Liquid biopsy for proliferative diabetic retinopathy: single-cell transcriptomics of human vitreous reveals inflammatory T cell signature

Purpose Current therapies for proliferative diabetic retinopathy (PDR) do not specifically target VEGF-independent, cell-type specific processes that lead to vision loss, such as inflammatory pathways. This study aims to identify targetable cell types, and corresponding signaling pathways, by elucidating the single cell landscape of the vitreous of patients with PDR. Design Case Series Samples Vitreous and peripheral blood obtained from five adult patients (six eyes) undergoing pars plana vitrectomy for vision-threatening PDR. Methods Single cell RNA sequencing (scRNA-seq) was performed on vitreous cells obtained from diluted cassette washings during vitrectomy from six eyes and peripheral blood mononuclear cells (PBMCs) (n=5). Droplet-based scRNA-seq was performed using the Chromium 10x platform to obtain single cell transcriptomes. Differences in tissue compartments were analyzed with gene ontology (GO) enrichment of differentially expressed genes and an unbiased ligand-receptor interaction analysis. Main Outcome Measures Single cell transcriptomic profiles of vitreous and peripheral blood Results Transcriptomes from 13675 surgically harvested vitreous cells and 22636 PBMCs were included. Clustering revealed four cell states consistently across all eyes with representative transcripts for T cells (CD2, CD3D, CD3E, GZMA), B cells (CD79A, IGHM, MS4A1 (CD20), HLA-DRA), myeloid cells (LYZ, CST3, AIF1, IFI30), and neutrophils (BASP1, CXCR2, S100A8, S100A9). Most vitreous cells were T cells (91.6%), unlike the peripheral blood (46.2%), while neutrophils in the vitreous were essentially absent. The full repertoire of adaptive T cells including CD4+, CD8+ and T regulatory cells (Treg) and innate immune system effectors (i.e. Natural Killer T cells) was present in the vitreous. Pathway analysis also demonstrated activation of CD4+ and CD8+ memory T cells and ligand-receptor interactions unique to the vitreous. Conclusions In the first single-cell transcriptomic characterization of human vitreous in a disease state, we show PDR vitreous is primarily composed of T cells, a critical component of adaptive immunity, with activity and proportions distinct from T cells within the peripheral blood, and neutrophils are essentially absent. These results demonstrate the feasibility of liquid vitreous biopsies via collection of otherwise discarded, diluted cassette washings during vitrectomy to gain mechanistic and therapeutic insights into human vitreoretinal disease.