI: ( /j.immuni )
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6. Figure 4 HEV Cells Act as Progenitors of All LN Blood ECs
(A) Representative pictures of monocolored capillary (arrows) and HEV (arrowheads) sprouts imaged in the inflamed LNs of Cdh5Ubow mice as in Figure 2C. Scale bar represents 50 μm.
(B) Monocolored capillary sprouts such as the ones displayed in (A) were identified on LN slices and used for determining the percentage of monocolored capillary sprouts connected to an HEV segment labeled with the same color.
(C) HEV cell and cBEC fractions were enriched from the peripheral LNs of tamoxifen-treated Cdh5RFP mice and separately injected in the two inguinal LNs of the same WT mouse immunized 3 days earlier with CFA at the base of the tail. Dot plots indicate the purity and viability of typical HEV cell and cBEC fractions isolated with this protocol.
(D) 3 weeks later, total LN cells were labeled with DDAO-SE and injected i.v. 10 min before the harvest of the LN. LNs were sliced (200 μm thickness), stained for CD146 expression, clarified, and imaged by confocal microscopy. Images are representative of four experiments (n = 3 mice). Scale bar represents 100 μm.
(E) Total numbers of RFP+ HEV cells and RFP+ capillaries generated by the injected RFP+ cBEC and RFP+ HEV cell fractions in three experiments (n = 2 mice per goup). Each RFP+ vessel contained at least ten contiguous RFP+ ECs.
See also Figures S2–S4.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
Return to LN Quiescence Relies on the Stochastic Death of Pre-existing and Neo-synthesized ECs
(A) 2 × 106 plaque-forming units of VSV were injected in the ears and footpads of AplnTOM mice. The day after, mice were treated with tamoxifen to label neo-synthesized ECs and their progeny. Images are representative of clarified thick LN slices harvested at infection day 3 and stained for CD146 expression. Abbreviations are as follows: S, sprout; and E, elongation. Scale bar represents 50 μm.
(B) Draining LNs were photographed at day 7 (peak of the LN expansion) and day 60 (return to quiescence). Images are representative of three experiments (n = 2 mice per group).
(C and D) Graphs indicate the corresponding total number of HEV cells (C) and percentages of Tomato+ HEV cells (D) determined by flow cytometry from three independent experiments (n = 4 mice per group). ∗∗p < 10−2; ∗∗∗p < 10−3; ns, not significant. Bars represent medians. Empty squares represent VSV, and filled circles indicate controls.
(E) Representative images of clarified thick LN slices stained for T cell (CD3) and B cell (B220) markers reveal the localization of Tomato+ neo-vessels in the inflamed LNs of VSV-infected mice. Images are representative of three experiments (n = 2 mice per group). Scale bars represent 200 μm.
Immunity  , DOI: ( /j.immuni )
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