Contribution of Programmed Death Ligand-1 Expression to Corneal

Avascularity

Yiping Jin,1 Sunil K. Chauhan, 1 Arlene Sharp, 2 Reza Dana1

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.

SupportNIH R01-EY12963

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

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.

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.

WT KO

C.

KO

CD31

% o

f cor

nea

area

Days after suture placement

NV

scor

e

0123456789

D3 D5 D7 D9 D11

Days

NV

scor

e

KO

WT

B.

D.

WT

*

WT KO

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

mR

NA

Rel

ativ

e ex

pres

sion

leve

l

WT KO WT KO

normal 24 hr 72

hr

3

6

9

*

TNF-α

mR

NA

Rel

ativ

e ex

pres

sion

leve

l

WT KO WT KO normal24 hr 72 hr

WT KO0.00

0.25

0.50

0.75

1.00

1.25

WT KO

IL-1α

mR

NA

Rel

ativ

e ex

pres

sion

leve

l

WT KO normal

24 hr 72 hr

0.00

0.25

0.50

0.75

1.00

1.25IL-1β

mR

NA

Rel

ativ

e ex

pres

sion

leve

l

WT KO WT KO24 hr 72 hr

0

100

200

300

400

normal

PDL-1 promotes the expression level of VEGFR-2, but not inflammatory cytokines, in suture-placed corneas

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

9.43 20.30

10 0 10 1 10 2 10 3 10 4100

101

102

103

104

8.84 22.01

10 0 10 1 10 2 10 3 10 4100

101

102

103

104

0.21 1.03

10 0 10 1 10 2 10 3 10 4100

101

102

103

104

Gr-1-Fitc

CD

11b-

PE

PDL-1KO WT

Fitc

PE

Isotype control104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

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.

Reference1. Albuquerque RJ, Hayashi T, Cho WG, et al. Alternatively spliced vascular endothelial growth factor

receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth, Nat Med 2009, 15:1023-1030

2. Ambati BK, Nozaki M, Singh N, et al. Corneal avascularity is due to soluble VEGF receptor-1, Nature 2006, 443:993-997

3. Streilein JW, Yamada J, Dana MR, et al. Anterior chamber-associated immune deviation, ocular immune privilege, and orthotopic corneal allografts, Transplant Proc 1999, 31:1472-1475

4. Greenwald RJ, Freeman GJ, Sharpe AH: The B7 family revisited, Annu Rev Immunol 2005, 23:515-548

5. Hayashi T, Yamagami S, Tanaka K, et al. Immunologic mechanisms of corneal allografts reconstituted from cultured allogeneic endothelial cells in an immune-privileged site, Invest Ophthalmol Vis Sci 2009, 50:3151-3158

6. Hori J, Wang M, Miyashita M, et al. B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts, J Immunol 2006, 177:5928-5935

7. Shen L, Jin Y, Freeman GJ, et al. The function of donor versus recipient programmed death-ligand 1 in corneal allograft survival, J Immunol 2007, 179:3672-3679

8. Stuart PM, Pan F, Plambeck S, et al. FasL-Fas interactions regulate neovascularization in the cornea, Invest Ophthalmol Vis Sci 2003, 44:93-98

9. Reed MJ, Iruela-Arispe L, O'Brien ER, et al. Expression of thrombospondins by endothelial cells. Injury is correlated with TSP-1, Am J Pathol 1995, 147:1068-1080

Contact Informationreza.dana@schepens.harvard.eduYiping.jin@schepens.harvard.edu