Open Access
Retina  |   June 2018
CD160 Expression in Retinal Vessels Is Associated With Retinal Neovascular Diseases
Author Affiliations & Notes
  • Adrien Henry
    Department of Ophthalmology, Hôpital Robert Debré, Reims, France
  • Camille Boulagnon-Rombi
    Department of Pathology, Hôpital Robert Debré, Reims, France
  • Thierry Menguy
    ElsaLys Biotech, Lyon, France
  • Jérôme Giustiniani
    INSERM U976, Hôpital Saint-Louis, UMR-S 976, Université Paris Diderot, Paris, France
    Department of Research, Institut Jean Godinot, Reims, France
    Derm-I-C Research Unit, EA-7319, Faculté de Médecine de Reims, Reims, France
  • Christian Garbar
    Department of Pathology, Hôpital Robert Debré, Reims, France
    Department of Research, Institut Jean Godinot, Reims, France
  • Corinne Mascaux
    Department of Research, Institut Jean Godinot, Reims, France
  • Marc Labrousse
    Department of Anatomy, Faculté de Médecine de Reims, Reims, France
  • Corentin Milas
    Department of Ophthalmology, Hôpital Robert Debré, Reims, France
  • Coralie Barbe
    Department of Clinical Research, Hôpital Robert Debré, Reims, France
  • Armand Bensussan
    INSERM U976, Hôpital Saint-Louis, UMR-S 976, Université Paris Diderot, Paris, France
  • Vincent Durlach
    Cardiovascular and Thoracic Division, Hôpital Robert Debré, Reims, France
  • Carl Arndt
    Department of Ophthalmology, Hôpital Robert Debré, Reims, France
  • Correspondence: Adrien Henry, Service d'Ophtalmologie, Hôpital Robert Debré, CHU de Reims, Avenue du Général Koenig, 51100 Reims, France; henry.reims@gmail.com
  • Footnotes
     AB, VD, and CA contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science June 2018, Vol.59, 2679-2686. doi:10.1167/iovs.18-24021
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      Adrien Henry, Camille Boulagnon-Rombi, Thierry Menguy, Jérôme Giustiniani, Christian Garbar, Corinne Mascaux, Marc Labrousse, Corentin Milas, Coralie Barbe, Armand Bensussan, Vincent Durlach, Carl Arndt; CD160 Expression in Retinal Vessels Is Associated With Retinal Neovascular Diseases. Invest. Ophthalmol. Vis. Sci. 2018;59(7):2679-2686. doi: 10.1167/iovs.18-24021.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Anti-angiogenic agents stand first in the treatment of neovascular diseases of the retina. CD160 appeared in several experimental studies as a marker of activated endothelial cells, suggesting it could represent a promising target for novel anti-angiogenic therapies. The aim of the present study was to assess the distribution of CD160 in the human eye, and to search for a possible correlation with retinal neovascular diseases.

Methods: The physiological distribution of CD160 in the normal eye was assessed with immunolabeling in 10 human donor eyes. Then, in a retrospective cohort of 75 surgical retinal specimens, the density of CD160+ microvessels was evaluated, along with immunolabeling on serial sections against ERG (pan-endothelial cell marker), CD105 (activated endothelial cell marker), and α-SMA (pericyte cell marker). The cohort was divided into two groups: 29 patients with neovascular disease (NV+) and 46 control patients (NV−).

Results: CD160 was physiologically expressed by several cell types: endothelial cells of retinal blood vessels, ganglion cells, macrophages, epithelial cells of the conjunctiva, ciliary body, and retinal pigment epithelium. In the patient cohort, the percentage of CD160+ vessels in the retina was significantly and independently higher in patients suffering from neovascular diseases (P = 0.04). On the contrary, the expression of CD105 was correlated neither with retinal neovascular diseases, nor with CD160 expression.

Conclusions: CD160 was expressed in some retinal vessels in both normal and pathologic eyes. CD160 expression by endothelial cells of retinal vessels was correlated with ocular neovascular diseases. CD160 could therefore represent an interesting target for novel anti-angiogenic therapies.

