The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene ( RGCC ), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

Purpose: Cutaneous T-cell lymphomas (CTCL), encompassing a spectrum of T-cell lymphoproliferative disorders involving the skin, have collectively increased in incidence over the last 40 years. Sézary syndrome is an aggressive form of CTCL characterized by significant presence of malignant cells in both the blood and skin. The guarded prognosis for Sézary syndrome reflects a lack of reliably effective therapy, due, in part, to an incomplete understanding of disease pathogenesis. Experimental Design: Using single-cell sequencing of RNA and the machine-learning reverse graph embedding approach in the Monocle package, we defined a model featuring distinct transcriptomic states within Sézary syndrome. Gene expression used to differentiate the unique transcriptional states were further used to develop a boosted tree classification for early versus late CTCL disease. Results: Our analysis showed the involvement of FOXP3⁺ malignant T cells during clonal evolution, transitioning from FOXP3⁺ T cells to GATA3⁺ or IKZF2⁺ (HELIOS) tumor cells. Transcriptomic diversities in a clonal tumor can be used to predict disease stage, and we were able to characterize a gene signature that predicts disease stage with close to 80% accuracy. FOXP3 was found to be the most important factor to predict early disease in CTCL, along with another 19 genes used to predict CTCL stage. Conclusions: This work offers insight into the heterogeneity of Sézary syndrome, providing better understanding of the transcriptomic diversities within a clonal tumor. This transcriptional heterogeneity can predict tumor stage and thereby offer guidance for therapy.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177⁺ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

Risk stratification of therapy for pediatric acute myeloid leukemia (AML) has been a focus of clinical protocol design to maximize treatment for high-risk groups while reducing therapeutic intensity for lower-risk groups. Molecular and cytogenetic markers have been used to define risk groups before therapy; however, 20% of pediatric cases lack all known markers1 and ~60% of cases lack markers that stratify outcome. The detection of measurable residual disease (MRD) by multidimensional flow cytometry (MDF) in the remaining ‘standard risk’ group has permitted the stratification of patients who lack genetic abnormalities linked to therapy outcomes. However, discrimination between good and poor-risk groups based on the immunophenotype at diagnosis has not been sufficiently robust to routinely stratify patients.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors. Choroidal endothelial and immune cells are implicated in the pathogenesis of age-related macular degeneration (AMD). In a cohort of 11 AMD and 10 control human donors, Voigt et al. used single-cell RNA sequencing to identify gene expression changes across the choroidal endothelial cell and immune subpopulations. The background depicts a whole mount of human choroidal endothelial cells (red) and IBA1-expressing immune cells (green).