Lions Eye Institute

Abstract Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40–50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes 1 . Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel 2 ) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10–20% (14–24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries.

Primary open-angle glaucoma (POAG), is a heritable common cause of blindness world-wide. To identify risk loci, we conduct a large multi-ethnic meta-analysis of genome-wide association studies on a total of 34,179 cases and 349,321 controls, identifying 44 previously unreported risk loci and confirming 83 loci that were previously known. The majority of loci have broadly consistent effects across European, Asian and African ancestries. Cross-ancestry data improve fine-mapping of causal variants for several loci. Integration of multiple lines of genetic evidence support the functional relevance of the identified POAG risk loci and highlight potential contributions of several genes to POAG pathogenesis, including SVEP1, RERE, VCAM1, ZNF638, CLIC5, SLC2A12, YAP1, MXRA5, and SMAD6. Several drug compounds targeting POAG risk genes may be potential glaucoma therapeutic candidates.

Primary angle-closure glaucoma (PACG) is a common cause of blindness. Angle closure is a fundamental pathologic process in PAGC. With the development of imaging devices for the anterior segment of the eye, a better understanding of the pathogenesis of angle closure has been reached. Aside from pupillary block and plateau iris, multiple-mechanisms are more common contributors for closure of the angle such as choroidal thickness and uveal expansion, which may be responsible for the presenting features of PACG. Recent Genome Wide Association Studies identified several new PACG loci and genes, which may shed light on the molecular mechanisms of PACG. The current classification systems of PACG remain controversial. Focusing the anterior chamber angle is a principal management strategy for PACG. Treatments to open the angle or halt the angle closure process such as laser peripheral iridotomy and/or iridoplasty, as well as cataract extraction, are proving their effectiveness. PACG may be preventable in the early stages if future research can identify which kind of angles and/or persons are more likely to benefit from prophylactic treatment. New treatment strategies like adjusting the psychological status and balancing the sympathetic-parasympathetic nerve activity, and innovative medicines are needed to improve the prognosis of PACG. In this review, we intend to describe current understanding and unknown aspects of PACG, and to share the clinical experience and viewpoints of the authors.

BACKGROUND: Retinal prosthesis systems (RPS) are a novel treatment for profound vision loss in outer retinal dystrophies. Ideal prostheses would offer stable, long-term retinal stimulation and reproducible spatial resolution in a portable form appropriate for daily life. METHODS: We report a prospective, internally controlled, multicentre trial of the Argus II system. Twenty-eight subjects with light perception vision received a retinal implant. Controlled, closed-group, forced-choice letter identification, and, open-choice two-, three- and four-letter word identification tests were carried out. RESULTS: The mean±SD percentage correct letter identification for 21 subjects tested were: letters L, T, E, J, F, H, I, U, 72.3±24.6% system on and 17.7±12.9% system off; letters A, Z, Q, V, N, W, O, C, D, M, 55.0±27.4% system on and 11.8%±10.7% system off, and letters K, R, G, X, B, Y, S, P, 51.7±28.9% system on and 15.3±7.4% system off. (p<0.001 for all groups). A subgroup of six subjects was able to consistently read letters of reduced size, the smallest measuring 0.9 cm (1.7°) at 30 cm, and four subjects correctly identify unrehearsed two-, three- and four-letter words. Average implant duration was 19.9 months. CONCLUSIONS: Multiple blind subjects fitted with the Argus II system consistently identified letters and words using the device, indicating reproducible spatial resolution. This, in combination with stable, long-term function, represents significant progress in the evolution of artificial sight.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1(+) NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1(-) NK cells are precursors of KLRG1(+) NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1(+) NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45(-/-) mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.

Although available empirically derived and theoretical formulas perform adequately for eyes of average axial length, both have been shown to be deficient for eyes that have unusually short and long axial lengths. I developed a formula based on a theoretical model eye in which anterior chamber depth is related to axial length and keratometry. A relationship between the A-constant and a "lens factor" is also used to determine anterior chamber depth. The location of the intraocular lens' principle planes of refraction is retained as a relevant variable in the formula, and the user need not know the material and construction of the lens and or its constant. I compared the new formula with the SRK II, Holladay, and SRK/T formulas in a group of 100 unselected patients and in selected subgroups of patients with average, short, and long axial lengths. The new formula was significantly more accurate than the other third-generation formulas and maintained its accuracy in the subgroups. The formula can be described as universal because it can be used for different lens styles and for eyes with short, medium, and long axial lengths.