Intravitreal (IVT) injections of anti-VEGF agents, such as ranibizumab, bevacizumab, or aflibercept, have emerged over the past decade as the first line of treatment in several retinal vascular diseases, such as retinal vein occlusion (RVO), macular edema,13 or diabetic retinopathy (DR) with diabetic macular edema (ranibizumab, February 2015; aflibercept, March 2015). These therapies are also considered as valuable adjuncts (albeit not approved by the Food and Drug Administration) in proliferative DR (PDR)4,5 and neovascular glaucoma (NVG).69 
Nevertheless, anti-VEGF therapies suffer some limitations, among which include a short time to recurrence (1 week to 3 months) of retinal neovascularization in PDR,5,1012 and an eroding biological effect after prolonged use, that adversely affect long-term efficacy, by tachyphylaxis or tolerance.1315 It was also suggested,16 then confirmed,1719 that anti-VEGF injections were associated with geographic atrophy progression, an advanced form of wet AMD. Moreover, pharmacokinetics studies revealed, in most animal models, a reduced half-life of intravitreal anti-VEGF drugs.20 Identifying new angiogenic factors could reveal additional mechanisms, uncover their regulation, and thus pave the way for novel therapies with complementary or slow-release modes of action,21 to reduce the number of IVT injections and their adverse effects. 
CD160 could represent such a new angiogenic factor. Initially described as BY55 in resting cytotoxic natural killer (NK) cells of the peripheral blood,22 CD160 is a 181 amino-acid glycosylphosphatidylinositol-anchored protein of the immunoglobulin superfamily. In healthy tissues it was shown to be mainly restricted to cytolytic lymphocytes, including NK cells, T cells, and intestinal intraepithelial CD8+ T cells,23,24 to some mast cells,25 to anti-inflammatory mononuclear phagocytes in the colonic lamina propria,26 and to some unstimulated CD4+ T cells.27 More recently, it was shown that some of the NK cells from CD160-deficient mice were unable to produce IFN-γ,28 which is known to be involved in angiogenesis.29 In cultured endothelial cells, CD160 was also shown to be expressed by growing activated but not quiescent cells.30 Furthermore, its specific engagement, either by MHC class Ib molecules or by CL1-R2, an agonist murine monoclonal antibody (mAb) directed against human CD160, reduced angiogenesis through the caspase-dependent apoptosis of endothelial cells.30 This anti-angiogenic mechanism is thus distinct from the current anti-angiogenic therapies that target the VEGF/VEGF-R pathway. The anti-angiogenic therapeutic efficacy of CL1-R2 has already been described in various in vivo models of oxygen-induced proliferative retinopathy and choroidal neovascularization.31 CL1-R2 treatment of mice with ischemic retinopathy was shown to restore the normal retinal vascularization, suggesting that this mAb has a dual activity, as it blocks neoangiogenesis and seems to restore a normal vascular structure.31 CD160 could therefore stand as a promising target for anti-angiogenic therapies; however, its distribution in human ocular tissues remains unknown. 
The aim of this study was to analyze CD160 expression in both normal and pathologic eyes from human adults, and to evaluate to what extent its presence on endothelial cells from blood vessels could be correlated to retinal vascular diseases. 
Materials and Methods
Case Selection
The computerized database of the Pathology Department, Reims University Hospital, France, was searched to identify all samples containing retina among all eye surgery procedure–related specimens, collected in adult patients between 2008 and 2016. All cases underwent surgical treatment in the Department of Ophthalmology at Reims University Hospital. Specimens had been previously 4% buffered formalin fixed and paraffin embedded (FFPE) and routinely stained with hematoxylin-eosin-saffron (HES). 
