Xeno-transplantation of the artificial corneal construct in nude mouse back skin. A,

<p>Hematoxylin-eosin (HE) staining images (100×) showed that the corneal constructs survived and grew well in 14 days on nude mouse back. <b>B.</b> Immunofluorescent staining images (400×) showed that the K3, p63, integrin β1 and EGFR were expressed by the artificial corneal constructs transplanted in 14 days.</p

Regeneration of a native-like corneal construct using limbal explants and donor corneal stromal discs.

<p><b>A.</b> Donor corneas stored in Optisol medium; <b>B.</b> Donor corneal epithelium was scraped; <b>C.</b> Horizen Microkeratome system used to fabricate corneal stromal lamella discs; <b>D.</b> A fabricated corneal stromal disc with 10–11 mm diameter and 200 µm thickness; <b>E.</b> The stroma disc was stored in culture medium; <b>F.</b> A fresh limbal explant cultured on the stromal disc that was placed in the culture insert; <b>G.</b> Xeno-transplantation of the corneal construct into the back skin of a nude mouse.</p

Morphology and structure of generated corneal epithelia on different substrates.

<p><b>A.</b> on basement membrane (BM) side of cornel stroma; <b>B.</b> on stromal side of cornel stroma; <b>C.</b> on denuded amniotic membrane (AM); <b>D.</b> on a hydrogel.</p

Location of intra-epithelial lymphocytes in conjunctiva before and after desiccating stress.

<p>Representative digital pictures of immunohistochemical staining of CD4⁺, CD8α⁺, CD103⁺, γδTCR⁺, NK⁺ in the conjunctivae of nonstressed (NS) C57BL/6 mice and after desiccating stress for 5 or 10 days (DS5, DS10). Inset in γδTCR row shows γδTCR in skin, which was used as positive control. Original magnification 40X; scale bar 20 µm. Number insets represent cell counts in the goblet cell rich of the conjunctiva in immunostained tissue sections in conjunctival epithelium of C57BL/6 mice. Data represents mean ± SD of cells/mm. Experiments were repeated three times with two mice per group per experiment. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.01 comparison vs. NS control.</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

Flow cytometry analysis of freshly isolated cells from the ocular surface.

<p>Data is presented as mean ±standard deviation of 5–6 different experiments per group/time point. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”), subsequently gated based on forward scatter height vs. forward scatter area and propridium iodide live/dead exclusion (“live gated cells”). Data presented represents percentage of positive (⁺) or negative (^-) cells after background subtraction.</p><p>NS = non-stressed, DS5 = desiccating stress for 5 days, DS10 = desiccating stress for 10 days.</p

Charcterization of intraepithelial lymphocytes (IELs) in the normal murine conjunctiva.

<p>Representative flow cytometry analysis of cells isolated from ocular surface (A) or spleen (B) stained with CD3 antibody and γδ (GDTCR), CD8α, NK1.1 and CD103 markers. (+) = positive cells, (–) =  negative cells. Lymphocytes were gated based on characteristic light-scatter properties (“gated cells”, circled population on far left panels), subsequently gated based on forward scatter height vs. forward scatter area (FSC-A) and propridium iodide live/dead exclusion (“live cells”, not shown). Numbers in the quadrants indicate the percentage of cells of one representative experiment. SSC-A = side scatter area.</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

Adoptive transfer results.

<p><b>A</b> Mean± SD of IL-17 ELISPOTs showing IL-17- producing cells isolated from the ocular surface (OS) and CD4⁺ T cells isolated from spleen and cervical lymph nodes (CLN) in donor mice that received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) antibody before (non-stressed, NS) and after 5 days of desiccating stress (DS5). Experiments were repeated two times with at least five mice per group per experiment. <b>B</b> Representative images of OGD corneal staining used to generate OGD intensity score in <b>C.</b> Bar charts show mean ± SD of three independent experiments with five mice for each group per experiment. <b>D-G-</b> Laser scanning immunofluorescent confocal microscopy of cornea immunostained for MMP-3 (in <b>D</b>) and MMP-9 (in <b>F</b>) in nude mice that received CD4⁺T cells isolated from donor mice treated with systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Bar graphs are mean±SD of fluorescence intensity measured in corneal epithelium for MMP-3 (E) and MMP-9 (G) of a total of two independent experiments with at least three mice per group per experiment. <b>H-K</b>-Gene expression analyses showing mean± SD (copies) of IL-17A (in <b>H</b>), CCL20 (in <b>I</b>), matrix metalloproteinases (MMP)-3 (in <b>J</b>) and MMP-9 (in <b>K</b>) mRNA transcripts in cornea epithelia of nude mice that received CD4⁺T cells isolated from donor mice that had received systemic injection of depleting antibody (NK1.1) to NK and NKT cells or isotype control (IC) after 5 days of desiccating stress (DS5). Data represents mean ± SD. Experiments were repeated two times with at least three mice per group per experiment.</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