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Heightened expression of MICA enhances the cytotoxicity of NK cells or CD8+T cells to human corneal epithelium in vitro.

Hong J, Qiu T, Qian T, Li G, Yu X, Chen J, Le Q, Sun X, Xu J - BMC Ophthalmol (2012)

Bottom Line: Cell cultures of HCE were harvested from human donor eyes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology, Eye, Ear, Nose, and Throat Hospital, School of Shanghai Medicine, Fudan University, 83 Fenyang Road, Shanghai, 200031, China.

ABSTRACT

Background: 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.

Methods: Cell 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.

Results: Surface 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.

Conclusion: MICA molecules may contribute to cytotoxic responses mediated by activated immune effector cells in corneal epithelium immunity.

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Related in: MedlinePlus

Contribution of MICA to NK cells and CD8+Tcells mediated killing of human corneal epithelium. (A)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added toIL-2-activated NK cells (effector) at a 3:1 (E:T) ratio. (B)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added to allogeneichuman CD8+T cells (effector) at a 10:1 (E:T) ratio.Nonparametric test comparing isotype and MICA antibody showed astatistical difference between groups for MICA-transfected cells (NKcells, P < 0.05; CD8+T cells,P < 0.05), but not for non-transfectedcells. Data are presented as percentage of specific killing. Each assaywas repeated three times and presented asmean ± standard error.
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Figure 3: Contribution of MICA to NK cells and CD8+Tcells mediated killing of human corneal epithelium. (A)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added toIL-2-activated NK cells (effector) at a 3:1 (E:T) ratio. (B)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added to allogeneichuman CD8+T cells (effector) at a 10:1 (E:T) ratio.Nonparametric test comparing isotype and MICA antibody showed astatistical difference between groups for MICA-transfected cells (NKcells, P < 0.05; CD8+T cells,P < 0.05), but not for non-transfectedcells. Data are presented as percentage of specific killing. Each assaywas repeated three times and presented asmean ± standard error.

Mentions: We evaluated the cytotoxicity mediated by allogeneic NK cells andCD8+T cells to MICA-transfected human corneal epithelium.IL-2-activated NK cells and CD8+T cells purified from allogeneic PBMCwere used as effector cells in killing assays, respectively. Empty vectortransfected corneal epithelium was used as the target cells control. Cornealepithelium was preincubated with an isotype control, anti-MICA antibody or antiMHC-I antibody for 1 h before being added to effector cells (NK cells: E:Tratio = 3:1; CD8+T cells: E:T ratio = 10:1).As illustrated in Figure 3, the specific killing wassignificantly inhibited only in the transfected cell group preincubated withanti-MICA. Blocking MHC class I molecules did not affect the killing, confirmingthat the MICA blocking was not caused by steric hindrance. Our observationsdemonstrated that ectopic expression of MICA on human corneal epithelium couldcontribute to its susceptibility to the killing mediated by NK cells orCD8+T cells.


Heightened expression of MICA enhances the cytotoxicity of NK cells or CD8+T cells to human corneal epithelium in vitro.

Hong J, Qiu T, Qian T, Li G, Yu X, Chen J, Le Q, Sun X, Xu J - BMC Ophthalmol (2012)

Contribution of MICA to NK cells and CD8+Tcells mediated killing of human corneal epithelium. (A)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added toIL-2-activated NK cells (effector) at a 3:1 (E:T) ratio. (B)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added to allogeneichuman CD8+T cells (effector) at a 10:1 (E:T) ratio.Nonparametric test comparing isotype and MICA antibody showed astatistical difference between groups for MICA-transfected cells (NKcells, P < 0.05; CD8+T cells,P < 0.05), but not for non-transfectedcells. Data are presented as percentage of specific killing. Each assaywas repeated three times and presented asmean ± standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733519&req=5

Figure 3: Contribution of MICA to NK cells and CD8+Tcells mediated killing of human corneal epithelium. (A)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added toIL-2-activated NK cells (effector) at a 3:1 (E:T) ratio. (B)Human MICA-transfected corneal epithelium or empty vector transfectedcorneal epithelium were preincubated for 4 h with either anisotype control (white bars), anti-MICA antibody (black bars) oranti-MHC class I antibody (gray bars) before being added to allogeneichuman CD8+T cells (effector) at a 10:1 (E:T) ratio.Nonparametric test comparing isotype and MICA antibody showed astatistical difference between groups for MICA-transfected cells (NKcells, P < 0.05; CD8+T cells,P < 0.05), but not for non-transfectedcells. Data are presented as percentage of specific killing. Each assaywas repeated three times and presented asmean ± standard error.
Mentions: We evaluated the cytotoxicity mediated by allogeneic NK cells andCD8+T cells to MICA-transfected human corneal epithelium.IL-2-activated NK cells and CD8+T cells purified from allogeneic PBMCwere used as effector cells in killing assays, respectively. Empty vectortransfected corneal epithelium was used as the target cells control. Cornealepithelium was preincubated with an isotype control, anti-MICA antibody or antiMHC-I antibody for 1 h before being added to effector cells (NK cells: E:Tratio = 3:1; CD8+T cells: E:T ratio = 10:1).As illustrated in Figure 3, the specific killing wassignificantly inhibited only in the transfected cell group preincubated withanti-MICA. Blocking MHC class I molecules did not affect the killing, confirmingthat the MICA blocking was not caused by steric hindrance. Our observationsdemonstrated that ectopic expression of MICA on human corneal epithelium couldcontribute to its susceptibility to the killing mediated by NK cells orCD8+T cells.

Bottom Line: Cell cultures of HCE were harvested from human donor eyes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology, Eye, Ear, Nose, and Throat Hospital, School of Shanghai Medicine, Fudan University, 83 Fenyang Road, Shanghai, 200031, China.

ABSTRACT

Background: 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.

Methods: Cell 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.

Results: Surface 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.

Conclusion: MICA molecules may contribute to cytotoxic responses mediated by activated immune effector cells in corneal epithelium immunity.

Show MeSH
Related in: MedlinePlus