Isolation, Identification And Target Cell Selection Of Chicken NK Cells

The natural killer(NK) cell is one of the most important lymphocytes, which plays critical roles in pathogenscontrol andgenetic resistance to diseases. However, until now, most of our knowledge about NK cells comes from mammalian, and we do not know whether it is applicable for other species. Chicken is the most extensively studied avian species, until now,we do not know much about chicken NK cells.Currently, the chicken NK cell is mainly isolated and cultured from14-day old embryonic spleens, and the identification is heavily depend on morpholog and natural killingfunction. BNK,located in the core region of major histocompatibility complex (MHC),is the C-type lectin receptor expressed on chicken NK cells.Therefore,it is predicted that chicken NK cells play critical roles in MHC-mediated genetic resistance to avian diseases.In this study, the established SPF MHC B19and B21haplotype chickens were used.Spleens isolated from14-day old embryos were disaggregated by cold trypsin. The isolated spleno lymphocytes were cultured in complete IMDM medium plus condition medium and chicken recombiant IL-2for48h. The suspensible cellswere harvested(i.g. chicken NK cells).The flow cytometry assay-based forward scatter and side scatter indicated that85.1%of them were morphologically similar. The Wright-Giemas’ staining assay showed large eosinophilic granules in one side of the cells. The immunophenotypic analysis indicated that the chNK cells did not express T eell and B cell markers of CD3, CD4and Bu-1, whereas,97.4%cells were stained brightly for MHC class I molecules and30.8%dimly for CD8a. These results demonstrated that chNK cells had been successfully isolated and cultured.The flow cytometry assay was established to detect chicken NK cells’natural killing function, and to detect the chicken NK cells cytotoxity to mouse and human NK cells target cell lines YAC-1and K562separately.The result showed that YAC-1was sensitive to the chNK cells lysis, but K562was not. This result showed that chicken NK cell could target mammalian cell line.What’s more,we found that MHC B21haplotype chNK cells specificly killed more YAC-1than B19haplotypes.The result suggested that chNK cells may affectchicken genetic resistance to diseases.Probing the pathways for chicken NK cells cytoxocity to YAC-1, MHC class I molecules and activating ligands RAE-1and H60were detected expressed on the surface of YAC-I, however, blocking the recognition of these molecules could not inhibit the specific cytotoxicity activity, which indicates that these molecules might not intervere with chicken NK cells cytotoxicity activity.BNK is NK cells inhibitory receptorgene. To benefit NK cells biological function study, BNK genes of B19and B21haplotypes were inserted and expressed with prokaryotic expression vector pET-30a and rabbit antiserum wereprepared, respectively. Western blot result showed that the antiserum could specifically detect BNK protein.The study will provide a solid foundation for further study chicken NK cells.