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Show Neuronal Programmed Cell Death-1 Ligand Expression Regulates Retinal Ganglion Cell Number in Neonatal and Adult Mice Caroline W. Sham, MD, PhD, Ann M. Chan, MS, Jacky M. K. Kwong, PhD, Joseph Caprioli, MD, Steven Nusinowitz, PhD, Bryan Chen, BS, Janice G. Lee, MD, Nishant M. Gandhi, MD, Loise M. Francisco, PhD, Arlene H. Sharpe, MD, PhD, Ling Chen, MD, PhD, Jonathan Braun, MD, PhD, Lynn K. Gordon, MD, PhD Objectives: During mouse retina maturation, the final number of retinal ganglion cells (RGCs) is determined by highly regulated programmed cell death. Previous studies demonstrated that the immunoregulatory receptor pro-grammed cell death-1 (PD-1) promotes developmental RGC death. To identify the functional signaling partner(s) for PD-1, we identified retinal expression of PD-1 ligands and examined the effect of PD-1 ligand expression on RGC number. We also explored the hypothesis that PD-1 signaling promotes the development of functional visual circuitry. Methods: Characterization of retinal and brain programmed cell death-1 ligand 1 (PD-L1) expression were examined by immunofluorescence on tissue sections. The contribution of PD-ligands, PD-L1, and programmed cell death-1 ligand 2 (PD-L2) to RGC number was examined in PD-ligand knockout mice lacking 1 or both ligands. Retinal architecture was assessed by spectral-domain optical coherence tomography, and retinal function was analyzed by electroretinography in wild-type and PD-L1/L2 double-deficient mice. Results: PD-L1 expression is found throughout the neonatal retina and persists in adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PD-ligands, there is a significant numerical increase in RGCs (34% at postnatal day 2 [P2] and 18% in adult), as compared to wild type, and PD-ligands have redundant function in this process. Despite the increased RGC number, adult PD-L1/L2 dou-ble- knockout mice have normal retinal architecture and outer retina function. Conclusion: This study demonstrates that PD-L1 and PD-L2 together impact the final number of RGCs in adult mice and supports a novel role for active promotion of neuronal cell death through PD-1 receptor-ligand engagement. Journal of Neuro-Ophthalmology 2012;32:227-237 doi: 10.1097/WNO.0b013e3182589589 © 2012 by North American Neuro-Ophthalmology Society In the developing central nervous system (CNS), many more neurons are created than are necessary to form functional circuits. Excess neurons are eliminated by care-fully regulated apoptotic programmed cell death (PCD) in a process called developmental cell death (1). Murine post-natal retina maturation is an example of ontogenic cell death, providing a model to study PCD in the CNS (2). PCD has been particularly explored well in retinal ganglion cells (RGCs), as about half of RGCs born will die by this process (3). To survive and form functional circuitry, RGCs must project axons to the brain, synapse on target cells, and avoid elimination by synaptic pruning (2). A variety of cell surface interactions are known to positively and negatively influence this developmental sequence. Cell surface adhe-sion g-protocadherin proteins (4), neurotrophic support (5), and electrical activity (6) are all required for proper Departments of Pathology and Laboratory Medicine (CWS, JB) and Ophthalmology (AMC, JMKK, JC, SN, BC, JGL, NMG, LKG), Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; Department of Pathology (LMF, AHS), Harvard Medical School, Boston, Massa-chusetts; Department of Pathology (LMF, AHS), Brigham and Women's Hospital, Boston, Massachusetts; Department of Ophthal-mology (LC), Eye and ENT Hospital, Shanghai Medical School, Fudan University, Shanghai, China; and Ophthalmology Section (LKG), Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California. This work was supported by the UCLA Medical Scientist Training Program NIH T32 GM08042 (CWS), National Institutes of Health/ National Institute of Allergy and Infectious Diseases Grant RO1 AI021256 (CWS), JSEI Vision Science Training Grant NIH/NEI T32 EY007026 (CWS), P01AI56299 (AHS), Pujiang Talent Foundation 10PJ1401900 (LC), and National Science Foundation of China 81000381 (LC). The authors declare no conflict of interest. Address correspondence to Lynn K. Gordon, MD, PhD, Jules Stein Eye Institute, David Geffen School of Medicine at UCLA, 100 Stein Plaza, Los Angeles, CA 90095; E-mail: lgordon@mednet.ucla.edu Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 227 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. RGC survival. In contrast to these influences, molecules decorating cell processes, such as complement initiating factor C1q (7), CD3z, and MHCI (8) and neuronal pen-traxins (9), act to promote synaptic refinement. Addition-ally, the p75NTR neurotrophin (10) and programmed cell death-1 (PD-1) (11) receptors act to promote RGC death. PD-1 is a membrane-associated coinhibitory receptor, expressed on a number of hematopoietic cells including T cells, B cells, and macrophages (12) and on neurons of the CNS (11,13). In the immune system setting, PD-1 engage-ment with 1 of the 2 ligands, PD-L1 or PD-L2, acts to attenuate immune responses and thus has broad immuno-regulatory roles in peripheral tolerance, as well as immunity to chronic infections, tumors, and organ transplants (12). We previously reported that PD-1 receptor ligation pro-motes RGC apoptosis during early postnatal development and identified PD-L1 and PD-L2 gene expression through-out retina maturation (11), suggesting that either one or both PD-ligands engage functionally with PD-1 in the ret-ina. In this study, we identify the cellular expression of PD-ligand proteins and demonstrate an increased RGC number in the absence of PD-L1 and PD-L2. METHODS Animals Knockout mice were constructed in the C57BL/6 back-ground, as previously described: PD-12/2, PD-L12/2, PD-L22/2, and PD-L1/L22/2 (14-16). Animals used for each experiment are described in Table 1. Wild-type (WT) embryonic and adult age-matched C57BL/6 mice were pur-chased from Charles River Laboratory. All animal experi-ments were reviewed and approved by the University of California, Los Angeles, Chancellor's Animal Research Committee, in adherence to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Animal Perfusion Mice were deeply anesthetized with intraperitoneal sodium pentobarbital (80 mg/kg of Nembutal) and perfused trans-cardially with 10 mL of 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 5 minutes. Immunofluorescence Staining Paraformaldehyde-fixed, frozen vertical retina sections of 7-mm thickness were prepared from postnatal day 2 (P2) and adult (P56) animals. Staining and epifluorescent imaging were performed as previously described (11). Antibodies used, including PD-L2 antibodies tested, are listed in Table 2. For brain sections, adult (P56) WT male mice were perfused, brains were dissected, postfixed in 4% PFA/PBS overnight at 4°C, cryoprotected in 30% sucrose/PBS, and embedded in optical coherence tomography (OCT) medium (Sakura Finetek). Serial coronal sections of 25 mm were collected at the level of the superior colliculus (SCs) at TABLE 1. Summary of experimental animal (mice) studies showing the effect of programmed cell death-1 ligands on retinal ganglion cell number Experiment Strain Age Age Rationale Result Retinal PD-L1 expression by IF WT P2 Age-match RGC quantification Expressed in GCL cell bodies and neuroblast layer neuropil WT P56 Age-match brain expression Expressed in RGCs, bipolar cells, and Müller glia Brain PD-1 and PD-L1 expression by IF WT P56 Age-matched to reference mouse brain atlas (ref) PD-1 and PD-L1 expressed in SCs RGC quantification by IF WT P2 Peak RGC death Increased RGC number in PD-12/2 and PD-L1/L22/2 PD-12/2 PD-L1/L22/2 WT P56 Age-match brain expression Increased RGC number in PD-L1/L22/2 PD-12/2 PD-L1/L22/2 PD-L12/2 PD-L22/2 Axon counting by electron microscopy WT P28 Establish baseline for future experiments Increased RGC number in PD-L1/L22/2 PD-12/2 PD-L1/L22/2 ERG and SD-OCT WT P42 Knockout animal availability Normal outer retina function and architecture in PD-12/2 and PD-L1/L22/2 PD-12/2 PD-L1/L22/2 