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Abstract
BackgroundMajor-histocompatibility-complex class I-related chain A (MICA) antigens are the ligands of NKG2D, which is an activating or coactivating receptor expressed on human NK cells and CD8+T cells. We sought to determine whether MICA expression in human corneal epithelium (HCE) could affect the cytotoxicity mediated by NK cells or CD8+T cells.MethodsCell cultures of HCE were harvested from human donor eyes. Flow cytometric analysis and ELISA was performed to determine the levels of MICA expression on HCE. Then, HCE was transfected with a lentivirus vector expressing MICA and GFP. Flow cytometric analysis, RT-PCR, western blot and ELISA were performed to check the levels of MICA expression. For cytotoxicity testing, allogeneic NK cells and CD8+T cells were isolated from peripheral blood mononuclear cells of healthy volunteers by magnetic cell sorting. The cytolytic activity of NK cells and CD8+T cells was assessed against MICA-transfected HCE (NK cells: E:T ratio = 3:1; CD8+T cells: E:T ratio = 10:1) using the nonradioactive cytotoxicity detection kit lactate deshydrogenase.ResultsSurface expression of MICA on corneal epithelium was identified at a low level. A cell line of stable human MICA-transfected corneal epithelium was successfully established. Heightened expression of MICA on HCE was found to promote the cytotoxicity mediated by NK cells or CD8+T cells, which could be blocked by an anti-MICA antibody.ConclusionMICA molecules may contribute to cytotoxic responses mediated by activated immune effector cells in corneal epithelium immunity.
Highlights
Major-histocompatibility-complex class I-related chain A (MICA) antigens are the ligands of NKG2D, which is an activating or coactivating receptor expressed on human natural killer (NK) cells and CD8+T cells
Low levels of surface MICA could be detected on human cultured corneal epithelium in vitro We performed flow cytometric analyses and ELISA to determine whether human corneal epithelium cells express MICA
Surface MICA expression on corneal epithelium was identified by flow cytometry (5.7 ± 0.6%, Figure 1C), while sMICA in the supernatant could not be detected by ELISA
Summary
Major-histocompatibility-complex class I-related chain A (MICA) antigens are the ligands of NKG2D, which is an activating or coactivating receptor expressed on human NK cells and CD8+T cells. We sought to determine whether MICA expression in human corneal epithelium (HCE) could affect the cytotoxicity mediated by NK cells or CD8+T cells. Major-histocompatibility-complex class I-related chain A (MICA) manifest homology with classical human leukocyte antigen molecules, yet they neither engage β2 microglobulin [1], nor bind peptides and are not expressed on normal circulating lymphocytes [2,3]. We found low levels of surface MICA expression in corneal epithelium. Anti-MICA antibodies could further block this process These findings might represent a possible mechanism of immune-mediated damage in conditions of corneal stress. We established stable human MICA-transfected corneal epithelium with a lentivirus vector to determine the susceptibility of MICA-transfected human corneal epithelium to the killing mediated by NK cells or CD8+T cells
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