Positively stained cells were counted in the stroma of the cornea because the epithelium showed nonspecific background signal in all the cross-sections investigated, including the isotype-matched controls, over a length of 4 mm and to a depth of 100 m (the thickness of the cornea)

Positively stained cells were counted in the stroma of the cornea because the epithelium showed nonspecific background signal in all the cross-sections investigated, including the isotype-matched controls, over a length of 4 mm and to a depth of 100 m (the thickness of the cornea). multiple CC and CXC chemokine ligands and receptors. Epifluorescence microscopy was used to evaluate corneal infiltration of CD3+T cells after immunohistochemical staining. == Results. == The increased expression of specific chemokine ligands and receptors in PD-L1/corneas of normal mice is associated with significant increases in T-cell homing into these corneas. Similar, and more enhanced, increases in T-cell infiltration were observed in PD-L1/DED mice or DED mice treated with antiPD-L1 antibody compared with controls. In addition, the authors found significantly decreased expression of PD-L1 by corneal epithelial cells in DED and significantly increased corneal fluorescein staining score with PD-L1 functional blockade using antiPD-L1 antibody. == Conclusions. == Downregulation of corneal epithelial PD-L1 amplifies dry eyeassociated corneal inflammation and epitheliopathy by increasing the expression of chemokine ligands and receptors that promote T-cell homing to the ocular surface. Dry eye disease (DED) affects many millions of persons, in particular women, in the United States alone.1The current literature on the immunopathogenesis of DED focuses on the inflammatory milieu of the tear film or conjunctiva, whereas it is corneal inflammation that is the most clinically recognizable and important ocular manifestation of DED.28There is growing evidence that CD4+T cellmediated inflammation plays a critical role in amplifying the pathogenesis of DED.915Still, the manner by which this inflammation can induce corneal pathology remains poorly understood. PD-L1 is a member of the B7 family of receptors and has a role in regulating T cellmediated immunity.16In vivo studies using PD-L1/mice and antiPD-L1 blocking antibody have provided evidence for the inhibitory functions of PD-L1 in both autoimmunity and alloimmunity. For example, it has been Gnb4 shown that tissue-specific PD-L1 expression protects against autoimmune diabetes, ocular inflammation, and corneal allograft rejection by inhibiting autoreactive and alloreactive T cells.1720In DED, there is increased T-cell infiltration into the conjunctiva, but, remarkably, the cornea remains relatively resistant to this infiltration. 913 Herein we test the hypothesis that decreased PD-L1 expression is associated with increased chemokine expression, increased T-cell infiltration, and increased corneal fluorescein staining. Galanin (1-30) (human) The purpose of the present study was to determine the effect of PD-L1 on modulating the expression of chemokine gene transcripts in the cornea. Additionally, we investigated the potential role of PD-L1 in the pathogenesis of DED by inducing DED in PD-L1/mice and in mice treated with antiPD-L1 blocking antibody to determine how the blockade or elimination of PD-L1 affects the expression of the principal T-cell chemokines and the clinical aspects of DED. == Materials and Methods == == Mouse Model of Dry Eye == Eight- to 12-week-old female C57BL/6 mice were obtained from Taconic Farms (Germantown, NY), and Charles River Laboratory (Boston, MA). Similarly aged PD-L1/C57BL/6 mice were generated as previously described.16In all the experiments performed, the mice were age-matched among the different groups. The protocol was approved by the Institutional Animal Care and Use Committee, and all animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Dry eye was induced by placement of mice in a controlled-environment chamber modified with subcutaneous administration of scopolamine to maximize ocular dryness as previously described.1315,21,22Age-matched mice not placed in the controlled-environment chamber were used as controls. == PD-L1 Blockade == To block PD-L1mediated signaling, five mice were treated Galanin (1-30) (human) 1 day before dry eye induction and every other day thereafter for 10 days with antimurine PD-L1 (10F.9G2, rat IgG2b; 150 g/mouse intraperitoneally) or control rat IgG (MP Biomedicals, Santa Ana, CA).17,23,24 == Measurement of Galanin (1-30) (human) Corneal Fluorescein Staining == Corneal fluorescein staining was performed at baseline (day 0) and then at days 2, 5, 7, and 9. One microliter of 2.5% fluorescein was applied to the lateral conjunctival sac of the mice, as previously described.1315,21,22Eyes were examined.