CCR6KO and CXCR3KO mice are resistant to dry eye disease.

<p><b>A.</b> Corneal permeability - Mean±SD of Oregon green dextran (OGD) permeability into the corneal epithelium of wild-type (WT), CCR6KO and CXCR3KO mice without (NS) or with 5 days of desiccating stress (DS5). OGD permeability was measured in five to seven mice per strain/time point/experiment. The graph represents the combined results of two independent experiments with a total of 10-14 mice per experimental group. <b>B.</b> Representative pictures of OGD staining of each strain. <b>C.</b> CD4⁺ T cell density – Mean±SD of the number of CD4⁺ cells per 100 µm of conjunctival epithelium of WT, CCR6KO, and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. <b>D.</b> Representative pictures of CD4⁺ T cell infiltration in each strain. * - indicates goblet cells; ↓ - indicates CD4⁺ T cells; CJ – conjunctiva; K – cornea. <b>E.</b> Goblet cell density - Mean±SD of the number of goblet cells per mm of conjunctival epithelium of WT, CCR6KO and CXCR3KO mice without (NS) or with 5 (DS5) or 10 (DS10) days of desiccating stress. Three sections from three animals were examined (N = 9) for each time point/mouse strain for both CD4⁺ T cell and goblet cell density. <b>F.</b> Representative pictures of PAS staining of each strain.</p

Inflammatory cytokines are not produced in response to DS in the cornea and conjunctiva of CCR6KO and CXCR3KO CD4⁺ T cell recipient mice.

<p>Wild-type (WT), CCR6KO, or CXCR3KO CD4⁺ T cells from NS or DS5 mice were adoptively transferred to T cell deficient RAG1KO mice. mRNA analysis for inflammatory cytokines and matrix metalloproteinases (MMP) was performed on the corneal (A) and conjunctival (B) tissues of CD4⁺ T cell RAG1KO recipient mice. Mean±SD of transcript levels of corneal and conjunctival tissues. Results represent the mean±SD expression of 2-3 animals per strain/time point per experiment in two independent experiments for a total of 5-6 mice. *, P<0.05, **, P<0.001, ***, P<0.0001.</p

Th1 and Th17 associated cytokines are produced in the cervical lymph node but not the ocular surface.

<p><b>A.</b> Gene expression of IFN-γ, IL-17A, and IL-13 in the superficial cervical lymph node (CLN) of wild-type C57BL/6, CCR6KO, and CXCR3KO exposed to DS for 5 days (DS5) or not exposed (NS). <b>B.</b> Gene expression of IFN-γ, IL-17A, and IL-13 the ocular surface of wild-type C57BL/6, CCR6KO, and CXCR3KO exposed to DS for 5 days (DS5) or not exposed (NS). Results represent the mean±SD expression 2-3 animals per strain per time point in two independent experiments with a total of five mice. *, P<0.05, **, P<0.01.</p

Increased expression of CCR6⁺ and CXCR3⁺ cells in the cervical lymph node and at the ocular surface in response the desiccating stress.

<p><b>A.</b> The percentage of CCR6⁺CD4⁺ T cells in the superficial cervical lymph node (CLN) in mice exposed to no stress (NS) or five (DS5) days of desiccating stress (DS) was determined by flow cytometry. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>B.</b> The percentage of CCR6⁺CD4⁺ T cells in the ocular surface (OS). The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>C.</b> The percentage of CXCR3⁺CD4⁺ T cells at the CLN. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>D.</b> The percentage of CXCR3⁺CD4⁺ T cells at the OS. The results represent mean±SD of five samples per time point for a total of ten samples (N = 10) in two independent experiments. <b>E.</b> A representative dot plot of the percentage of CD4⁺ cells that are CCR6⁺ or CXCR3⁺ in the CLN.</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

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

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