Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Corneal tissue-resident memory T cells form a unique immune compartment at the ocular surface.

The eye is considered immune privileged such that immune responses are dampened to protect vision. As the most anterior compartment of the eye, the cornea is exposed to pathogens and can mount immune responses that recruit effector T cells. However, presence of immune memory in the cornea is not defined. Here, we use intravital 2-photon microscopy to examine T cell responses in the cornea in mice. We show that recruitment of CD8+ T cells in response to ocular virus infection results in the formation of tissue-resident memory T (TRM) cells. Motile corneal TRM cells patrol the cornea and rapidly respond in situ to antigen rechallenge. CD103+ TRM cell generation requires antigen and transforming growth factor ß. In vivo imaging in humans also reveals highly motile cells that patrol the healthy cornea. Our study finds that TRM cells form in the cornea where they can provide local protective immunity.

Altered Corneal Epithelial Dendritic Cell Morphology and Phenotype Following Acute Exposure to Hyperosmolar Saline.

Purpose: The purpose of this study was to assess the morphological and phenotypic responses of corneal epithelial dendritic cells (DCs) to acute topical hyperosmolar stress, given a pathogenic role for tear hyperosmolarity in dry eye disease (DED). Methods: C57BL/6J mice were anesthetized and received 350 mOsm/L (physiological; n = 5 mice), 450 mOsm/L (n = 6), or 600 mOsm/L (n = 6) saline on a randomly assigned eye. Corneas were harvested 2 hours later. Immunofluorescent staining was performed using CD45, CD86, and CD68 antibodies to investigate DC morphology (density, viability, field area, circularity, and dendritic complexity) and immunological phenotype. Flow cytometry was used to confirm CD86 and CD68 expression in CD11c+ DCs, using C57BL/6J mice that received topical applications of 350 mOsm/L, 450 mOsm/L, or 600 mOsm/L (n = 5 per group) bilaterally for 2 hours. Results: Following exposure to 450 mOsm/L topical saline for 2 hours, DCs in the central and peripheral cornea were larger (field area: Pcentral = 0.005, Pperipheral = 0.037; circularity: Pcentral = 0.026, and Pperipheral = 0.013) and had higher expression of CD86 compared with 350 mOsm/L controls (immunofluorescence: P < 0.0001; flow cytometry: P = 0.0058). After application of 600 mOsm/L saline, DC morphology was unchanged, although the percentage of fragmented DCs, and phenotypic expression of CD86 (immunofluorescence: P < 0.0001; and flow cytometry: P = 0.003) and CD68 (immunofluorescence: P = 0.024) were higher compared to 350 mOsm/L controls. Conclusions: Short-term exposure to mild hyperosmolar saline (450 mOsm/L) induced morphological and phenotypic maturation in corneal epithelial DCs. More severe hyperosmolar insult (600 mOsm/L) for 2 hours appeared toxic to these cells. These data suggest that hyperosmolar conditions activate corneal DCs, which may have implications for understanding DC activation in DED.