The Investigation On Biological Functions Of Natural Killer Receptors NKp80 And NKG2F

Natural killer (NK) cells play important roles in innate immunity. They are able to distinguish malignant cells or pathogen-infected cells from normal cells and kill these abnormal cells without prior sensitization, as a result, natural killer cells react quickly to dangerous signals and act as the first line of defense against pathogens and transformed cells.NK cells are able to distinguish the abnormal cells from normal cells effectively, which is mediated by the NK cell receptors expressed on the NK cell surface.Currently accepted theory to explain this phenomenon as the "Missing Self" mechanism:NK cells received both activating signals and inhibitory signals through their surface receptors. Normal cells expressed sufficient HLA molecules, which was able to bind to the inhibitory receptors on the membrane of NK cell (human leukocyte antigen-specific inhibitory receptors) and transduce inhibitory signals, resulting in the inhibition of NK cell cytotoxicity. As a result, NK cells won't attack normal cells. However, expression of HLA molecules of pathogen-infected cells or transformed cells was greatly down-regulated. As a result, NK cells couldn't receive sufficient inhibitory signals, and NK cells were staying at activating state with strong cytotoxicity, resulting in elimination of these target cells.NK cell receptors were divided into two groups as activating receptors and inhibitory receptors depending on the signal they transduced. NK cell receptors play crucial roles in the biological functions of NK cells. A lot of NK cell receptors have been identified, some of which has been deeply investigated. The ligands and the 3-D structure information of these receptors has been identified, moreover, the specific monoclonal antibodies against these receptors has also been produced.However, few studies have been conducted for some newly identified NK cell receptors, whose biological functions were not clear yet.NKp80 and NKG2F were newly identified, whose biological functions were not clear. This work focused on the two NK cell receptors, aiming at a further progress of the biological functions.NKp80 has been reported to transduce activating signals, leading to NK cell activation and increase of NK cell cytotoxicity. AICL has been identified as the ligand of NKp80. But the interaction sites of NKp80-AICL were not clear. In this study,3-D model of AICL was constructed with the online server 3D-JIGSAW based on the crystal structure of CD69 which shared the highest sequence homology among released protein structures in PDB.7 proteins with highest sequence homology to AICL were listed by BLAST. Multiple sequence alignment of the amino acid sequence of these proteins was conducted by the tool Clustal W2, and 7 conserved sequences of AICL were found. Among these conserved sequences, three sequences were on the surface of the 3-D model of AICL which was constructed based on the crystal structure of CD69. Flow cytometric analysis demonstrated that these peptides were able to compete with anti-NKp80 mAb on NKp80 binding activity in a dose-dependent manner. Moreover, two peptides reduced NKp80-AICL mediated cytotoxicity of both fresh PBMCs and purified NK cells in 51Cr release cytotoxicity assay. Considering both the bioinformatic analysis and experimental results, we concluded that the P1 and P2 sequences on AICL might be the potential sites of NKp80-AICL interaction.Previous studies have demonstrated that NKG2F contained an immunoreceptor tyrosine-based inhibition motif (ITIM) like sequence in cytoplasmic domain, however, NKG2F also associated with DAP12 which contained an immunoreceptor tyrosine-based activation motif (ITAM). It was controversial whether NKG2F is an activating receptor or an inhibitory receptor.In this study, we recombinantly expressed the human NKG2F protein with the E. coli for the first time. Anti-NKG2F polyclonal antibody was produced by immunizing BALB/c mice with the recombinant NKG2F.With the anti-NKG2F polyclonal antibody, real-time PCR and flow cytometry were performed. The results demonstrated that IL-2 and IL-15 stimulation up-regulated the expression of NKG2F expression on the surface of peripheral blood NK cells. As NK cell were activated by IL-2 and IL-15 stimulation, we considered that NKG2F as an activating receptor. Furthermore, the flow cytometry results demonstrated that NKG2F was expressed on the surface of peripheral blood NK cells for the first time, which was consistent with other members of NKG2 family.