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

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

Cytokine burst from NK positive populations after DS.

<p>mRNA levels in NK/NKT positive (+) and NK/NKT negative (–) cells isolated from nonstressed (NS) spleen and ocular surface (OS) and at different time points after desiccating stress (DS; DS1 = DS for 1 day, DS5 = DS for 5 days, DS10 = DS10 for 10 days). Unfractionated spleen was used as calibrator. Experiments were repeated two times with at least three samples per group per experiment. Because the standard deviation is relatively small compared to the levels of IL-6, IL-23 and IL-17A expression, the error bars do not show in the graph. * indicates p<0.05, *** indicates p<0.001 comparison vs. NS control NK/NKT+. ^∧ indicates P<0.05, ^∧∧∧ indicates p<0.001 comparison vs. NS control NK/NKT−.</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

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