Immune Landscape and Intercellular Crosstalk in the Cornea of an Autoimmune-Mediated Dry Eye Disease

Purpose: The purpose of this study was to comprehensively characterize the identity and signaling profiles of resident and infiltrating immune cells, and their interactions with non-immune cells, in a spontaneous mouse model of autoimmune-mediated dry eye disease (DED). Methods: Corneal tissues from wild-type (WT) and autoimmune regulator-deficient (Aire-/-) mice were analyzed by whole-mount immunofluorescence to define immune cell composition and spatial distribution within the limbus and central cornea. Immune cell identities and cell-cell communication were further characterized using single-cell RNA sequencing (scRNA-seq). Intercellular signaling pathways among immune cells, epithelial, and stromal cells were analyzed using CellChat. Results: Immunofluorescence revealed extensive infiltration of CD3⁺ and CD4⁺ T cells, CD45R⁺ B-cells, and IBA1⁺ macrophages in Aire-/- cornea compared with WT controls. The scRNA-seq identified diverse immune subsets, including cytotoxic T cells, γδ17 cells, regulatory T cells, NK T cells, B-cells, plasma cells, and multiple macrophage subtypes. Monocyte-derived macrophages demonstrated distinct transcriptional programs, including pro-inflammatory and antigen-presenting phenotypes, with CD40 to CD40L signaling linking T cells, B-cells, and macrophages. CellChat analysis revealed coordinated upregulation of signaling pathways associated with immune activation, inflammation, apoptosis, and loss of immune tolerance. Additionally, epithelial cells and keratocytes in Aire-/- corneas upregulated antigen-presenting and pro-inflammatory gene signatures, suggesting active participation of non-immune cells in immune activation and recruitment. Conclusions: Single-cell transcriptomics reveals a complex, multicellular inflammatory environment in the Aire-/- cornea involving diverse immune subsets and activated resident epithelial and stromal cells. These findings suggest that non-T cell mechanisms contribute to chronic dry eye pathogenesis and may underlie the limited efficacy of currently US Food and Drug Administration (FDA)-approved T cell-targeted therapies. Further characterization of these multi-cellular interactions and signaling pathways may inform the development of broader therapeutic strategies for autoimmune-mediated dry eye disease.