The Role of DNAM-1/Tigit Axis of NK Cells in the Regulation of Alloreactive T Cell Responses and the Potential Mechanism

Objective: Previous studies has showed the important role of NK cell in the regulation of alloreactive T cell response and negative regulation of GVHD. The paired receptors DNAM-1 and TIGIT, which bind the same ligands but have opposite effects on NK cell function, might present as potential targets for the GVHD treatment. This study is designed to explore the role of TIGIT/DNAM-1 balance of NK cells in the regulation of alloreactive T cell responses and the potential mechanism.

Methods: Antibodies blocking of TIGIT or DNAM-1, over-expression of TIGIT or DNAM-1 via lentiviral transduction and knockdown of TIGIT or DNAM-1 by lentiviral shRNA were used to manipulate the TIGIT/DNAM-1 balance on NK cell. Cytotoxicity assay using alloantigen activated T cells as targets were used to evaluate the regulating function of NK cell on alloreactive T cell responses. Western blot and small molecule inhibitors against PI3K were combined to investigate whether the PI3K-Akt-ERK signaling cascade is involved in the signal transduction process following TIGIT/DNAM-1-PVR engagement.

RESULTS: Blocking of DNAM-1 by an anti-DNAM-1 antibody and knockdown of DNAM-1 expression by lentiviral shRNA both resulted in deceased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of DNAM-1 via lentiviral transduction resulted in enhanced cytotoxicity. Blocking of TIGIT by an anti-TIGIT antibody and knockdown of TIGIT expression by lentiviral shRNA both resulted in increased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of TIGIT via lentiviral transduction resulted in decreased cytotoxicity. Increases in NK cytotoxicity against activated T cells through TIGIT knockdown could be overcome by blocking DNAM-1 signaling. Simultaneously, over-expression of DNAM-1 or knockdown of TIGIT expression resulted in an increase of the phosphorylation levels of Akt and ERK1/2 in NK cells after contacted with activated T cells, which could be overcome by pretreating NK cells with anti-DNAM-1 or PI3K small molecule inhibitor. Pretreating alloantigen activated T cells with anti-PVR also resulted in deceased cytotoxicity and Akt and ERK1/2 phosphorylation in DNAM-1 over-expression NK cells.

Conclusion: The paired receptor DNAM-1/TIGIT on the surface of NK cells compete the same PVR ligand on the surface of activated T cells and the DNAM-1/TIGIT axis is involved in the regulation of cytotoxicity of NK cells on alloantigen activated T cells through PI3K-Akt-ERK cascade phosphorylation. The DNAM-1/TIGIT expression balance may present as biomarkers for aGVHD and potential targets for aGVHD therapy.