The Mechanism Of NK Cell Receptors Interacting With Their Ligands In Nectin/Necl Family

NK cells play important roles in immune surveillance. They could detect and kill tumor cells or virus infected cells directly through lymphocyte cytotoxicity. The recognition and killing of target cells depend on activating and inhibiting receptors expressed on NK cells, which activates or inhibits the cytotoxity respectively. Besides common receptors such as NKG2D、NKp30、NKp44and NKp46, NK cells also express a specific kind of receptors recognizing proteins which belongs to Nectin/Necls family members whose major role are mediating cell adhesion. There are9members in this family, Nectin1,2,3,4and Necl-1,2,3,4,5.Nectin-2, Necl-2and Necl-5are also ligands for a spefic kind of NK cell receptors, which includes CRTAM, DNAM-1,CD96and TIGIT. Though DNAM-1and TIGTI are stimulating and inhibitory receptors respectively, they share the same ligands nectin-2and necl-5. CRTAM is an activating receptor which recognize Necl-2. CD96recognize Necl-5. Whether CD96is an activating or inhibitory receptor is still controversial.Our research aims to delineate the mechanism of how CRTAM,CD96and DNAM-1recognize their ligand.The interaction of CRTAM and Necl-2stimulate NK cells and T cells to kill tumor cells. Besides, binding between CRTAM and Necl-2faciliates CD8+T cells to secrete interferon y as well as regulates their retention in the lymph nodes.The excessive interaction beween these two proteins often results in autoimmune diseases. In mouse autoimmune alopecia model, activating CD8+T cells express CRTAM and kills necl-2positive hair follicle keratinocytes.But mechanism of the interaction between these two proteins is not clear. In addition, CRTAM and Necl-2are also involved in cell adhesion. What is relationship between cell adhesion and immune recognition both mediated by these proteins is also unkown.In our research, we expressed CRTAM/Necl-2complex in vitro, found a "double lock and key" region located at the interface of CRTAM and necl-2, which was responsible for binding of these two proteins.Key amino acids in this region are identied and futher validated by biochemical assays such as surface plasmon resonance and tetramer staining. The same interafaces both in CRTAM and necl-2were also identified,which mediated CRTAM and necl-2self-homodimerization as well as their cross heterodimerization. Since the former interaction was involved in cell adhesion whereas the latter one contributed to immune recognition, the competion between them might also reflect their competing roles in vivo. This competing mode might be significant in vivo, as necl-2expressed on tumor cells facilitated their adhesion, aggregation, binding to endothelial cells and transferring to tissues. Binding between necl-2and CRTAM resulted in weaker tumor adhesion and their killing by immune cells.The major function of DNAM-1is to stimulate the cytotoxity of NK cells. For instance, the interaction between DNAM-1and its ligands nectin-2and necl-5stimulates NK cells to kill colon cancer cells, colorectal cancer cells, cervical cancer cells and melanoma. Besides, they also contribute to other immune process. DNAM-1also participtes in regulating T cell development and differenciation, the interaction between NK cells and antigen presenting cells, binding of leukocytes to endothelial cells. DNAM-1and its ligands are involved in the immnopathogy of graft-versus-host disease, Systemic Lupus Erythematosus and Refractory Celiac Disease Type Ⅱ. However, how DNAM-1interact with its ligands nectin-2and necl-5is unclear. Besides, DNAM-1, TIGIT and CD96share the same receptor necl-5, whether they use common mechanism is unkown. This are the questions we want to solve. We prepare DNAM-1, CD96, necl-5and nectin-2. We successfully get the complex of DNAM-1with its two ligand. By comparing the the anino acid sequences and the crystal structures of DNAM-1, CD96with the previously reported TIGIT/Necl-5. Conserve amino acids that might be responsible for complex formation.were identified in DNAM-1, CD96and TIGIT as well as their corresponding ligands. Our analysis might reveal a common receptor-ligand binding mechasm in this specific family of NK cell receptors.In summary, our research delineate our NK cell receptor CRTAM, CD96and DNAM-1recognize their ligands, which provides foundation for research on NK cell recognition and drug design...