Mapping Resident Immune Cells in the Murine Ocular Surface and Lacrimal Gland by Flow Cytometry

Abstract

ABSTRACT Background The ocular surface and lacrimal gland have a frontline position in mucosal immunology. However, there have been few updates to the immune cell atlas of these tissues in recent years. Purpose To map the immune cells in murine ocular surface tissues and lacrimal gland. Methods Central and peripheral corneas, conjunctiva, and lacrimal gland were dissociated into single cell suspensions, followed by flow cytometry. Discrepancy of immune cells between the central and peripheral corneas was compared. In the conjunctiva and lacrimal gland, myeloid cells were clustered by tSNE and FlowSOM based on the expression of F4/80, Ly6C, Ly6G, and MHC II. ILCs, type 1 immune cells, and type 3 immune cells were analyzed. Results The number of immune cells in peripheral corneas was about 16 folds of that in central corneas. B cells accounted for 8.74% of immune cells in murine peripheral corneas. In the conjunctiva and lacrimal gland, most myeloid cells tended out to be monocytes, macrophages, and classical dendritic cells (cDCs). ILC3 were 6.28% and 3.63% of ILCs in the conjunctiva and lacrimal gland, respectively. Th1, Tc1, and NK cells were predominant type 1 immune cells. γδ T17 cells and ILC3 outnumbered Th17 cells among type 3 T cells. Conclusion B cells resident in murine corneas were reported for the first time. Additionally, we proposed a strategy of clustering myeloid cells to better understand their heterogeneity in the conjunctiva and lacrimal gland based on tSNE and FlowSOM. Furthermore, we identified the ILC3 in the conjunctiva and lacrimal gland for the first time. Compositions of type 1 and type 3 immune cells were summarized. Our study provides a fundamental reference and novel insights for ocular surface immune homeostasis and diseases.