Fusion Expression And Functional Investigation Of NK Cell Activating Receptor NKp80

Natural Killer (NK) cells are an important subset of lymphocytes that mainly participate in innate immunity and are thought to play a relevant role in host defense because they are involved in killing of tumor or virally infected cells. The capability of NK cells to kill tumor or virally infected cells mainly depends on the synthesis of stimulatory and inhibitory signals mediated by various NK cell surface receptors.NK cell surface membrane protein is very important for the tumor or virally infected cells recongnition by NK cells and it’s hotspot in immunology research all the time that study on the recongnition and killing mechanism of membrane protein. NKp80, also termed KLRF1 (killer cell lectin-like receptor subfamily F, member 1), is a type-Ⅱ transmembrane glycoprotein with a C-type lectin domain in ectodomain and belongs to the family of C-type lectin-like receptors like NKG2D. NKp80 is specially expressed on all human NK cells and a small subset of effector memory CD8+T cells as an 80 kDa homodimeric surface molecule.NKp80 stimulates NK cell cytotoxicity and cytokine release and appears to cooperate with other triggering receptors to induce optimal NK cell activation upon interaction with potential target cells. The ligand of NKp80, AICL, the abbreviation for activation-induced C-type lectin, also called CLEC2B, is a myeloid-specific activating receptor expressed by monocytes, macrophages and granulocytes. NKp80 engagement by AICL is discussed not only to promote cytolysis of myeloid cells, but is also critically involved in the mutual activation of NK cells and monocytes.The major results in our study were summarized as follows:1. Activation and enhancement of NK cell-mediated cytotoxicity against leukemia by NK receptor NKp80 via its ligand AICLNK cells are innate immune effector cells that are important for early and effective immune responses against transformed cells and infection. Unlike cytotoxic T lymphocytes (CTL), NK cells are capable of killing cancer and viral-infected cells without prior sensitization or immune memory. Activation and lysis of target cells is triggered mainly by natural cytotoxicity receptors (NCRs) and NKG2D. However, additional activating receptors or costimulatory molecules also modulate NK cell activation. In this paper, we described that NKp80 representing an activating NK receptor was mainly expressed by human NK cells, but not by NK cell lines. After NKp80 antibody stimulation, the expression of activated marker CD69 showed a remarkable increase on NK cells. In redirected lysis assays, cross-linking of NKp80 triggered degranulation and cytotxicity activity of NK cells, even to the same level of common NK activating receptors like NCR, NKG2D and CD226. Also, Induction of NKp80 coss-linking potentiated the direct NK cell-mediated cytotxicity against leukemia cells. In addition, soluble NKp80 protien specifically bound its ligand AICL, which was frequently highly expressed on malignant leukemia cells. Cell binding assays showed that blockade of NKp80-AICL interaction using AICL antibody or peptide impaired conjugation between NK and leukemia cells apparently. Meanwhile, Improving NKp80-AICL interaction by over-expression of AICL significantly increased binding rate between NK and AICL-negative cancer cells. In functional analyses, blockade of NKp80-AICL interaction using antibodies to NKp80 or AICL or soluble proteins both attenuated periphery NK cytotoxicity against leukemia cells. Thus, our results demonstrate that NKp80 can trigger NK activation and cytotoxicity via AICL ligand. NKp80-AICL interaction can enhance NK cell-mediated cytotoxicity against leukemia cells through promoting conjugation formation, suggesting the possibilities for NK cell therapy for leukemia.2. Generation and preclinical characterization of an NKp80-Fc fusion protein for redirected cytolysis of NK cells against leukemiaThe capacity of NK cells to mediate Fc-receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for NK activating receptor NKp80, is frequently highly expressed on malignant leukemia cells in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), and given the current lack of therapeutic AICL-specific antibodies, we aimed to explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-NKp80-hIgG1-Fc (NKp80-Fc) fusion proteins. Binding tests using flow cytometry indicated that NKp80-Fc chimera specifically bound to leukemia cell lines in an AICL-specific and concentration-dependent manner. Moreover, cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK-target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced an NK cell ADCC effect. Cell apoptosis and lysis assays showed that the fusion proteins not only promoted NK-mediated target cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in an AICL-dependent manner in the relatively late stage of cell conjugation. The bifunctional recombinant fusion proteins re-directed the NK cells toward the leukemia cells by binding to both the tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Taken together, our results demonstrate that NKp80-Fc fusion proteins can effectively target AICL-expressing leukemia cells for tumor lysis by NK cells. This method will be potentially useful in further research on molecular targeted therapy, and the fusion proteins may thus constitute a promising means for immunotherapy of leukemia.