228 Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Bregma 24.1 to 24.4 mm; coordinates were determined using the Allen Mouse Brain reference atlas (26). Immuno-fluorescence staining was performed for PD-1 and PD-L1, using the same method as for retina sections, with the excep-tion that all slide washing was performed with agitation. Optic Nerve Axon Analysis Adult (P28) WT, PD-12/2, and PD-L1/L22/2 mice were perfused. Optic nerves were dissected, postfixed in 1% osmium tetraoxide, stained with 1% potassium ferricyanide, dehydrated in ethanol and acetone, and embedded in epoxy resin. Ultrathin (70 nm) optic nerve sections were obtained with a microtome (Reichert Ultracut) at approximately 400 mm posterior to the eye globe, placed on slotted grids, and counterstained with 1% uranyl acetate and Sato lead. Images were obtained using transmission electron micros-copy (Tecnai; FEI). For each section, 20 micrographs were taken at ·21,000 at representative optic nerve regions: cen-tral (4), midperipheral (8), and peripheral (8). No positional adjustments were made. Images included blood vessels and glial cells. Micrographs were digitized using a 2K charge-coupled device (Photometrics Inc). Analysis was performed according to published methods (27). Semi-thin (1 mm) optic nerve sections were stained with 1% toluidine blue (Sigma Aldrich). To determine axon density, axons located within a counting frame (8 · 8 mm) and intersecting the upper and right frame edges were counted manually in a blinded manner. Mean axon density was calculated as total axons divided by total area. Nissl Staining To stain all neuronal cell bodies, every fourth coronal brain section was Nissl stained as previously described (28), with 0.1% cresyl violet (Sigma-Aldrich). Structures were local-ized using Nissl stains in conjunction with the Allen Mouse Brain reference atlas (26). Imaging and Quantification of Immunofluorescence Staining Retina sections were imaged at ·400 magnification, yielding an image width of 262 mm. Quantification of ganglion cell layer (GCL) cell types, RGCs and displaced amacrine cells, and statistical analysis were performed as previously described (11). Semithin optic nerve sections were imaged at ·400 magnification, and cross-sectional area was mea-sured by outlining the nerve border with image acquisition software (DP2-BSW, Olympus) and repeated on 3 consec-utive sections. Brain sections were imaged at ·100 magnifi-cation. All images were obtained with light or epifluorescent microscopy (BX51, Olympus). Retina Architecture Following euthanasia, eyes were rapidly enucleated and immediately fixed in 4% PFA. Paraffin embedding, sec-tioning at 4 mm thickness, and hematoxylin and eosin (H&E) staining were performed by UCLA's Translational Pathology Core Laboratory (TPCL). Images were obtained at ·400 magnification by light microscopy, as above. Electroretinogram Adult (6 week-old) WT and PD-L1/L22/2 male mice were dark-adapted overnight for full-field flash electroretinogram (ERG) recording as previously described (29). Briefly, ERGs were recorded using a gold electrode touching the corneal surface referenced to a gold electrode in the mouth. Scotopic rod-mediated responses were obtained to blue TABLE 2. Primary antibodies used for immunofluorescence staining (13,17-25). Antibody Dilution Source Anti-mouse PD-1 (clone 29F.1A12, rat IgG2a) (17) 1:200 Gift of Gordon Freeman, MD, PhD (Boston, MA) Anti-PD-L1/CD274 (rabbit polyclonal) (17) 1:750 Lifespan Biosciences, #LS-B480 Anti-mouse Brn3a (clone 5A 3.2, mouse IgG1) (18) 1:200 Millipore/Chemicon Anti-mouse AP2a (clone 3B5, mouse IgG2b) (19) 1:20 Developmental Studies Hybridoma Bank Anti-Calbindin D28K (clone CB-955 (formerly CL-300), mouse IgG1) (20) 1:1000 Sigma-Aldrich Anti-PKCa (clone H-7, mouse IgG1) (21,22) 1:4000 Santa Cruz Biotechnology Anti-G protein Goa (clone 2A, mouse IgG1) (23) 1:1000 Millipore/Chemicon Anti-glutamine synthetase (clone GS-6, mouse IgG2a) (24,25) 1:1000 Millipore/Chemicon Species- or isotype-specific Alexa Fluor 488 or 594 1:1000 Invitrogen Anti-mouse PD-L2 (clone