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

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

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

Locus specific analysis of whole-blood DNA identified similar methylation patterns in healthy and psoriatic individuals.

<p>(A) The CGI encompassing the minimal promoter and the first two exons <i>of HLA-C</i> was investigated by methyl-specific PCR, following treatment of whole blood DNA with sodium bisulfite. The DNA of a healthy donor was modified with the M.SssI CpG methyl-transferase and used as a positive control (C+). (B) The CGIs lying upstream of <i>HCG27</i> (top left), <i>HLA-C</i> (top right) and <i>POU5F1</i> (bottom) were analysed by CoBRA, exploiting the loss of restriction nuclease sites in unmethylated and bisulphite converted samples. A sample that did not undergo bisulfite treatment was used as positive control for the enzymatic digestion (C+). The methylation status of the CGIs was further validated by direct sequencing of cloned PCR products, generated from the DNA of one healthy and one affected individual (data not shown).</p

Distribution of <i>PSORS1</i> candidate variants.

<p>The number of changes that are unique to <i>PSORS1-Cw0602</i> haplotypes is plotted against GRCh37/hg19 coordinates.</p

High-resolution analysis of the <i>PSORS1</i> interval reveals multiple regions of LD conservation.

<p>LD decay was measured using the D’ index, which was derived based on the genotypes of 4,803 UK controls. Markers occurring with a minor allele frequency (MAF) <0.1 were excluded from the analysis, as variation at these loci is likely to be the result of recent mutational events. The plot documents the analysis of 147 SNPs, randomly selected from the 373 variants that had a MAF >0.1 (see Methods for details). The top panel shows the position of <i>PSORS1</i> genes (the <i>HCG27</i> pseudogene is shaded in grey), with the region of LD conservation around <i>HLA-C</i> highlighted by a double arrow.</p

Epigenetic analysis of the refined <i>PSORS1</i> locus in patient T-lymphocytes.

<p><b>A)</b> H3K4me1 and H3K27ac ChiP-Seq peaks are shown for a representative sample. The size of each peak is plotted as a black box immediately below the Chip-Seq track. The figure besides each box indicates the False Discovery Rate (FDR) associated with the identification of the peak (e.g. 0.47 indicates a FDR of 0.47%). <b>B)</b> A detailed view of the <i>HLA-C</i> gene region is shown. The dotted line box highlights the boundaries of the active regulatory region defined by the overlap of an unmethylated CpG island with H3K4me1 and H3K27ac peaks.</p