Anonymous human donor eyes were provided from the Department of Anatomy, Medicine University of Reims, France, within up to 48 hours after death. Only macroscopically normal eyes were included. Eyes underwent FFPE processing. Ten normal-appearing eyes from 10 different donors were prepared as described. 
Clinical Data
Patient data, such as age at the time of surgery; sex; history of arterial hypertension, dyslipidemia, or diabetes mellitus; clinical or B-mode echography arguments for retinal detachment (RD); and time interval between primary event that led to eye loss and surgery, were obtained from patients' medical files. Patients were excluded when data were missing. According to their clinical history and eye examination, patients were classified into two groups. The neovascular group (NV+) included patients with DR, RVO complicated with iris rubeosis (IR) or NVG, ocular ischemic syndrome (OIS), NVG secondary to any ocular event, or IR secondary to RD. The non-neovascular control group (NV−) comprised patients without those neovascular diseases or complications but who underwent events leading to eye loss and surgery, such as trauma, endophthalmitis, cellulitis, anterior perforation, corneal abscess, or RD. Classifications were performed by a retinal expert (CA) and a resident in ophthalmology (AH). Altogether, a total of 75 patients were included in the study. 
The study was performed in accordance with the ethical standards of the Declaration of Helsinki. Patients' records and samples were anonymized before histology, immunochemistry, and analysis. The database was built in accordance with the reference methodology MR003 of the Commission Nationale de l'Informatique et des Libertés (no. 2016198, December 12, 2016). 
Ten eyes were obtained from 10 different human Caucasian donors: six women and four men with a mean age of 88.1 ± 7.05 years. Two eyes had a posterior chamber intraocular lens and one had calcified senile scleral plaques. No other morphologic macroscopic abnormalities were observed. The cadavers were donated to the Department of Anatomy, Reims Faculty of Medicine and University Hospital, for anatomical education and research. They were studied following all ethical rules of work on cadaver material. 
Histopathological Procedures
Eight 4-μm-thick serial sections from FFPE blocks of each eye were cut and mounted on SuperFrost Ultra Plus adhesive slides (Thermo Scientific, Dominique Dutscher SAS, Brumath, France). First and last sections were mounted on Starfrost slides (Thermo Scientific) and stained with HES. Five other sections were used for immunohistochemistry using antibodies directed against CD160, ERG (endothelial cell marker), CD68 (macrophage marker), CD105 (endoglin, activated endothelial cell marker), or α-smooth muscle actin (α-SMA) (pericyte cell marker). Antibodies and their working dilutions are detailed in Table 1. The anti-CD160 CL1-R2 antibody (murine IgG1) was produced as previously described31 in the laboratory of AB, who developed it. The immunolabeling was performed with the BenchMark XT automated slide stainer (Ventana Medical Systems, Tucson, AZ, USA). After deparaffinization, sections were incubated with Cell Conditioner 1 (EDTA, pH 8.4) for 30 minutes for α-SMA, CD160, and ERG antibodies and 8 minutes for CD68 antibody and with Cell Conditioner 2 (Citrate, pH 6) for 8 minutes for CD105 antibody. Then, the endogenous preprimary peroxydase was inhibited by a 32-minute incubation at 37°C for CD68, ERG, CD105, and α-SMA or a 60-minute incubation with CD160. The staining reaction was then performed with the ultraView Red v3 Kit for CD160, CD105, α-SMA, CD68, and control isotype, and with the ultraView DAB v3 Kit (Ventana Medical Systems) for ERG immunolabeling. The counterstain and post counterstain were composed of hematoxylin and bluing reagents. In each eye, positive and negative controls were performed in parallel with the same procedure as CD160 immunolabeling, using the anti-CD160 CL1-R2 antibody on tonsil tissue sections for positive control, and using a murine IgG1 isotype control on serial section for negative control. 
Table 1
 