TY25, rat IgG2a) (13,17) - eBioscience Anti-mouse PD-L2 (clone 19G.4H3, rat IgG2b) - Gift of Gordon Freeman, MD, PhD (Boston, MA) Anti-human PD-L2 (clone 24F.7G12, mouse IgG2a) - Gift of Gordon Freeman, MD, PhD (Boston, MA) Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 229 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. FIG. 1. PD-L1 expression in the neonatal mouse retina and brain by immunofluorescence staining. A. PD-1 (green) and PD-L1 (red) expression at P2 in the central retina, at the level of the optic nerve. B. PD-L1 (green) and the following retinal cell type markers (all in red): Brn3a for RGCs, AP2a for amacrine cells, Calbindin D28K (D28K) for horizontal cells, PKCa for rod bipolar cells, Goa for optic nerve bipolar cells, glutamine synthetase (GS) for Müller glia. White arrows indicate cells shown at higher magnification inset into each panel. All images were taken at the level of the optic nerve, at the midpoint between the optic nerve head (ONH) and periphery. C. Coronal section of the superior colliculus at Bregma 24.3 mm, after Allen Mouse Brain Atlas coordinates. Panels from left to right show: 1) Nissl stain, 2) PD-1 (green) and PD-L1 (red) expression by immunofluorescence, with DAPI counterstain (blue), 3) same as second image, without DAPI, and 4) secondary only negative control, where PD-1 and PD-L1 primary antibodies were omitted, and the white box indicates area shown at higher 230 Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. flashes (Wratten 47A, lmax = 470 nm, Eastman Kodak). After a 5-minute light adaptation, cone-mediated responses were recorded. Analysis was performed using custom soft-ware (29). Amplitude of dark-adapted b wave was plotted against flash intensity and resulting intensity-response func-tion was fit with a Naka-Rushton equation to derive Vmax (maximum saturated amplitude) and k (semisaturation intensity.) Spectral-Domain Optical Coherence Tomography Spectral-domain OCT (SD-OCT) imaging (Bioptigen) was performed. Animals were anesthetized and left pupils dilated with atropine (1% wt/vol atropine sulfate ophthal-mic solution). Recordings were taken with a 50° field of view, yielding a 1.5-mm-diameter image. En face view C-scans were recorded with the optic nerve head (ONH) centered in the image and consisted of 100 two-dimensional B-scans, which in turn consisted of 800 one-dimensional A-scans. Total scan time was approximately 5 seconds. Ret-inal layers were measured at 0.2 mm eccentricity from the ONH with analysis software (Software Suite; Bioptigen). At 0.6 mm eccentricity from the ONH, in both temporal and superior orientations, no differences were noted between WT and PD-L1/L22/2 total retina thickness (P = 0.83 and 0.43, respectively), thus representative central measure-ments are reported. Retina layer analysis was performed referencing published mouse OCT images (30). "Inner ret-ina" was defined as the distance from the inner limiting membrane to the inner nuclear layer (INL), and "outer retina" was defined as the distance from the outer plexiform layer (OPL) to the retinal pigment epithelium. Statistical Analysis A graphing program (Prism 5; GraphPad) was used for data visualization and statistical analysis. For immunofluores-cence quantification, optic nerve axon counts, and retina architecture analysis, a Student t test was used to compare groups. For ERG data, mean and standard error of the mean (SEM) were plotted at each frequency, and Boltz-mann sigmoidal curves were fitted to the data. For scotopic ERG, an unpaired t test was used to compare Vmax and k between WT and PD-L1/L22/2 mice. For timing and photopic ERG amplitude, 2-way analysis of variance was used to evaluate difference between WT and PD-L1/ L22/2 mice. RESULTS PD-L1 Is Expressed in RGCs, Bipolar Interneurons, and Müller Glia of the Mature Mouse Retina Retinal expression of Pdcd1lg1 (PD-L1 gene) has been reported (11), identifying it as a neuronal PD-ligand. To determine potential locations for PD-1 ligation, PD-L1 protein localization was characterized in the mouse retina and SCs, the major