1. Contribution of Programmed Death Ligand-1 Expression to Corneal Avascularity Yiping Jin, 1 Sunil K. Chauhan, 1 Arlene Sharp, 2 Reza Dana 1 1.Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, 2. Department of Pathology, Harvard Medical School, The authors have no financial interest in the subject matter of this poster. Support NIH R01-EY12963

2. The cornea possesses an extensive array of mechanisms by which immune effectors and neovascularization (NV) are regulated, or even silenced, including an intact corneal epithelial layer, epithelial expression of membrane and soluble VEGF receptors and the high expression of FasL and thrombospondin-1. Recently, an immunoregulatory molecule, programmed death ligand-1 (PDL-1, CD274, B7-H1), has been shown to be expressed at high levels by corneal tissue by our lab and others. PDL-1 is a member of the B7-CD28 family, which plays important roles in regulating T cell activation and tolerance. Several immunoregulatory factors, such as FasL and thrombospondin-1, also possess a regulatory function on corneal NV. However, the anti- angiogenic effect of PDL-1 has not been demonstrated to date. Here, we provide novel evidence for the regulatory function of PDL-1 in corneal angiogenesis in vivo. Introduction

3. Methods Animals: PDL-1 -/- C57BL/6 mice, C57BL/6 mice Three intrastromal sutures were placed to induce corneal neovascularization. The numbers of infiltrating CD11b + and Gr-1 + cells were measured by flow cytometry. The expression levels of VEGR-2, IL-1α, IL-1β and TNF-α in corneas were analyzed by real-time PCR. Angiogenesis was quantified by biomicroscopically and by measuring the blood vessel area spanned by CD31 + cells on whole-mount corneas.

4. Figure 1. (A) Corneal neovascularization (NV) was induced in WT and PDL-1 KO mice after suture- placement. (B) In a masked fashion, corneal NV was scored biomicroscopically with a slit-lamp using a grid system. Values are expressed as the mean (+ SEM) of 6 corneas. (C) On Day 14, whole corneas were dissected and stained with anti-CD31-FITC (Green) (D) The density of blood vessels (CD31+) covering the cornea was analyzed. Values are expressed as the mean (+ SEM) of 6 corneas of per treatment group (* P< 0.001 vs. vehicle-treated group). PDL-1 suppresses corneal neovascularization Results A.A. WT KO C.C. CD31 % of cornea area B. D. WT * KO

5. Results Figure 2. Three days after suture placement, PDL-1 KO mice showed a significantly higher mRNA expression level of VEGFR-2, comparing to the WT mice. However, there were no significant differences in the mRNA expression levels of IL-1α, IL-1β or TNF-αbetween both groups. 0 ** VEGFR2 mRNA Relative expression level WT KO normal 24 hr 72 hr 3 6 9 * TNF-α mRNA Relative expression level WT KO normal 24 hr72 hr WT KO 0.00 0.25 0.50 0.75 1.00 1.25 WT KO IL-1α mRNA Relative expression level WT KO normal 24 hr72 hr 0.00 0.25 0.50 0.75 1.00 1.25 IL-1β mRNA Relative expression level WT KO 24 hr72 hr 0 100 200 300 400 normal PDL-1 promotes the expression level of VEGFR-2, but not inflammatory cytokines, in suture-placed corneas

6. Figure 3. Corneas were harvested from WT and PDL-1 KO mice at 72 hr after suture placement and digested by collagenase D. Cells were double-stained with anti-CD11b-PE and anti-Gr-1-FITC Abs, and analyzed by flowcytometry. Gr-1 + CD11b + neutrophil and Gr-1 - CD11b + macrophage populations were compared between WT and PDL-1 KO groups. PDL-1 does not affect the infiltration of innate immune cells in suture-placed corneas Results 9.4320.30 10 0 1 2 3 4 0 1 2 3 4 8.8422.01 10 0 1 2 3 4 0 1 2 3 4 0.211.03 10 0 1 2 3 4 0 1 2 3 4 Gr-1-Fitc CD11b-PE PDL-1KOWT Fitc PE Isotype control 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4

7. Conclusion PDL-1 expression in the cornea contributes to the inhibition of corneal angiogenesis. The anti-angiogenic effect of PDL-1 is not related to its immune regulatory function.

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