Antibody staining panel.

<p>Antibody staining panel.</p

Particle-uptake within CALT.

<p>A) Fluorescing <i>E. coli</i> bacteria (green) were transported through the lymphoepithelium of CALT and detected close to MHC II positive cells (red) within CALT and around and within lymphatic vessels (asterisk) 60 minutes after application. (<i>Ex vivo</i> confocal microscopy, image stack, steps 1 µm, scale bar 55 µm). B) Intravital two-photon microscopy of CALT lymphoepithelium. An intraepithelial fluorescent microsphere (arrowhead) is approached by a DAPI-labeled lymphocyte (arrow). (Time course in minutes). C+D) Transmission electron microscopy of <i>E. coli</i> particle (arrow) during approach (C) and within (D) a macrophage (asterisk). E) Scanning electron microscopy of lymphoepithelium. Cellular surface villi with typical features of M-cells.</p

Antibody combinations used and related cellular targets (note that antibodies do not stain the quoted cells exclusively).

<p>Antibody combinations used and related cellular targets (note that antibodies do not stain the quoted cells exclusively).</p

Classification of CALT compartments and according cellular velocities.

<p>A) Schematic drawing of CALT microcompartment with CALT follicle (1), lymphoepithelium (2) and scattered lymphocytes within subepithelial space (3). B) Cells within the lymphoid follicle featured median velocities of 8.0 µm/min (n = 46 cells, 874 individual measurements). Intraepithelial lymphocytes (lymphoepithelium) demonstrated median velocities of 6.7 µm/min (n = 68 cells, 2101 individual measurements) whereas cells within the subepithelial space demonstrated median velocities of 8.4 µm/min (n = 47 cells, 2416 individual measurements). Statistical analysis demonstrated significant differences of the velocities between lymphoepithelium and subepithelial space (*p<0.05). The data derives from 6 individual experiments with 9 time series/separate sets of data. Statistical analysis included One-Way ANOVA (p = 0.02) for testing of normal distribution, followed by Bonferroni Multiple Comparison Test. Significance values below p<0.05 were considered to be significant. Bartlett-Test was used to analyze data variances (p = 0.4). (p>0.05 n.s., p≤0.05 *). C) Intravital two-photon microscopy of CALT and consecutive tracing of individual cells within the follicle (zone 1, compare to 7A). White spheres represent motile cells, blue spheres represent non motile cells with distinct differing autofluorescence properties and dendritic cell-like morphology (compare supplemental Video 3). D) Lymphoid vessel (Ly) and high endothelial venule (HEV) located in close proximity to a CALT follicle demonstrate cellular transmigration. A lymphocyte migrates into a lymphatic vessel (black arrow), whereas another lymphocyte migrates from a high endothelial venule (white arrow).</p

Development of CALT under SPF housing conditions.

<p>A) Immunhistological analysis of conjunctiva-associated lymphoid tissue (CALT). Mice aged 10 days until 24 weeks, kept under SPF housing conditions, were investigated using a panel of antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082355#pone-0082355-t001" target="_blank">table 1</a>. Spatial distribution of lymphocytes, dendritic cells and follicular dendritic cells was analyzed. B) Schematic CALT development: 10 days after birth only CD4+ T-cells were sparsely present, followed by an influx of B-cells and formation of first follicles at 4 weeks of age. At 8 to 12 weeks CD8+ T-cells and CD4+CD25+ Tregs appeared, altogether forming a complex lymphoid follicle. After 16 weeks of age spatial organization diminished. C) CALT expression rate: CALT was not present at 10 days of age. 25% of the eyes at 4 weeks of age contained CALT, with a further increase to 43% CALT at 10 and 12 weeks of age. This was followed by a decrease to 31% at 16 weeks and an increased to 50% at 24 weeks of age. Numbers of follicles increased until 25% of the eyes contained 2 follicles at 12 weeks of age. At 24 weeks of age 25% of the eyes contained 3 follicles (n = number of eyes).</p

B-cell and T-cell activation markers in CALT following stimulation with KLH/CtB and OVA/CtB in 12 week old animals.

<p>CD22 positive B-cells were found in all sections from all animals. CD28 positive T-cells were restricted to animals stimulated with KLH/CtB or OVA/CtB respectively.</p