target of rodent optic nerve axons (31). During postnatal retina maturation, a wave of RGC death occurs, peaking at P2-P5 (32). At P2, coincident with this peak, PD-L1 expression is found in the GCL in a non-nuclear distribution and in the neuroblast layer distributed on the neuropil colocalizing with PD-1 receptor expression (Fig. 1A). In addition, PD-L1 is expressed in cell processes of the incipient inner plexiform layer (Fig. 1A). In the mature retina, PD-L1 expression persists and is restricted to the inner retina layers (Fig. 1B). Since PD-L1 was found across the INL, inner plexiform layer, and GCL, immunofluores-cence (IF) double labeling with GCL and INL cell type markers was performed. PD-L1 was found to be expressed in many but not all cell types. PD-L1 expression did not colocalize with 2 types of interneurons: AP2a-positive ama-crine cells and calbindin D28K-positive horizontal cells (Fig. 1B). However, PD-L1 expression did colocalize with 2 neuronal cell types: Brn3a-positive RGCs, and both pro-tein kinase C (PKC) a-positive rod bipolar axon terminals and Goa-positive optical nerve (ON) bipolar dendrites, and in addition, 1 glial cell type: glutamine synthetase-positive Müller cell bodies and processes (Fig. 1B). Within the OPL, PD-L1 expression was observed at ON cone bipolar termi-nals of cone pedicle synapses, as evidenced by colocalization with the ON bipolar marker Goa, which labels both ON cone and rod bipolars (33) but not with the rod bipolar marker PKCa. Although neuronal Pdcd1lg2 (PD-L2 gene) expression has been previously described (11), PD-L2 pro-tein expression was below the threshold of detection by IF using several anti-PD-L2 antibodies (Table 2). Since RGCs express both PD-1 and PD-L1 and extend axons to the brain via the optic nerve, we next explored expression of the PD-1 signaling system in the superficial layers of the SCs, the location to which the majority of mouse RGCs project (31). Both PD-1 and PD-L1 are expressed across the SCs, including the stratum opticum, which con-tains afferent optic nerve fibers of RGCs (Fig. 1C, D). This magnification in D. D. Higher magnification of SCs. Panels from left to right show: 1) PD-L1 (red) expression, 2) PD-1 (green) expression, 3) PD-L1 and PD-1 expression colocalization (yellow), and 4) PD-L1 and PD-1 expression with DAPI (blue) counterstain. Scale bars = 50 mm (A, B, D) and 200 mm (C). For each expression pattern shown, 3 animals were examined. Nuclei were visualized with DAPI (blue) counterstain. Yellow represents colocalization of PD-1 and PD-L1. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IPLi, incipient inner plexiform layer; NBL, neuroblast layer; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; op, stratum opticum; PAG, periaqueductal gray; SCs, superior colliculus superficial layers; SCd, superior colliculus, deep layers; sg, stratum griseum superficiale; zo, stratum zonale. Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 231 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. expression pattern supports the possibility that PD-1:PD-L1 engagement can also occur at the axon terminals of RGCs. Within the SCs, higher resolution imaging will be necessary to determine the subcellular localization of PD-1 and PD-L1 and also the spatial relationship of cells expressing each molecule. Absence of Both PD-Ligands Results in Persistent Increase in RGC Number To test the importance of PD-L1 GCL expression during peak RGC apoptosis, we next tested the functional role of PD-ligands in developmental RGC death and predicted that similar to that reported previously for PD-1 receptor knockout (PD-12/2) mice (11), the genetic absence of PD-ligands would increase RGC survival in the neonatal period. GCL layer compartments were assessed during the development of the PD-L1 and PD-L2 double-knockout (PD-L1/L22/2) mouse retina, in comparison to WT con-trols, by immunofluorescent staining for the RGC and amacrine cell markers Brn3a and AP2a, respectively (Fig. 2A). At P2, the PD-L1/L22/2 retina has a significant increase in RGC number compared to WT (34 ± 2.9%; P = 0.006; Fig. 2B), measured by the number of Brn3a-positive nuclei per millimeter. Given that these observations for PD-L1/L22/2 mirror the previously reported PD-12/2 phenotype (11), we were surprised to observe that in con-trast to the PD-12/2 adult retina, PD-L1/L22/2 adult ret-inas have a significant increase in RGC number compared to WT (18 ± 5.4%; P = 0.04; Fig. 2C). To confirm this increase, optic nerve axon counting was performed and showed that adult PD-L1/L22/2 had significantly increased mean axon density, as compared to WT (Fig. 3C; P = 0.01). To determine which PD-ligand contributes to the adult phenotype, we examined retinas from single PD-ligand knockouts, PD-L12/2, and PD-L22/2 and found that nei-ther had a difference in RGC number as compared to WT (P = 0.07 and 0.14, respectively; Fig. 2C). A significant number of displaced amacrine cells also reside in the GCL FIG. 2. Increased RGCs in the neonatal and adult retina of PD-L1/L22/2 mice. A. Immunofluorescence staining for Brn3a (green) and AP2a (red) in P2 and adult (P56) retina of WT, PD-12/2, and PD-L1/L22/2 mice, with DAPI nuclear counterstain (blue). Scale bars = 50 mm. B. Quantification of RGC number (Brn3a/mm) in WT, PD-12/2 mice at P2. Quantification of RGC number (Brn3a/mm, B, C) and amacrine cell number (AP2/mm, D) in the adult retina. Three mice per strain were analyzed at each time point. For each quantified readout, the mean and standard error of the mean were plotted. *Significant P value in comparing each knockout to WT; *P # 0.04, **P = 0.006. 232 Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. (34), so we also examined amacrine cell number in the absence of PD-1 ligation. In the adult retina, there were no significant differences in amacrine cell number between any of the knockout strains, as compared to WT (P $ 0.4 for all knockout strains; Fig. 2D). These findings suggest that PD-L1 and PD-L2 have redundant roles in RGC cell death, and their concurrent absence is associated with a per-sistent increase in RGC number of the mature retina. Normal Gross Retina Architecture and Function in the PD-L1/L22/2 Adult Mouse The observed increase in the numbers of RGCs in the PD-L1/L22/2 animals prompted further evaluation of the functional significance of this observation. We first explored if the normal highly organized, laminated retina structure was present in the mature adult PD-L1/L22/2 retina by H&E stain and found the gross architecture to be normal, as compared to WT (Fig. 4A). Retina architecture of 6-week-old PD-L1/L22/2 and WT mice was visualized in vivo by SD-OCT. Reconstructed vertical sections (Fig. 4B, D) and en face fundus images (Fig. 4C, E) both appeared grossly normal in PD-L1/L22/2 mice (Fig. 4D, E). There were no significant differences in inner or outer retina layer thickness between PD-L1/L22/2 and WT (Fig. 4F, G), and concor-dantly, no significant differences when either the inner (Figs. 3, 4H) or outer (Figs. 3, 4I) retina was broken down to nuclear and nonnuclear layer components. Since we observed PD-L1 expression colocalizing with PKCa-positive rod bipolar axon terminals and Goa-positive ON bipolar dendrites, we next investigated middle and outer retina function using standard ERG. Representative ERGs recorded from PD-L1/L22/2 mice are shown in Figure 5A. No statistically significant differences in rod-mediated retinal function were observed between PD-L1/L22/2and WT adult mice (Fig. 5B, C), although the PD-L1/L22/2 dem-onstrated a weak trend in the direction of reduced response amplitude (Vmax) and photoreceptor sensitivity (k) (P = 0.68 for Vmax and P = 0.07 for k; Fig. 5B). Correspondingly, there were no statistically significant differences in cone-mediated function (Fig 5C; P = 0.46). Finally, we analyzed the timing of peak components of the rod-mediated and cone-mediated ERGs and found no statistically significant differences (P = 0.16 and 0.71, respectively). These data collectively demon-strate that retinal structure and outer retinal function are both normal in adult PD-L1/L22/2 mice. DISCUSSION Developmental