Perivascular immune cell clusters in aged mice have characteristics of tertiary lymphoid organs (TLOs).

<p>Representative photomicrographs of a perivascular cell cluster from C57L/J mouse serial sections containing (A) Ki67⁺ proliferating cells (magnification 200x) and (B) peripheral node addressin (PNAd) expressing high endothelial venules (HEVs) (magnification 400×). Insets show higher-power magnifications of the indicated areas. Arrowheads indicate proliferating lymphocytes (A) and PNAd⁺ HEVs (B). (C) Podoplanin expression on lymphatic endothelial cells in a lymph vessel in the close proximity of a perivascular cell cluster (left panel: magnification 200x, right panel: 400x). (D) Immunofluorescent double labeling for LYVE-1/podoplanin (upper row) and VEGFR3/podoplanin (bottom row) on C57Bl/6 mouse kidney sections. Abbreviations: i: infiltrate; lv: lymph vessel; v: vein.</p

Phenotypic characterization of perivascular immune cell clusters in the aged mouse kidney.

<p>(A) PAS staining was used for computerized morphometric analysis. Left panel: low-power magnification (40x) showing representative perivascular infiltrates (arrows) in a male C57L/J mouse. Middle panel: higher-power magnification (100x) of the left panel showing three individually measured perivascular cell clusters. The cell clusters were encircled and the surface area was calculated and expressed in μm² as indicated. Right panel: high-power magnification (400×) of the middle panel. (B) The immune cell clusters consisted of CD45⁺ cells of which the majority was CD3⁺ T cells (C) and B220⁺ B cells (D). Panels B, C & D display serial sections (magnification: 200×). Insets show high-power magnifications (500x) of the indicated areas. Arrows indicate immune cell clusters. a: arteriole, v: vein.</p

Genome-wide haplotype association mapping in aged mice.

<p>In both female (A) and male (B) mice, binary data were used based on the threshold at 0.15 of relative cluster size. Strains with relative cluster size less than 0.15 were marked as “0”, and those higher than 0.15 were marked as “1”. Associations with a P-value of less than 10⁻⁶ were considered significant. Results are displayed in Manhattan plots (left graphs) and Q-Q plots (right graphs).</p

Distribution of relative immune cell cluster size in 20-months-old male and female mice across the different mouse strains.

<p>Perivascular immune cell clusters were measured in 20-months-old male and female mice and expressed as relative cluster size (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091850#s2" target="_blank"><i>Materials & Methods</i></a>). Only those strains containing both female mice and male mice analyzed were listed (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091850#pone-0091850-t001" target="_blank">Table 1</a>). Data are expressed as mean ± SEM. Numbers indicated above each bar represent the number of mice analyzed.</p

Lymphocytes in renal TLOs express WISP2 and CTNNBIP1, but not TNFRSF8.

<p>(A) H&E staining on a kidney section from a 20 month old male LP/J mouse with perivascular infiltrates (TLOs). Magnification: 20x (left panel) and 400x (right panel). (B) Immunohistochemistry revealed expression of WISP2 (left panel) and CTNNBIP1 (middle panel), but not TNFRSF8 (right panel). Magnification: 400x. v: vein.</p

Mean perivascular cell cluster number and cumulative cluster size identified in male and female mice of the various mouse strains at the age of 20 months.

<p>*Cluster #: number of perivascular cell clusters present per renal cross-section.</p><p>**“0”: relative cluster size <0.15; “1”: relative cluster size >0.15.</p>§<p>strains included in the kinetics analyses: 6, 12 and 20 months.</p>†<p>strains without TLOs included in lymphatics and liver infiltration analyses.</p>‡<p>strains with TLOs included in lymphatics and liver infiltration analyses.</p><p>n: number of kidneys/mice analyzed; nd: not determined.</p

Characterization of the glomerular deposits.

<p>Elektron microscopy of an affected glomerulus of a 20-month-old NOD.B10 mouse shows filling of the intracapillary lumina with optically lucent areas, suggestive of lipid vacuoles. Intracapillary lumina (closed arrow), nucleus (*) and podocyte (dashed arrow).</p

Summary of the HAM peaks for both sexes at the age of 20 months with a P-value <10⁻⁶.

<p>Candidate genes are in bold. M: males; F: female.</p><p>*NCBIm37 Assembly.</p

Expression levels of the five associated genes.

<p>Rt-PCR for <i>Spata17, Gpatch2, Esrrg, Ush2a,</i> and <i>Kctd3</i> (n = 3–5). The results represent the mean ± SE. Comparing all animals (regardless of strain) with and without deposits using a t-test showed no significant difference for any of the genes.</p

Strains with TLO formation are characterized by reduced numbers of lymph vessels.

<p>(A) Representative photomicrograph of peri-arteriolar lymph vessels in a C57BLKS/J mouse kidney (left panel). The right panel shows a high-power magnification of the indicated framed area. Arrowheads indicate podoplanin⁺ lymph vessels. Abbreviations: a: arteriole; g: glomerulus; lv: lymph vessel; v: vein. (B) The number of podoplanin⁺ lymph vessels was quantified in selected strains based on the relative absence (BALB/cByJ, C3H/HeJ, RIIIS/J, C57BLKS/J [black bars]) and abundance (BTBR T+ tf/J, C57L/J, P/J, LP/J [white bars]) of TLOs at the age of 20 months. Numbers indicated within each bar represent the number of mice analyzed. (C) The mean number of lymph vessels in the 4 strains without TLOs (BALB/cByJ, C3H/HeJ, RIIIS/J, C57BLKS/J [black bars]) and with TLOs (BTBR T+ ^tf/J, C57L/J, P/J, LP/J [white bars]) was calculated. Strains with TLO formation had overall significantly lower numbers of lymph vessels compared with strains without TLOs (*P<0.05).</p