The knowledge on the genetic background of refractive error and myopia has expanded dramatically in the past few years. This white paper aims to provide a concise summary of current genetic findings and defines the direction where development is needed. We performed an extensive literature search and conducted informal discussions with key stakeholders. Specific topics reviewed included common refractive error, any and high myopia, and myopia related to syndromes. To date, almost 200 genetic loci have been identified for refractive error and myopia, and risk variants mostly carry low risk but are highly prevalent in the general population. Several genes for secondary syndromic myopia overlap with those for common myopia. Polygenic risk scores show overrepresentation of high myopia in the higher deciles of risk. Annotated genes have a wide variety of functions, and all retinal layers appear to be sites of expression. The current genetic findings offer a world of new molecules involved in myopiagenesis. As the missing heritability is still large, further genetic advances are needed. This Committee recommends expanding large-scale, in-depth genetic studies using complementary big data analytics, consideration of gene-environment effects by thorough measurement of environmental exposures, and focus on subgroups with extreme phenotypes and high familial occurrence. Functional characterization of associated variants is simultaneously needed to bridge the knowledge gap between sequence variance and consequence for eye growth.

// Peter Johansson 1 , Lauren G. Aoude 1 , Karin Wadt 2 , William J. Glasson 3 , Sunil K. Warrier 3 , Alex W. Hewitt 4, 5 , Jens Folke Kiilgaard 6 , Steffen Heegaard 6, 7 , Tim Isaacs 5 , Maria Franchina 5 , Christian Ingvar 8 , Tersia Vermeulen 9 , Kevin J. Whitehead 10 , Christopher W. Schmidt 1 , Jane M. Palmer 1 , Judith Symmons 1 , Anne-Marie Gerdes 2 , G&ouml;ran J&ouml;nsson 8 , Nicholas K. Hayward 1 1 QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia 2 Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark 3 The Terrace Eye Centre, Brisbane, QLD, Australia 4 Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia 5 Lions Eye Institute, University of Western Australia, Perth, WA, Australia 6 Department of Ophthalmology, Rigshospitalet-Glostrup Hospital, University of Copenhagen, Copenhagen, Denmark 7 Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark 8 Department of Clinical Sciences, Lund University, Lund, Sweden 9 The Royal Perth Hospital, Perth, WA, Australia 10 Sullivan Nicolaides Pathology, Brisbane, QLD, Australia Correspondence to: Peter Johansson, e-mail: Peter.Johansson@qimrberghofer.edu.au Keywords: uveal melanoma, recurrent mutation, PLCB4, copy number variation, structural variants Received: September 14, 2015&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: November 26, 2015&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: December 14, 2015 ABSTRACT Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ , GNA11 , EIF1AX , SF3B1 and BAP1 . To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ , consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm's canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.

Purpose: To evaluate the impact of image magnification correction on superficial retinal vessel density (SRVD) and foveal avascular zone area (FAZA) measurements using optical coherence tomography angiography (OCTA). Methods: Participants with healthy retinas were recruited for ocular biometry, refraction, and RTVue XR Avanti OCTA imaging with the 3 × 3-mm protocol. The foveal and parafoveal SRVD and FAZA were quantified with custom software before and after correction for magnification error using the Littman and the modified Bennett formulae. Relative changes between corrected and uncorrected SRVD and FAZA were calculated. Results: Forty subjects were enrolled and the median (range) age of the participants was 30 (18-74) years. The mean (range) spherical equivalent refractive error was -1.65 (-8.00 to +4.88) diopters and mean (range) axial length was 24.42 mm (21.27-28.85). Images from 13 eyes were excluded due to poor image quality leaving 67 for analysis. Relative changes in foveal and parafoveal SRVD and FAZA after correction ranged from -20% to +10%, -3% to +2%, and -20% to +51%, respectively. Image size correction in measurements of foveal SRVD and FAZA was greater than 5% in 51% and 74% of eyes, respectively. In contrast, 100% of eyes had less than 5% correction in measurements of parafoveal SRVD. Conclusions: Ocular biometry should be performed with OCTA to correct image magnification error induced by axial length variation. We advise caution when interpreting interocular and interindividual comparisons of SRVD and FAZA derived from OCTA without image size correction.