RGC death in mice is a highly regulated process, generating the correct number of surviving RGCs FIG. 3. Optic nerve axon analysis in PD-1 and PD-ligand knockout mice. A. Electron micrographs (·21,000) of the central region. Scale bars are 1 mm. B. Optic nerve cross-sectional area: WT (53,624 ± 982), PD-12/2 (52,865 ± 2137), and PD-L1/ L22/2 (48,008 ± 363). C. Total axon number: WT (72,107 ± 2113), PD-12/2 (68,141 ± 3574), PD-L1/L22/2 (81,658 ± 3356) (*P = 0.05). D. Axon density: WT (1.35 ± 0.057), PD-12/2 (1.29 ± 0.089), PD-L1/L22/2 (1.70 ± 0.059), *P = 0.01 (WT vs PD-L1/L22/2), P = 0.02 (PD-12/2 v. PD-L1/L22/2). E. Schematic representation of location for 20 micrographs taken from each nerve. Central region includes the 4 squares at the center, peripheral region includes the 8 outer most squares, and midperipheral region includes the 8 remaining squares. F. Central region axon number: WT (17,901 ± 582), PD-12/2 (17,337 ± 1409), PD-L1/L22/2 (20,158 ±1081). G. Central region axon density: WT (0.334 ± 0.015), PD-12/2 (0.329 ± 0.031), PD-L1/L22/2 (0.419 ± 0.020), *P = 0.03. Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 233 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. FIG. 4. Retina architecture in PD-1 and PD-ligand knockout mice. A. Hematoxylin and eosin stain showing gross retina architecture of WT and PD-L1/L22/2 adult mice. Representative B-scan images at the level of the optic nerve head showing a retina cross-section from WT (B) and PD-L1/L22/2 (D) mice. White arrows indicate Bergmeister papilla, a remnant of fetal hyaloid vasculature commonly observed in C57BL/6 mice on spectral-domain optical coherence tomographic imaging. Representative en face C-scans showing fundus images from WT (C) and PD-L1/L22/2 (E) mice, with the horizontal green line indicating orientation of respective B-scans. F. Cropped view of WT (B) and PD-L1/L22/2 (D) B-scans, labeled to indicate retinal layers and landmarks used for quantification of layer thickness. Quantification of inner, outer, and total retina thickness (G), and nuclear layer and nonnuclear layer components of the inner or outer retina (H or I, respectively). GC/IPL, ganglion cell and inner plexiform layers; INL, OPL, ONL, as defined above in Fig. 1; IS/OS, photoreceptor inner and outer segments; OLM, outer limiting membrane; ns, not significant; RPE/ CC, retinal pigment epithelium and choriocapillaris. Scale bars = 50 mm (A), 100 mm (both x- and y-axes, B, D, F), or 200 mm (C, E). 234 Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. necessary to form functional connections with the optic tectum and visual cortex (3). According to the neurotrophic hypothesis, RGC death results from the lack of available extracellular neurotrophic support (35). The discovery of a neurotrophic death-promoting signal, nerve growth factor engagement with p75NTR, introduced the additional hypothesis that soluble neurotrophic ligands can provide an active death trigger for retinal neurons (36). Our work introduces an important refinement to this idea, where receptor engagement with a cell-associated ligand can also promote developmental RGC death. We refer to this hypothesis as "active selection" of neuronal elimination. PD-1 ligation blockade in vitro was found to decrease RGC apoptosis during peak RGC death (11), supporting a role for PD-1 receptor-ligand interaction in this process. This work, summarized in Table 1, presents strong evidence that PD-1 ligands, PD-L1, and PD-L2 are the functional neuronal ligands for PD-1, acting within the GCL to influence RGC-specific developmental cell death. Further-more, PD-L1 is expressed in the developing and mature retina, suggesting potential for engagement with PD-1 receptor during these times. We hypothesize several poten-tial locations for PD-1 ligation within the retina. First, developmental ligation between GCL cell types could lead to cell death. Second, in the mature retina, PD-L1 on rod bipolar could potentially interact with PD-1 on AP2a-positive glycinergic AII amacrine cells (11,37). Third, PD-L1 on Müller glia, which ensheath all