Expression And Functional Research Of Activating Receptor NKp30 On NK Cells

As the front line of host defense, the innate immunity can quickly response to the invasion of pathogens, resulting in non-specific immunity against infection, while also plays important roles in adaptive immune response.Natural killer (NK) cells are important components of innate immunity and play important roles in immune response to viral infection and tumors. The immune response of NK cell is regulated collaboratively by its surface activating and inhibitory receptors. As unique NK cell activating receptors, natural cytotoxicity receptors (NCRs) play important roles in cytotoxicity when NK cell inhibitory receptors losing "Self" recognition. As one important member of NCRs, NKp30 (NCR3) expressed on all NK cells and plays an important role in NK cell activation and cytotoxicity to tumor cells. In recent years, acculamated evidences have shown that NKp30 plays an important role in the crosstalk between the NK cells and dendritic cells (DCs), resulting in regulation of adaptive immunity. The functions of NKp30 have been studied widely in the past 10 years, however, the roles of NKp30 in NK cell activation and formation of NK cell immunological synapse are still not clear. In this study, we expressed the extracellular domain of human NKp30 with bacteria and prepared the soluble protein through dilution refolding procedure. We immunized mice with NKp30 recombinant protein as antigen to prepare anti-NKp30 monoclonal antibodies. Using immunofluorescence, Flow cytometry (FACS), Western blotting and 51Cr release assays, we investigated the roles of the NKp30 molecule involved in NK cell activation and formation of NK cell immunological synapse.The major results in our study were summarized as follows:1, Recombinant expression and purification of extracellular domain of human NKp30We constructed prokaryotic recombinant expression vector of extracellular domain of NKp30 and induced the expression of recombinant protein. The recombinant protein was expressed as inclusion bodies and identified by Western blotting and Mass Spectrometry as recombinant human NKp30(rhNKp30). After dilution refolding procedure and purification with nickel affinity chromatography, the purity of rhNKp30 can reach more than 95%. FACS and NK92 cytotoxicity results showed that the soluble rhNKp30 was functional and may be used as a potential diagnostic or therapy tool. 2, Preparation and application of murine monoclonal antibodies against NKp30With the soluble rhNKp30 protein as antigen, several strategies were applied to immunize 8-10 weeks old female BALB/c mice. After the fifth immunization, splenocyte were fused with SP2/0 cell. After screening and subcloning, four hybridoma strains were obtained and all can stably secret anti-NKp30 monoclonal antibodies (mAbs). One of the four mAbs, mAb 3G5 was highly specific against NKp30. Furthermore, mAb 3G5 can be widely used in Western blotting, Immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and FACS assays. Moreover, mAb 3G5 do not affect the binding of NKp30 to its ligands.3, NKp30 play important roles in NK cell activation and formation of NK cell immunological synapse51Cr release assay showed that NKp30 blocking antibody(clone,P30-15) significantly inhibited NK cell or NK92 cell line killing of various tumor cells, indicating that NKp30 plays an important role in NK cytotoxicity. After screening different target cells, we found NKp30 play a dominate role in NK92 cell cytotoxicity to Hela cells. So we choose NK92 cells as effector cells and Hela cells as target cells for further study about the roles of NKp30 in synapse formation and activation of NK cells. Confocal results showed that NKp30 and CD11a accumulated at the interface of NK92-Hela conjugates, suggesting that NKp30 participated in the formation of NK cell immunological synapse. FACS and immunofluorescence results showed that in a certain period of time (0-15min), the binding rate of NK92 and Hela cells was positively correlated and reached peak at 15 min. However, NKp30 blocking antibodies could not inhibit the binding rate of NK92 cells with Hela cells, indicating that NKp30 does not affect the formation of NK92-Hela conjugates. FACS results showed that Hela cells could stimulate NK92 cell degranulation and NKp30 blocking antibodies could obviously inhibit the degranulation effect. ELISA results showed that NKp30 blocking antibodies could inhibit the IFN-γsecretion of NK92 cells. Furthermore, Western blotting and 51Cr release assay showed that PI3K-Erk1/2 signaling pathway was involved in the NKp30 mediated NK cell activation.Conclusion: We prepared soluble and funtional rhNKp30 which provides a useful tool for the structure and functional research of NKp30 and may be used as a potential diagnostic or therapy tool. We obtained one mAb against NKp30 which was highly specific and could be widely used in Western blotting, Immunoprecipitation, and ELISA and FACS assays. We investigated the importane roles of NKp30 in in synapse formation and activation of NK cells. We observed that NKp30 molecules participated in the formation of NK cell immunological synapse, but did not affect the binding and formation of NK-target conjugates. NKp30 recognize its ligands on target cells leading to the activation of intracellular signal pathway such as PI3K-Erk1/2, thereby results in NK cell degranulation and cytokine secretion, which lead to NK cell cytolysis of target cells.