Antibody staining panel.

<p>Antibody staining panel.</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

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

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

Desiccating stress-induced changes in wild-type (WT) and DNTGFβRII mice at 14 weeks of age (14W).

<p>Desiccating stress was induced for 5 days in both strains (DS5); nonstressed mice served as controls (NS). <b>A.</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>B.</b> Bar charts show mean ± SD of three independent experiments (final n = 12 for each group). <b>C.</b> CD4 counts in corneal epithelium (EPI) and corneal stroma (ST). Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). <b>D.</b> Representative images of conjunctiva sections immunostained for CD4 (in red) used to generate CD4 counts in <b>E</b>, in conjunctiva epithelium Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). <b>F.</b> PAS+ conjunctival goblet cells counts. Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). <b>G–I.</b> Relative fold of expression in cornea (I), conjunctiva, (CJ, in J) and lacrimal gland (LG, in K) using the WT-NS of each strain as the strain-calibrator. Bar charts show mean ± SD of three independent experiments (final n = 8 for each group). **P<0.01;*** P<0.001 indicate within WT comparison (NS vs. DS5) ∧ P<0.05;∧∧∧P<0.01 indicates DNTGFBRII comparison (NS vs. DS5).</p

Age-related changes in wild-type (WT) and DNTGFBRII mice, from 8 to 14 weeks of age (8W and 14W, respectively).

<p><b>A.</b> Representative images of cornea sections immunostained for CD4 (in red) used to generate CD4 counts in B, in corneal epithelium (EPI) and corneal stroma (ST) in wild-type and DNTGFβRII mice at 8 and 14 weeks of age (8W,14W). Bar charts show mean ± SD of two independent experiment (final n = 5 for each group). <b>C.</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>D.</b> Bar charts show mean ± SD of three independent experiments (final n = 12 for each group). <b>E.</b> Corneal smoothness score. Bar charts show mean ± SD of three independent experiments (final n = 13 for each group). <b>F.</b> Representative images of conjunctiva sections immunostained for CD4 (in red) used to generate CD4 counts in <b>G.</b> Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). <b>H.</b> PAS+ conjunctival goblet cells counts. Bar charts show mean ± SD of three independent experiments (final n = 5 for each group). <b>I–K.</b> Relative fold of expression in cornea (I), conjunctiva, (CJ, in J) and lacrimal gland (LG, in K) using the WT-8W as the calibrator. Bar charts show mean ± SD of three independent experiment (final n = 8 for each group). In graphs B, D,E, G, H **P<0.01;*** P<0.001 indicate within strain comparison In I–K graphs,*** P<0.001 indicates interstrain comparison.</p

CD4+ T cell proliferation.

<p>Proliferation of CD4⁺ T cell in CD4⁺ T cells co-cultured in the presence of cornea and conjunctival tissues (CN and CJ, respectively) before (nonstressed [NS]) or after desiccating stress for 5 days [DS5] from wild-type (WT) and CD4-DNTGFβRII mice (DN). Bar charts show mean ± SD of two independent experiments (final n = 5 for each group).</p

Expression of chemokine receptors and adoptive transfer results in RAG1KO mice.

<p><b>A–B</b> Flow cytometry analysis of freshly isolated cells from ocular surface (OS, in A) and cervical lymph nodes (CLN, in B) from wild-type (WT) and DNTGFβRII mice before (nonstressed, NS) and after 5 days of desiccating stress (DS5).*P<0.05, within strain comparison. Bar charts show mean ± SD of three independent experiments (final n = 4 for each group for OS, final n = 6 for each group for CLN). <b>C.</b>Conjunctival sections stained for CD4⁺ T cells (in red) of RAG1KO recipient mice that received CD4+ cells isolated from WT and DNTGFβRII DS5 mice. Note CD4+T cell infiltration in goblet cell rich area of conjunctiva in WT-DS5 recipient mice. The black dotted circle is a higher magnification of area demarcated by blue dotted circle. Original magnification: 20×. Scale bar = 50 µm. <b>D and E</b>. Conjunctival CD4+T cell <b>(in D)</b> and PAS+ conjunctival goblet cells <b>(in E)</b> counts in RAG1KO recipient mice that received cells from CD4+ cells isolated from WT and DNTGFβRII mice after 5 days of desiccating stress (DS5). Bar charts show mean ± SD of three independent experiments (final n = 5 for each group) <b>F</b>.Relative change of IL-17A, IFN-γ and IL-13 mRNA transcripts in conjunctiva of RAG recipients. Asterisks indicate comparison to strain-specific controls. Bar charts show mean ± SD of two independent experiments (final n = 6 for each group).</p