retinal cell types (38), could engage PD-1-positive RGCs and/or amacrine cells (11). PD-L1 expression at cone bipolar terminals sug-gests a potential role for PD-L1 in cone pathway visual responses. The presence of PD-L1 in the mature retina, along with PD-1 receptor (11), suggests additional roles for PD-1 ligation beyond developmental RGC cell death. Since the PD-L1/L22/2 retina has normal gross function by ERG, PD-ligands are not required for wiring functional circuitry within the retina. Further studies are necessary to assess whether PD-1 ligation has a role in defining retina-brain projections, perhaps through synaptic refinement, sim-ilar to other cell surface immune molecules in the CNS (8). Perturbations of neurotrophic support (39-41), neuro-trophic death (10,42), or PD-1 signaling (11) cause tran-sient changes in RGC survival in vivo. However, none of these cell surface survival modulators are sufficient to influence the final number of RGCs in the mature adult retina. Here, we provide the first evidence for a receptor-ligand signal having a more persistent effect on RGC num-bers. Genetic absence of both PD-ligands yields an 18% increase in RGCs in the mature retina. However, this dif-ference is modest compared to inactivation of intracellular apoptotic machinery, where RGC numbers can be increased 50%-226%, as reviewed (3). The observation that the absence of both PD-ligands had a persistent effect on RGC survival, while the absence of FIG. 5. PD-L1/L22/2 mice have normal outer retina function. A. Representative raw electroretinogram traces for WT and PD-L1/ L22/2 mice. Maximum b-wave amplitude in PD-L1/L22/2 (n = 3, open triangles) and WT (n = 5, open circles) mice, over a range of stimulus intensities, for both scotopic (B) and photopic (C) conditions. Amplitudes are presented as mean ± standard error of the mean. Scotopic response characteristics for WT: Vmax = 883.6 ± 60.1 mV, k = 0.055 ± 0.003 c-s/m2; and for PD-L1/L22/2: Vmax = 832.5 ± 122.1 mV, k = 0.066 ± 0.004 c-s/m2. Sham et al: J Neuro-Ophthalmol 2012; 32: 227-237 235 Basic Science in Neuro-Ophthalmology Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. PD-1 receptor only has a transient early developmental effect (11) led us to hypothesize the presence of another neuronal PD-ligand receptor functioning during the final phase of developmental cell death. One strong candidate is another B7 family costimulatory ligand B7-1, shown to interact with PD-L1 (but not PD-L2) in lymphocytes, with ligation resulting in similar immunoinhibitory effects as PD-1 ligation (43). Defining additional PD-ligand interact-ing molecules in the retina, including B7-1, and which elicit an "active selection" death signal are questions for future investigation. PD-L1 expression in Müller glia is intriguing, as this cell type has both protective and detrimental roles in retinal disease (44). We hypothesize that PD-1 ligation could act to downregulate Müller glia activity in settings of retinal damage. Although PD-L1 expression was characterized in this study, we were unable to reliably detect PD-L2 protein expression by immunofluorescence in the developing and mature mouse retina. Genetic evidence that PD-L2 is func-tionally redundant with PD-L1 in promoting RGC death, in combination with identification of PD-L2 gene expres-sion, will require further characterization of neuronal PD-L2 protein expression. In summary, our work solidifies the role of PD-ligands in RGC developmental cell death and provides impetus to explore the role of the PD-1 sig-naling pathway in neurodegenerative retinal disease. ACKNOWLEDGMENTS The authors thank Dr. Xian-Jie Yang for valuable dis-cussions throughout this work, Dr Gordon Freeman for the gift of the PD-1 monoclonal antibody, Marianne Cilluffo at the UCLA Brain Research Institute Electron Microscopy core laboratory for extensive tissue processing and imaging assistance, and the UCLA Translational Pathology Core Laboratory for tissue processing assistance. The 3B5 mono-clonal antibody, developed by T. 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