Primary Antibodies Used for Immunohistochemistry
Table 1
 
Primary Antibodies Used for Immunohistochemistry
Immunostaining Analyses
The immunolabeling was evaluated independently by two observers (CBR, AH) blinded to clinical data. In the event of a discrepancy, a common decision was reached after collegial discussion. The cell type and the staining intensity (SI; 0: absent, 1: faint, 2: moderate, 3: strong) was assessed for both CD160 and CD105 immunolabeling. 
Tissue sections stained with anti-ERG antibody were used for microvessel density (MVD) evaluation. The slides were examined under ×40 magnification for the retina or gliosis in the most vascularized region, and MVD quantification was performed under ×400 magnification (0.196 mm2/field), averaged over five consecutive fields, as previously described.32 For each field, serial section comparisons were made to determine in the same area with the same counting method the number of ERG+ microvessels stained with CD160 only (CD160-MVD), CD105 only (CD105-MVD), and with both markers (CD160/CD105-MVD). Values were expressed as the percentage of CD160+, CD105+, or CD160+/CD105+ vessels, calculated respectively as the ratios of CD160-MVD, CD105-MVD, or CD160/CD105-MVD on the MVDs. 
Statistical Analyses
Data were described using mean and SD for quantitative variables, and number and percentage for qualitative variables. Comparisons between the NV+ and the NV− groups were then performed using univariate analysis (χ2 tests or Wilcoxon tests, as appropriate), and using multivariate analysis (logistic regression with factors significant at P = 0.10 in univariate analysis included). For multivariate analysis, collinearity between histological parameters was studied. Association between the percentages of CD160+ and CD105+ vessels was evaluated using Spearman's correlation coefficient. For all analyses, differences were declared statistically significant when P < 0.05. All statistical analyses were performed using SAS version 9.4 (SAS Inc., Cary, NC, USA). 
Results
CD160 Expression in Healthy Human Donor Eyes
As detailed in Table 2, CD160 was moderately expressed in ciliary body epithelium (Fig. 1A), retinal pigmented epithelium (RPE), epithelial cells of the conjunctiva, and iris sphincter muscles (Fig. 1B). Ciliary body muscle cells were faintly positive for CD160, whereas lenses and sclera were negative. Some endothelial cells of retinal blood vessels expressed CD160 in 4 of 10 eyes (Fig. 1C). Of note, CD160 was strongly and constantly expressed by ganglion cells (Fig. 1C) and macrophages (Fig. 2), as revealed by the CD68-CD160 cross section. CD160 was not expressed in choroidal blood vessels except in macrophages surrounding normal-appearing choroidal blood vessels (Fig. 1D). Control isotypes were negative in all cases, whereas tonsil tissue sections stained with the anti-CD160 antibody showed intense staining of some cells in germinal centers (Supplementary Fig. S1), where B-cells are located, among which CD160-expressing NK cells.23 
Table 2
 
CD160 Staining Intensity Scores in Structures of Human Donor Eyes
Table 2
 
CD160 Staining Intensity Scores in Structures of Human Donor Eyes
Figure 1
 
Overview of CD160 distribution in nonpathologic donors' eyes. CD160 red staining evidence in (A) ciliary epithelium, (B) iris sphincter muscle, (C) retina with ganglion cell layer (asterisk) and retinal microvessels (caret), and macrophages surrounding choroidal blood vessels (D). Original magnifications are indicated. Left column, scale bar: 200 μm; right column, scale bar: 50 μm. Zoom insets are displayed in the right column.
Figure 1
 
Overview of CD160 distribution in nonpathologic donors' eyes. CD160 red staining evidence in (A) ciliary epithelium, (B) iris sphincter muscle, (C) retina with ganglion cell layer (asterisk) and retinal microvessels (caret), and macrophages surrounding choroidal blood vessels (D). Original magnifications are indicated. Left column, scale bar: 200 μm; right column, scale bar: 50 μm. Zoom insets are displayed in the right column.
Figure 2
 
CD160 expression in macrophages using serial sections in nonpathologic donors' eyes. CD160-positive cells (anti-CD160) and macrophages (anti-CD68) are co-detected with red markers in ciliary body of a human healthy donor. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Figure 2
 
CD160 expression in macrophages using serial sections in nonpathologic donors' eyes. CD160-positive cells (anti-CD160) and macrophages (anti-CD68) are co-detected with red markers in ciliary body of a human healthy donor. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
CD160 Expression in a Cohort of Patients
As detailed in Table 3, 29 patients were classified in the neovascular group (NV+) and 46 patients in the non-neovascular control group (NV−). The NV+ group included patients with DR (n = 6); RVO complicated with NVG or IR (n = 5); OIS (n = 1); NVG secondary to any ocular event but RVO, DR, or OIS (n = 6); RD complicated with NVG or IR (n = 11). 
Table 3
 
