MCMV Splenic Tropism

<div><p>All analysis was performed 3 d post virus infection.</p><p>(A) Splenic stromal cells from mock or MCMV (2 × 10⁵ pfu, Smith strain) infected spleens were separated from hematopoietic cells by extrusion through a nylon mesh filter (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020016#s4" target="_blank">Materials and Methods</a>). Total cell RNA was then isolated from either the stromal cells (S) or hematopoietic cells (H) fractions, and quantitative RT-PCR was performed to detect expression of CCL21-ser, CCL19, and CXCL13 as well as expression of the MCMV immediate early-1 (IE1) transcript.</p><p>(B) Spleens from MCMV-GFP infected mice (2 × 10⁵ pfu) were examined by immunohistochemistry. Panels are color-coded with the text for the antigen examined, and field magnification is shown to the right of the panels. No background staining was seen in uninfected mouse spleen when directly detecting GFP expression or when using an anti-GFP antibody (αGFP) for signal amplification.</p><p>WP, white pulp.</p></div

MCMV-Induced Suppression of CCL21 Abolishes T Cell Localization to the White Pulp

<div><p>Mock- or MCMV-infected B6 mice (1.5 × 10⁵ pfu) were injected with CFSE labeled naive T cells, and spleens were removed for analysis 2 h later.</p><p>(A) Flow cytometry was used to assess the total number of CFSE-labeled cells in the spleen after adoptive transfer into mice 3 d post-infection.</p><p>(B) Immunohistochemical analysis of T cell localization in the spleen at 3 d post-infection using the MOMA-1 marker to identify MMM (shown in blue, magnification 10×). WP, white pulp; RP, red pulp.</p></div

Reduced CCL21-ser Expression after MCMV Infection

<div><p>Spleens from B6 mice mock-infected or infected with 3.2 × 10⁵ (A,B,D) or 1.5 × 10⁵ (C) pfu of MCMV were harvested at day 3 post-infection. Gene expression was measured by quantitative PCR and was normalized to 18S RNA and presented as relative mRNA levels compared to uninfected mice (A and D) or as absolute values (C). When presented as relative values, mRNA levels from three uninfected mice (mock) were determined and averaged for comparison for each experiment.</p><p>Open symbols in (A) represent mice infected with UV-inactivated MCMV (equivalent to 3.2 × 10⁵ pfu).</p><p>(B) Immunohistochemical analysis of CCL21 expression in the spleen of a mock or MCMV-infected B6 mouse 3 d post-infection; the location of the periarteriolar lymphoid sheath “PALS” and B-cell follicles “B” is noted, and the interrupted white line indicates the outer edge of the MZ. This section is representative of three experiments (<i>n</i> = 3 mice per group).</p><p>In (C), primers used for analysis of CCL21 levels amplified either the serine (CCL21-ser), leucine (CCL21-leu), or both (CCL21u) isoforms.</p><p>The data in (D) is an average of three infected mice ± SEM (<i>p</i> < .001).</p></div

MCMV Modulates Splenic Architecture

<div><p>(A) Spleens from PBS (mock) or MCMV-infected B6 mice (3.2 × 10⁵ pfu) were harvested at day 3 post-infection for immunohistochemical analysis. Spleen sections were incubated with markers specific to MMM (MOMA-1, MMM, blue), MZM (ER-TR9, MZM, red), granulocytes (Gr-1/Ly-6G, green), stromal cell populations (BP-3, blue, to identify B-cell-zone stromal cells and gp38, red, to identify T-cell-zone stromal cells, in uninfected mice), and B (B220, blue), and T lymphocytes (Thy1.2, red) (magnification 20×). These sections are representative of four independent experiments with <i>n</i> = 3 mice per group with two or more sections taken from each spleen. White pulp (WP) and red pulp (RP) and the location of the central arteriole (ca) are indicated.</p><p>(B) Similar immunohistochemical analysis performed on Balb/c mice, 3 d post-MCMV-infection (1 × 10⁵ pfu).</p></div

Pharmacologic Activation of LTβR Signaling Partially Restores CCL21 Expression and T Cell Localization in the Spleen of MCMV-Infected Mice

<div><p>(A) Quantitative PCR was used to determine the relative levels of CCL21 gene expression 3 d post-MCMV infection in spleens from control- or virus- (V) infected mice ± agonistic anti-LTβR antibody (Ab) treatment.</p><p>Left panel, 3.2 × 10⁵ pfu of MCMV was used for infection, and only total CCL21 mRNA levels were analyzed using CCL21u primers (see below).</p><p>Right Panel, mice were infected with 1.5 × 10⁵ pfu MCMV and expression of the various CCL21 isoforms was determined. CCL21u, universal CCL21 primers that amplify both the leucine (leu) and serine (ser) isoforms; <i>p</i> = 0.02</p><p>(B) Shown are representative spleen sections from the same mice that were analyzed in the right panel of (A). Sections were incubated with an antibody to detect MMM (MOMA-1, MMM, blue), and adoptively transferred, CFSE-labeled T cells are green. The number of CFSE labeled T cells located per WP area after transfer was analyzed (<i>n</i> = number of WP regions analyzed per spleen), and differences between groups of infected mice ± antibody is statistically significant to a <i>p-</i>value < 0.0001.</p></div