Immunity, Volume 47

Supplemental Information

Lymphatic Endothelial Cells Control

Initiation of Lymph Node Organogenesis

Lucas Onder, Urs Mörbe, Natalia Pikor, Mario Novkovic, Hung-Wei Cheng, ThomasHehlgans, Klaus Pfeffer, Burkhard Becher, Ari Waisman, Thomas Rülicke, JenniferGommerman, Christopher G. Mueller, Shinichiro Sawa, Elke Scandella, and BurkhardLudewig

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Figure S1, relates to Figure 1. Transgene activity and secondary lymphoid organ

development in Cxcl13-Cre/Tdtomato mice.

(A) Schematic design of the Cxcl13-Cre/Tdtomato transgene. Cxcl13-Cre/Tdtomato mice have been

crossed to the R26R-EYFP reporter strain (Cxcl13-EYFP). (B) Flow cytometric analysis of viable

CD45-depleted cells from peripheral LNs from 6 wk old Cxcl13-EYFP mice (left dot plot).

Quadrants indicate LN stromal cell subpopulations: CD31+PDPN- blood endothelial cells (BEC),

CD31+PDPN+ lymphatic endothelial cells (LEC), CD31-PDPN+ fibroblastic reticular cells (FRC),

CD31-PDPN- double negative cells (DNC). Transgene expressing EYFP+ cells were further

analyzed for CD31 or PDPN expression by backgating (right dot plot). (C) Distribution of EYFP+

cells among LN stromal cell subpopulations, as percentages from quadrants in (B) (n = 5-6 per

group from 2 independent experiments). (D) Confocal microscopic analysis of the T cell zone in

iLNs of adult Cxcl13-EYFP mice using the indicated staining (scale bar = 10 m). (E) Confocal

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microscopic analysis from the B cell zone and subcapsular region in iLNs of Cxcl13-EYFP mice.

Arrowheads indicate EYFP+RFP+CXCL13+ marginal zone reticular cells (MRC) and arrow shows

EYFP+RFP+CXCL13+ follicular dendritic cell (FDC) (scale bar = 10 m). (F) Confocal

microscopic analysis of a B cell follicle (upper panel) and an activated B cell follicle (lower panel)

containing a germinal center (GC) in iLNs of Cxcl13-EYFP mice (scale bar = 20 m). (G)

Formation of Peyer’s patches in adult Cxcl13-Cre Ltbrfl/fl and Cre-negative control (Ctrl) mice.

Pooled values from more than 3 independent experiments representing numbers of Peyer’s patches

per mouse, horizontal bar indicates mean. (H) Confocal microscopic analysis of splenic cross-

sections from adult Cxcl13-EYFP and Cxcl13-EYFP Ltbrfl/fl mice using the indicated staining (scale

bars = 500 m). (I) Percent reduction of CD45+ and B220+ cell numbers in spleens of adult Cxcl13-

Cre Ltbrfl/fl mice determined by flow cytometry (n = 5-6 mice per group from two independent

experiments, mean ± SEM). Microscopy and flow cytometry data are representative for two or more

independent experiments (n ≥ 3 per group).

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Figure S2, relates to Figure 3. Assessment of iLN anlage development in E17 embryos.

(A) Micrograph shows embryo after fixation and dotted line indicates region of the inguinal fat pad

used for further analysis. (B) Whole mount analysis of inguinal fat pad containing iLN anlage

located at the major branch of the subepigastric vessels. Fat pad tissue was stained with indicated

antibodies, analyzed by confocal microscopy and reconstructed in 3-D (scale bars = 500 m). (C)

High resolution confocal microscopy analysis of whole mount E17 iLN anlage from Cre-negative

control (Ctrl) embryo with indicated staining; major lymphatic vessels were reconstructed and

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pseudo-colored in yellow. Symbols indicate subepigastric artery (asterisk) and vein (hash), scale bar

= 30 m. Boxed area shows region of confocal z-plane in (D). (D) Confocal z-plane extracted from

z-stack in (C) showing a quadrant of the iLN anlage. CD4+ LTi cells were found in close proximity

of CD31+LYVE1+ LECs (arrows) and CD31+LYVE1- BECs (arrowheads), scale bar = 10 m. (E)

High resolution confocal microscopy analysis and reconstruction of whole mount E17 iLN anlage

from Cdh5-Cre Ltbrfl/fl and Cdh5-Cre Ltbr

fl/flNik

fl/fl embryos. Major lymphatic vessels have been

reconstructed and pseudo-colored in yellow, scale bar = 30 m. (F-I) High resolution confocal

microscopy analysis and reconstruction of mesenchymal stromal cells in E17 iLN anlage from Cre-

negative control (Ctrl) and Cdh5-Cre Ltbrfl/fl embryos, boxed areas show region of confocal z-

planes in (G) and (I), scale bar = 30 m. (G) and (I) Confocal z-plane extracted from z-stack in (F)

and (H), showing the ICAM1+VCAM1+ mesenchymal cell network in a quadrant of the iLN anlage,

scale bar = 10 m. Microscopy data are representative for two or more independent experiments.