Comparison of Clinical Characteristics and Histological Parameters in Neovascular (NV+) and Control (NV−) Groups
Table 3
 
Comparison of Clinical Characteristics and Histological Parameters in Neovascular (NV+) and Control (NV−) Groups
In univariate analyses, patients in the NV+ group were significantly older (65.6 ± 15.2 years vs. 56.1 ± 19.7 years; P = 0.04) and suffered more frequently from arterial hypertension (62.1% vs. 34.8%; P = 0.02) and dyslipidemia (55.2% vs. 19.6%; P = 0.001) than patients in the NV− group. The sex ratio, time interval before surgery, presence of RD, and history of diabetes mellitus were not significantly different between the two groups. 
Like in healthy donors, CD160 labeling was observed in some endothelial cells of the cohort specimen (Fig. 3, Table 3). Microvascular densities (MVD) evaluated with the pan-endothelial marker ERG were not significantly different between the two groups (26.4 ± 24.9 vessels/mm2 versus 26.7 ± 23.0 vessels/mm2; P = 0.82). The percentage of vessels positive for CD160 was significantly higher in the NV+ group, compared with the NV− group in univariate analysis (33.8% ± 27.9% versus 19.4% ± 25.7%; P = 0.008). CD160 staining intensity scores (CD160-SI) were not significantly different between NV+ and NV− groups (1.1 ± 0.6 vs. 1.0 ± 0.9; P = 0.64). The percentage of vessels positive for CD105, an established marker of active angiogenesis in both solid human tumors and ischemic retinopathies,33 was not statistically different between NV+ and NV− groups (58.5% ± 28.9% versus 52.9% ± 31.6%; P = 0.55), and their staining intensity did not differ between NV+ and NV− groups (CD105-SI : 1.3 ± 0.7 vs. 1.1 ± 0.8; P = 0.53). The percentage of CD160+/CD105+ vessels was significantly higher in the NV+ group compared with the NV− group (24.5% ± 27.0% versus 14.9% ± 21.1%; P = 0.04). 
Figure 3
 
CD160 expression in inner retina microvessels using serial sections. Inner retina blood microvessel density marker is detected with a brown pan-endothelial marker (anti-ERG), whereas activated endothelial cells (anti-CD105), actin of pericytes (anti-α-SMA), and CD160-positive cells (anti-CD160) are all detected with red markers. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Figure 3
 