Fish catch color with rods Vertebrates are typically thought to have a consistent system for processing light, in which multiple cone opsins permit color vision during the day, but a single rod opsin provides only monochrome vision in the dark. Musilova et al. analyzed more than 100 deep-sea fish genomes and found a previously unknown proliferation of rod opsin genes that generate rod opsin photopigments that are tuned to different wavelengths of light. These receptors may allow the fish to perceive bioluminescent signals that pervade their deep-sea environment. These results reveal a previously undescribed visual system that allows for color vision in the dark. Science , this issue p. 588

OBJECTIVE: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

AIM: To determine the sub-macular Bruch's membrane (BrM) macrophage count and the choroidal and BrM macrophage immunophenotype in normal eyes and in eyes with early and advanced age-related macular degeneration (AMD). METHODS: BrM macrophages were counted in 125 human eyes (normal, normal aged, early AMD and geographical atrophy), and CD68 and inducible nitric oxide synthase (iNOS) immunohistochemistry was performed on 16 human eyes (normal, normal aged, early AMD, geographical atrophy and disciform scarring). All eyes were examined clinically ante mortem. Results were correlated with histopathological features, including basal laminar deposit and membranous debris, and with clinical fundus appearance. RESULTS: CD68(+) macrophages were found in the choroid of normal human eyes, and did not express iNOS. Expression of iNOS by choroidal macrophages (as well as endothelial cells and pericytes) was associated with: (1) recruitment of macrophages to BrM in early AMD eyes with soft drusen or thick continuous basal laminar deposit, corresponding to clinically detectable soft drusen or pigment changes; and (2) active disciform scarring. iNOS expression was absent in BrM macrophages, suggesting immunomodulatory differences between the choroid and BrM. The highest BrM macrophage counts were found in eyes with subclinical choroidal neovascularisation. CONCLUSION: The presence of extracellular deposits (soft drusen and thick continuous basal laminar deposit) is associated with macrophage recruitment to BrM and alteration in the immunophenotype of resident choroidal macrophages.

PURPOSE: To measure the tissue pressure gradient through the optic disk and to determine the relationship between intraocular, cerebrospinal fluid, and retrolaminar tissue pressures. The relationship of optic nerve subarachnoid space pressure to intracranial cerebrospinal fluid pressure also was explored. METHODS: Micropipettes coupled to a pressure transducer were passed through pars plana and vitreous to enter the optic disk in the anesthetized dog. Using a micromanipulator, pipettes penetrated the optic disk in steps while pressure measurements were taken. In some animals, pipettes also were passed into the optic nerve subarachnoid space. Lateral ventricle cerebrospinal fluid pressure, intraocular pressure, and arterial blood pressure were measured concurrently, and the effect of raising CSF pressure was explored. RESULTS: Retrolaminar tissue pressure was largely dependent on the surrounding cerebrospinal fluid pressure, which was on average 8.6 +/- 3.5 mm Hg (SD, n = 8) higher, and was independent of intraocular pressure. Most (85% +/- 15% [SD, n = 8]) of the pressure drop between intraocular pressure and retrolaminar pressure occurred across the anterior 400 microns of disk tissue. When the intraocular pressure was 21 mm Hg and the cerebrospinal fluid pressure was zero, retrolaminar tissue pressure averaged 7 mm Hg and the translaminar pressure gradient was 3.08 +/- 0.29 mm Hg/100 microns tissue (SD, n = 3). Optic nerve subarachnoid space pressure was equivalent to lateral ventricular pressure. CONCLUSIONS: These results show that cerebrospinal fluid pressure largely determines retrolaminar tissue pressure; hence, along with intraocular pressure, it is of major importance in setting the translaminar tissue pressure gradient. Results also demonstrate hydrostatic continuity between the optic nerve subarachnoid space and the lateral ventricle. That the translaminar pressure gradient can vary independently of intraocular pressure may be of importance in understanding the pathophysiology of glaucoma.

PURPOSE: To measure the effects of cerebrospinal fluid pressure (CSFp) on retrolaminar tissue pressure (RLTp) and the translaminar pressure gradient (TLPG), particularly at low CSFp, which is the normal situation in erect posture. METHODS: Micropipettes coupled to a servonull pressure system were passed into eyes of anesthetized dogs to the optic disc and advanced in steps through the lamina cribrosa to the optic nerve subarachnoid space (ONSAS), while pressure measurements were taken. Cerebrospinal fluid pressure and intraocular pressure (IOP) were monitored and controlled. The TLPG was measured at varying IOPs and CSFps. The RLTp and ONSAS pressure (ONSASp) were measured at varying CSFps. In separate experiments, the optic nerve dura was incised, and pressure measurements were taken across the pia mater. RESULTS: The TLPG was strongly correlated to the difference between IOP and CSFp (r=0.93; n=18) when CSFp was more than zero. Mean RLTp was 3.7+/-0.2 mm Hg (SEM; n=15) when CSFp was 0 mm Hg. The ONSASp and RLTp were largely dependent on the presence of CSFp higher than break point pressures of -0.5 mm Hg and 1.33 mm Hg, respectively. However, below these break points, RLTp (slope 0.07) and ONSASp (slope 0.18) were little influenced by CSFp. Separate measurements across the pia mater revealed that 95% of the pressure drop occurred within 100 microm of the pial surface. CONCLUSIONS: The TLPG and RLTp are dependent on CSFp when CSFp is more than -0.5 mm Hg. Below this level, there is no hydrostatic continuity between the intracranial and optic nerve subarachnoid space. In this range, RLTp is stable and is little influenced by CSFp changes.

