1. Volume 30, Issue 3, Pages 408-420 (March 2009)
The Lymphotoxin LTα1β2 Controls Postnatal and Adult Spleen Marginal Sinus Vascular Structure and Function 
Carlene L. Zindl, Tea Hyun Kim, Meiqin Zeng, Angela S. Archambault, Mitchell H. Grayson, Kyunghee Choi, Robert D. Schreiber, David D. Chaplin 
Immunity 
Volume 30, Issue 3, Pages (March 2009)
DOI: /j.immuni
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Flk-1 Marks Marginal Sinus Smooth Muscle-Associated Endothelial Cells in the Mouse Spleen
Frozen sections (10 μm thick) from C57BL/6 spleens (n > 10) were stained with anti-B220 (green) and either (A) anti-Flk-1 (red) or (B) anti-CD144 (VE-cadherin) (red) (magnification 200×). In (C) and (D), staining was with anti-Flk-1 (green) and either (C) anti-PECAM-1 (red) or (D) anti-MOMA-1 (red). For (E) and (F), spleen sections were cut at 20 μm and stained with (E) anti-SMA alone (red) (magnification 100×) or together with (F) anti-B220 (green) (magnification 200×). For (G) and (H), staining was with (G) anti-SMA alone (red) (magnification 100×) or together with (H) anti-Flk-1 (green) (magnification 630×). For (C), (D), (G), and (H), confocal microscopy was used to compile a series of Z stack images to reconstruct a 6 μm thick section. CA, central arteriole; ∗, central arteriole branching vessel; (←), MS connecting vessels. Similar data were obtained in two additional experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
LTβR Signaling Supports the Organization of Endothelial and Smooth Muscle Cells in the Marginal Sinus
Frozen sections (20 μm thick) from spleens (n ≥ 5) of the indicated mouse strains were stained with (A) anti-Thy1.2 (brown), either anti-Flk-1 (blue, top panels) or anti-CD144 (VE-cadherin) (blue, bottom panels), (B) anti-Thy1.2 (green), and anti-SMA (red) (magnification 200×). Inset images show magnified areas highlighting the Flk-1+ and CD144+ structures at the periphery of white pulp areas in the gene-targeted mice.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 Disturbed MS Endothelial Cell Network in Lta−/− Mice
(A–D) Spleens of Efnb2+/− (A and C) and Efnb2+/− Lta−/− (B and D) mice were cut to yield (A and B) 10 μm or (C and D) 120 μm sections and stained with anti-β-galactosidase (green) to visualize ephrinB2 expression via the expression of the in-frame knockin of the LacZ gene. Confocal images were compiled to yield a 50–60 μm section of spleen, permitting analysis of the 3D structure of a region of a single white pulp nodule (magnification 200×). Similar data were obtained by analyzing spleens from five additional pairs of mice.
(E) The percentage of ephrinB2+ and Flk-1+ MS structures per WP area was quantified from 10 μm sections with a 20× objective.
Graphs show mean + SE (n ≥ 40 WP nodules with ≥ 5 spleens per strain). Data were compiled from five independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Lymphotoxin Controls Organization of Flk-1+ Vessels and Expression of MAdCAM-1 during Neonatal Development
(A–C) (A) Frozen sections of spleens from C57BL/6 and Lta−/− mice harvested at the indicated ages (n ≥ 5 per strain per time point) were stained with anti-Flk-1 (red), anti-SMA (green), and anti-B220 (blue) (magnification 200×). Expression of MAdCAM-1 protein was analyzed by (B) immunoblotting and (C) ELISA from extracts of whole spleens (n = 10 spleens per strain per time point) of C57BL/6, Lta−/−, Ltbr−/−, and Tnfrsf1a−/− mice.
(D) Spleen sections from the indicated mouse strains (n = 10 spleens per strain per time point) harvested on the indicated postnatal days were stained with anti-MAdCAM-1 (red) and anti-B220 (green).
#p < 0.01 comparing C57BL/6 and Tnfrsf1a−/− mice; ∗p < and ∗∗p < for comparisons between C57BL/6 and Ltbr−/− mice; ϕp < for comparison between Tnfrsf1a−/− and Ltbr−/− mice. Graphs show mean + SE.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
Endothelial Cells Express the LTβR and Utilize MAdCAM-1 to Form Specialized Structures on Matrigel
(A–E) (A) Spleen sections (10 μm) from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n = 3). Endothelial cells isolated from WT spleen (sECs) with either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n = 3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR.
(F) sECs isolated from WT and Lta−/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days.
(G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 μg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hr.
(H) Protein extracts from transfected bEND.3 cells were prepared, and an ELISA was performed to assess MAdCAM-1 protein expression. The graph shows means + SE.
(I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T., and 8 μm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue).
Inset images show magnified areas highlighting the EC phenotype and the amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of three independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
Intact Marginal Sinus Organization in Adult Mice Is LT Dependent and Supports the Localization of Immune Cells within the Spleen following Challenge by Bacterial Antigens
(A) C57BL/6 adult mice were treated i.p. with either human IgG1 (huIg) or LTβR-Fc, and spleens were harvested 2 weeks later. Sections (10 μm) were stained with anti-Flk-1 (blue) and anti-B220 (brown). Similar results were obtained in four additional experiments.
(B) The percentage of Flk-1+ MS structures per WP area was quantified from 10 μm sections with a 20× objective (n ≥ 40 WP nodules from three independent experiments; graph shows mean + SE).
(C) C57BL/6 mice were treated i.p. with either huIg or LTβR-Fc and 2 weeks later were injected i.v. with 250 μg of TMR-labeled S. aureus bioparticles (red). Spleens were harvested at 3, 12, or 24 hr after injection with the particles, and sections were stained with anti-CD11b (green). Dashed lines outline WP areas.
(D) The numbers of CD11b+ cells localized in the WP was quantified from ≥ 40 WP areas from three independent experiments with three to five mice per group. Graph shows mean + SE. #p < comparing control C57BL/6 mice and hulg-treated mice; ∗p < comparing control C57BL/6 mice and LTβR-Fc-treated mice; ϕp < for comparison between hulg-treated mice and LTβR-Fc-treated mice at the same time interval after treatment.
(E) Expression levels of the indicated cytokines, chemokines, and adhesion molecules were analyzed by immunoblotting. Data are representative of at least three independent experiments with three to five mice per group.
Immunity  , DOI: ( /j.immuni )
Copyright © 2009 Elsevier Inc. Terms and Conditions