CD160 expression in inner retina microvessels using serial sections. Inner retina blood microvessel density marker is detected with a brown pan-endothelial marker (anti-ERG), whereas activated endothelial cells (anti-CD105), actin of pericytes (anti-α-SMA), and CD160-positive cells (anti-CD160) are all detected with red markers. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Variables included in the multivariate analysis were patient's age, arterial hypertension, dyslipidemia, and percentage of CD160+ vessels (Table 3). Because of collinearity, CD160-CD105-MVD (which depended on CD160-MVD) was not included in the multivariate analysis. Moreover, because the vascular expressions of CD105 and CD160 were correlated (r = 0.35, P = 0.002), we did not include CD105 vascular expression in multivariate analysis. In multivariate analyses, dyslipidemia (odds ratio [OR] 3.85 [1.1–13.1]; P = 0.03) and the percentage of CD160+ vessels (OR 1.02 [1.001–1.04]; P = 0.04) were significantly different between the NV+ and NV− groups (each 1% increase of CD160+ vessels was associated with a 2% increase in the risk of NV+). Patient age and arterial hypertension were not significantly different (P = 0.45 and P = 0.67, respectively; Table 3). 
CD160 was also strongly expressed by some macrophages, with the same staining intensity observed in healthy human donor eyes (Supplementary Fig. S1). On the contrary, no coexpression of CD160 and pericyte-specific marker α-SMA was observed. 
Discussion
This study is to our knowledge the first to describe CD160 expression in the human eye. CD160 was expressed by different cell types, including endothelial cells of retinal blood vessels, ganglion cells, macrophages, epithelial cells of the conjunctiva, ciliary body, and RPE. Our study revealed that the percentage of CD160+ retinal blood vessels was significantly higher in the eyes of patients suffering from vascular retinal diseases, compared with patients without such disease, whereas the total number of vessels was equivalent in both groups, as estimated by the comparison of MVDs. This is the first report of an association between CD160 expression in retina blood vessel endothelial cells and pro-neovascularizing diseases, in both univariate and multivariate analyses. On the contrary, we observed no evidence for CD160-specific staining in endothelial cells of the choriocapillaris, neither in healthy donor eyes nor in pathologic specimen retinas, suggesting that its expression could be restricted to particular subsets of endothelial cells or to specific conditions. Only one patient with wet AMD, receiving anticoagulants, was included in this study because of phthisis secondary to NVG after massive subretinal hemorrhage. In his retina specimen, the percentage of CD160 vessels reached 42.3%. No choroidal vessel was found positive, but it was not possible to assess the precise location of the sections used for immunochemistry within the retina. 
CD105 (also known as endoglin) is expressed by endothelial cells mostly during regenerating and its expression in tumors is restricted to newly formed activated vessels. Because CD105 is upregulated in response to hypoxia and VEGF blockade, it represents another anti-angiogenic alternative strategy.34 Indeed, TRC105 (Tracon Pharmaceuticals, San Diego, CA, USA), a chimeric anti-human endoglin antibody, is currently being tested in several clinical trials, including a phase I/II trial, as a treatment for wet AMD named DE-122 (Santen Pharmaceutical, Osaka, Japan) (see Ollauri-Ibáñez et al.33 and references therein). It therefore seemed pertinent to evaluate CD105 expression in our retinal specimens and its correlation with CD160. Interestingly, the percentages of CD105+ vessels were similar between the NV+ and the NV− groups, but only a weak association with CD160 vascular expression was found. 
Finally, we noticed an intense CD160 staining in ganglion cells and macrophages, both in our cohort of patients and in healthy donor eyes. Ganglion cells are known to have a high metabolic activity, and the precise subcellular location of CD160 has to be determined, to specify its role in these cells. Whether macrophages could be involved in the potential anti-angiogenic therapeutic efficacy of anti-CD160 antibodies, such as CL1-R2, needs to be further studied, because the mode of action of CL1-R2 seems to differ from that of the current anti-angiogenic therapies targeting the VEGF/VEGF-R pathway.31 There is in fact an increasingly recognized contribution of bone marrow–derived cells in tumoral and ocular neovascularization.3537 For example, macrophages seem to play a role in both retinal and choroidal neovascularizations, which could rely on the expression of the pro-angiogenic, stromal-derived factor-1 receptor CXCR4 and on the secretion of several pro-angiogenic and proinflammatory signals, including VEGF. The neovascularization process could be facilitated by the increased recruitment and retention of macrophages around sprouting retinal blood vessels, resulting from the increased production of SDF-1 in ischemic retina through retinal pigmentary epithelium stimulation.38,39 In addition, it is in accordance with CD160 expression in some macrophage subsets that has recently been described by Barman et al.26 
We acknowledge that our results are limited by the number of patients in each group. Patients in the NV− (control) group suffered from various ophthalmic diseases (e.g., phthisis induced by endophthalmitis, multioperated RDs), and the small number of patients in each group precluded any valid subgroup comparison. In the NV− group, the number of patients with DR, RVO, or OIS was relatively small. In the NV+ group, patients were older and suffered more frequently from arterial hypertension and dyslipidemia. These conditions are known to represent cardiovascular risk factors and might thus be associated with retinal neovascular diseases, including among others CVRO, OIS, and DR. 
As suggested by the time interval before surgery, ranging from 16 days to 57 years, histopathological feature calculations had to be made on heterogeneous specimens, some with perfectly conserved retina morphology, whereas others had sustained severe morphological changes. However, the variety of eye diseases and the number of patients included in our study result mostly from the difficulty to access human retina specimens. 
In conclusion, our study is the first to describe CD160 expression in human eyes. CD160 endothelial expression in retinal vessels is associated with ocular neovascular diseases. Whether the expression of CD160 has a causal role in neovascularization is currently unknown and has to be evaluated. If this were to be the case, CD160 could represent an interesting target for novel anti-angiogenic therapies. Further studies are needed to evaluate the potential of anti-CD160 antibody therapy, which could represent either an alternative or a complement to VEGF-targeted medications in ocular neovascular diseases. 
Acknowledgments
The authors thank Nicole Bouland, Stéphanie Vicente, and Saviz Nasri for their help with immunohistochemistry. The authors thank Isabelle Larre and Alain Ducasse for their contribution to the surgeries performed, and Jacques Mizrahi for his help in the preparation of the manuscript. 
Supported in part by grants from ElsaLys Biotech, Lyon, France. 
Disclosure: A. Henry, ElsaLys Biotech (F); C. Boulagnon-Rombi, None; T. Menguy, ElsaLys Biotech (I, E), P; J. Giustiniani, None; C. Garbar, None; C. Mascaux, None; M. Labrousse, None; C. Milas, None; C. Barbe, None; A. Bensussan, ElsaLys Biotech (C, R), P; V. Durlach, None; C. Arndt, ElsaLys Biotech (R) 
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Figure 1
 
