Identification And Function Analysis Of NK Cell Activating Receptor NKp30Ligands

Natural killer (NK) cells are important effect lymphocytes of the innate immune system and play critical role in tumor immunosurveillance. NK cells rapidly kill certain target cells without prior immunization or MHC restriction, whose activation is dependent on the balance between inhibitory and activating signals from invariant receptors. Natural cytotoxicity receptors (NCRs) are important activated receptors of NK cells. As one important member of NCRs, NKp30(NCR3) expressed on all NK cells and plays critical roles in NK lytic activity against virus-infected and tumor cells. Gal-3(Gal-3), one of the members of the p-galactoside-binding lectin family, contains either two carbohydrate recognition domains (CRDs), which have a high affinity for β-galactosides, expressed by many types of tumor cells, was reported to directly work as an immune regulator to induce apoptosis in activated T cells; however, its effect on NK cells is elusive. Here, we established a stable transfected cell line that effciencetly expresses the NKp30receptor and human IgG1Fc (NKp30-Fc) chimeric protein, by which we obtained the bioactive soluble NKp30-Fc proteins that were used in the further studies. Furtherly, human cervical cancer cell lines and their xenograft tumor NOD-SCID mice model are used to study the effect of Gal-3on NK cells anti-tumor.The major results in our study were summarized as follows:1. Preparation and functional identification of recombinant human NK cells activation receptors NKp30fusion proteins.Here we constructed the NKp30-Fc expression vectors including NKp30receptor ectodomain and the Fc portion of the human IgGl heavy chain. CHO-K1cells were transfected with lipofectamine transfection reagent, and G418-selected clones were screened for NKp30-Fc proteins expression. Then cell clones are further subcloned by limited dilution method for highest protein production. ELISA assay was performed to screen cell clones for very high secretion level of soluble NKp30-Fc protein. Serum-free cell culture supernatant were collected and purified by one step chromatography on a protein A/G column for soluble NKp30-Fc protein.Functionally, a flow cytometry assay showed that recombinant NKp30-Fc molecule had a sufficient ability to bind NKp30ligand-positive tumor cells. Moreover,51Cr release assays also demonstrated soluble NKp30-Fc protein could attenuate NKp30-mediated antitumor effects of human NK cells.2. Tumor-released Gal-3, a soluble inhibitory ligand of human NKp30, plays an important role in tumor escaping from NK cell attack.Using the recombinant human NK cell activating receptor NKp30fusion protein (NKp30-Fc) and a human cervical cancer model, we found that soluble NKp30-Fc could immunoprecipitate Gal-3. The direct interaction between NKp30and Gal-3was further confirmed using surface plasmon resonance (SPR) experiments. Because Gal-3was mainly released from tumor cells in a soluble form in our study, a flow cytometry assay was performed to show that soluble Gal-3specifically bound to NK cells, and NKp30on the surface of the NK cells bound the soluble Gal-3, as shown by biochemical analyses. Functionally, when soluble Gal-3was added to the NK-92-HeLa cell co-culture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by pre-incubation of soluble Gal-3with NKp30-Fc fusion protein. Moreover, genetic downregulation of Gal-3(shGal-3) resulted in HeLa cells being more sensitive to NK cell lysis, and reversely, Gal-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft NOD-SCID mice after NK cell adoptive immunotherapy, indicating that Gal-3strongly antagonizes human NK cell attack against tumors in vivo.In conclusion, our results clearly demonstrated that recombinant NKp30-Fc molecule produced through the approach described here had a sufficient ability to bind NKp30ligand-positive tumor cells and compete with the natural, membrane-bound form of the receptor. This protein may have general applications in defining interactions of NKp30and its ligands as well as searching for antagonists of NKp30receptor function. In addition, we demonstrate that the Gal-3secreted from the tumor works as a soluble inhibitory ligand of NK cell receptor NKp30to inhibit NK cell immune responses against tumors. The novel mechanism of how Gal-3regulates the anti-tumor immunity of human NK cells may provide a new therapeutic target for tumor treatment.