Abstract
Presentation Description :
Different imaging techniques, such as whole mount confocal microscopy (WMCM) and in vivo confocal microscopy (IVCM), have shown the presence of immune cell types in the epithelium and stroma of naïve and inflamed corneas. However, the approach has limitations in phenotyping the immune cell subsets and their effector function. We recently published a flow cytometry-based approach to quantitate immune cell types in the individual epithelium and stroma of uninfected and herpes simplex virus-1 infected corneas. In this procedure, using an enzymatic digestion approach, the corneal epithelium is separated from the underlying stroma and endothelium layer. This is followed by trypsin-EDTA digestion to prepare a single-cell suspension of the epithelium and Liberase-mediated digestion to prepare the single-cell suspension of the stroma. The single-cell suspension is used for flow cytometry study. Using this approach, we ascertained that the naïve corneal epithelium of C57BL/6J mice harbor a distinct CXCR4+ cell population in the corneal epithelium. These CXCR4+ cells do not express CD45 but a higher CD326 (EpCAM) level. A subset of CXCR4+CD45-ve cells express CD207 (langerin) molecule. Among many other cell types, CXCR4 is also expressed on hematopoietic stem and progenitor cells, especially in the bone marrow. After corneal HSV-1 infection, these CD45-CXCR4+ cells undergo proliferation, as evident from the Edu incorporation assay, that is measured by flow cytometry. The ongoing experiments are ascertaining if these CXCR4+CD45-ve cells have the potential to differentiate into hematopoietic colony-forming cells and fully mature myeloid cells. Using our stromal separation approach and flow cytometry assay, we determined the presence of myeloid progenitors in the stroma of HSV-1 infected corneas during the development of herpes stromal keratitis (HSK). Our flow cytometry results also showed the presence of CD31+ vascular endothelial cells, CD4 T cells and neutrophils in the stroma of HSK-developing corneas. Our results showed a flow-cytometry-based approach to quantitate immune and non-immune cell types in separated epithelium and stroma of HSV-1 infected corneas. However, this approach can also phenotype immune cells in the epithelium and stroma of any other model of corneal inflammation, thereby documenting its broader application.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.