In craniates, opsin-based photopigments expressed in the eye encode molecular 'light sensors' that constitute the initial protein in photoreception and the activation of the phototransduction cascade. Since the cloning and sequencing of the first vertebrate opsin gene (bovine rod opsin) nearly 30 years ago (Ovchinnikov Yu 1982, FEBS Letters, 148, 179-191; Hargrave et al. 1983, Biophysics of Structure & Mechanism, 9, 235-244; Nathans & Hogness 1983, Cell, 34, 807-814), it is now well established that variation in the subtypes and spectral properties of the visual pigments that mediate colour and dim-light vision is a prevalent mechanism for the molecular adaptation to diverse light environments. In this review, we discuss the origins and spectral tuning of photopigments that first arose in the agnathans to sample light within the ancient aquatic landscape of the Early Cambrian, detailing the molecular changes that subsequently occurred in each of the opsin classes independently within the main branches of extant jawed gnathostomes. Specifically, we discuss the adaptive changes that have occurred in the photoreceptors of craniates as they met the ecological challenges to survive in quite differing photic niches, including brightly lit aquatic surroundings; the deep sea; the transition to and from land; diurnal, crepuscular and nocturnal environments; and light-restricted fossorial settings. The review ends with a discussion of the limitations inherent to the 'nocturnal-bottleneck' hypothesis relevant to the evolution of the mammalian visual system and a proposition that transition through a 'mesopic-bottleneck' may be a more appropriate model.

Effective vaccine adjuvants must induce expression of major histocompatibility (MHC) class II proteins and the costimulatory molecule CD86 on dendritic cells (DCs). However, some adjuvants elicit production of cytokines resulting in adverse inflammatory consequences. Development of agents that selectively increase MHC class II and CD86 expression without triggering unwanted cytokine production requires a better understanding of the molecular mechanisms influencing the production and degradation of MHC class II and CD86 in DCs. Here, we investigate how CD83, an immunoglobulin protein expressed on the surface of mature DCs, promotes MHC class II and CD86 expression. Using mice with an N-ethyl-N-nitrosourea-induced mutation eliminating the transmembrane (TM) region of CD83, we found that the TM domain of CD83 enhances MHC class II and CD86 expression by blocking MHC class II association with the ubiquitin ligase MARCH1. The TM region of CD83 blocks interleukin 10-driven, MARCH1-dependent ubiquitination and degradation of MHC class II and CD86 in DCs. Exploiting this posttranslational pathway for boosting MHC class II and CD86 expression on DCs may provide an opportunity to enhance the immunogenicity of vaccines.

Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line-derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti-IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.

Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific markers rhodopsin, opsin, and recoverin in vitro. CD90+ cells were either transduced with recombinant adeno-associated virus expressing green fluorescent protein (GFP) or bromodeoxyuridine (BrdU) labeled and then injected into the subretinal space of adult Royal College of Surgeons rats. Fundus photography and angiography showed no adverse effects of CD90+ MSC transplantation. GFP-expressing cells or BrdU-positive cells covered approximately 30% of the entire retinal area. By 2 weeks after injection, CD90+ MSCs integrated into the host retina, forming structures similar to the photoreceptor layer and expressed a photoreceptor-specific marker. No teratoma formation was observed in the recipient retina. The subretinally delivered CD90+ MSCs did not stain for proliferating cell nuclear antigen, indicating that they primarily undergo differentiation rather than proliferation. In addition, we established that transplanted cells can attract synaptic vesicles and hence are potentially capable of signal transduction. This study demonstrates for the first time the partial differentiation of adult CD90+ MSCs into photoreceptors in vitro and in vivo. Our results establish a proof of concept for CD90+ MSC differentiation with autologous transplantation, which may provide a promising therapeutic strategy for the treatment of some forms of genetically inherited retinal degenerations.