Overview of CD160 distribution in nonpathologic donors' eyes. CD160 red staining evidence in (A) ciliary epithelium, (B) iris sphincter muscle, (C) retina with ganglion cell layer (asterisk) and retinal microvessels (caret), and macrophages surrounding choroidal blood vessels (D). Original magnifications are indicated. Left column, scale bar: 200 μm; right column, scale bar: 50 μm. Zoom insets are displayed in the right column.
Figure 1
 
Overview of CD160 distribution in nonpathologic donors' eyes. CD160 red staining evidence in (A) ciliary epithelium, (B) iris sphincter muscle, (C) retina with ganglion cell layer (asterisk) and retinal microvessels (caret), and macrophages surrounding choroidal blood vessels (D). Original magnifications are indicated. Left column, scale bar: 200 μm; right column, scale bar: 50 μm. Zoom insets are displayed in the right column.
Figure 2
 
CD160 expression in macrophages using serial sections in nonpathologic donors' eyes. CD160-positive cells (anti-CD160) and macrophages (anti-CD68) are co-detected with red markers in ciliary body of a human healthy donor. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Figure 2
 
CD160 expression in macrophages using serial sections in nonpathologic donors' eyes. CD160-positive cells (anti-CD160) and macrophages (anti-CD68) are co-detected with red markers in ciliary body of a human healthy donor. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Figure 3
 
CD160 expression in inner retina microvessels using serial sections. Inner retina blood microvessel density marker is detected with a brown pan-endothelial marker (anti-ERG), whereas activated endothelial cells (anti-CD105), actin of pericytes (anti-α-SMA), and CD160-positive cells (anti-CD160) are all detected with red markers. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Figure 3
 
CD160 expression in inner retina microvessels using serial sections. Inner retina blood microvessel density marker is detected with a brown pan-endothelial marker (anti-ERG), whereas activated endothelial cells (anti-CD105), actin of pericytes (anti-α-SMA), and CD160-positive cells (anti-CD160) are all detected with red markers. Original magnifications are indicated. Left column, scale bars: 200 μm; right column, scale bars: 50 μm. Zoomed insets are displayed in the right column.
Table 1
 
Primary Antibodies Used for Immunohistochemistry
Table 1
 
Primary Antibodies Used for Immunohistochemistry
Table 2
 
CD160 Staining Intensity Scores in Structures of Human Donor Eyes
Table 2
 
CD160 Staining Intensity Scores in Structures of Human Donor Eyes
Table 3
 
Comparison of Clinical Characteristics and Histological Parameters in Neovascular (NV+) and Control (NV−) Groups
Table 3
 
Comparison of Clinical Characteristics and Histological Parameters in Neovascular (NV+) and Control (NV−) Groups
Supplement 1
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