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Figure S3, relates to Figure 4. Chemokine expression during iLN anlage formation and

structure and development of ectopic LNs. (A) High resolution confocal microscopy analysis and reconstruction of whole mount E17 iLN

anlage from C57BL/6 embryo stained with antibodies against the indicated markers. Major

LYVE1+ lymphatic vessels have been reconstructed and pseudo-colored in yellow, scale bar = 30

m. Inguinal fat pads of (B) E14 and (C) E17 Ccl19-EYFP embryos stained with the indicated

antibodies, analyzed by confocal microscopy and 3-D reconstructed in whole mount; boxed area

indicates region shown in main Figure 4C. (D) C57BL/6 embryos were treated with FTY720 at E14

and inguinal fat pads were isolated at E16. Whole mount fat pads were stained with antibodies of

the indicated specificity, analyzed by confocal microscopy and reconstructed in 3-D (scale bar =

200 m). Boxed area indicates location of ectopic LN anlage formation and is shown at high

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magnification in main Figure 4H. (E) Cxcl13-EYFP mice were treated with FTY720 at E14 and E16

and ectopic LNs were analyzed in 6 wk old mice by confocal microscopy using the indicated

antibodies (scale bar = 100 m). (F) FTY720 (E14 and E16) treated C57BL/6 mice at the age of 6

weeks were injected with MECA-79 antibody to stain the high endothelial (HEV) network in vivo.

Isolated ectopic LNs were cleared and analyzed by confocal microscopy in whole mount (scale bar

= 30 m). (G) Histological sections of ectopic LNs from FTY720 (E14 and E16) treated C57BL/6

mice at the age of 6 weeks were stained with the indicated antibodies. Boxed area indicates region

of high power images on the right (scale bar = 100 m). (H) FTY720 (E14 and E16) treated

C57BL/6 mice at the age of 6 wk were injected subcutaneously (s.c.) with FITC-dextran (MW 40

kDa). Histological sections of ectopic LNs were stained with the indicated antibodies and analyzed

for FITC-dextran distribution by confocal microscopy (scale bar = 20 m). Microscopy data are

representative for two or more independent experiments.

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Figure S4, relates to Figure 5. Organogenesis of iLNs in mice lacking Rank

expression in lymphatic endothelial cells.

(A) High resolution microscopic analysis of LTi cells interacting with LYVE1+ lymph vessels in the

vicinity of E14 LN anlagen from Lyve1-EYFP embryos. Scale bar = 10 m. (B) High resolution

confocal microscopy analysis and reconstruction of E17 iLN anlagen from C57BL/6 (Ctrl) and

Prox1-CreERT2 Rankfl/fl embryos, which have been treated with Tamoxifen at E11 and 12. 3D

datasets were used to calculate volume of CD4+ LTi cell clusters (right graph). Data are

representative for two independent experiments. (C) ICAM1 and VCAM1 expression in C57BL/6

(Ctrl) and Prox1-CreERT2 Rankfl/fl E17 iLN anlagen (scale bar 30 m). Microscopy data are

representative for two or more independent experiments.

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Figure S5, relates to Figure 6. BEC activation in E18 LN anlagen.

(A) Confocal planes acquired from E18 iLN anlagen (Ctrl) covering arterial, post-capillary-venular

(PCV) and venular regions, stained with antibodies against CD31 and ICAM1. Arrowheads indicate

CD31+ICAM1+ postcapillary venules (scale bar = 10 m). (B) Whole mount iLN anlagen from E18

Cdh5-Cre Ltbrfl/fl

Nikfl/fl embryos stained with antibodies directed to CD31 and ICAM1 and analyzed

by confocal microscopy (scale bar = 20 m). SEA = subepigastric artery, SEV = subepigastric vein.

Microscopy data are representative for 2 or more independent experiments (n ≥ 3 per group).