Role Of CD207/Langerin、CD8α And Functional Identification Of Langerhans Cells In Grass Carp

CD8α and CD207 are important phenotypic molecules on the surface of mammalian dendritic cells(DCs),which play an essential role in dendritic cell-mediated adaptive immunity,and CD207 is a characteristic surface marker molecule of Langerhans cells(LCs).This study aimed to elucidate the role of CD8α and CD207 in the anti-bacterial immune response of grass carp dendritic cells and characterize the phenotypic features of grass carp Langerhans cells and their biological functions.Proceed to the next step,exploring the connection between grass carp dendritic cells and the immune function of the organism.The main research contents and results of the experiment are as follows:1.Study on the expression of cd8α and CD207 and their anti-bacterial immunityThe CD8α and CD207 recombinant proteins were obtained by constructing the prokaryotic expression plasmid p ET-32a-CD8α/CD207,induced by IPTG.The polyclonal antibodies against CD8α and CD207 were prepared by immunizing New Zealand White Rabbit(Oryctolagus cuniculus).Western Blot verification showed that the antibodies could detect endogenous proteins at the tissue and cell levels in grass carp.CD8α was highly expressed in the head kidney,spleen,and intestine of grass carp,while CD207 was highly expressed in the spleen,skin,and gills of grass carp.After grass carp dendritic cells were treated with inactivated Aeromonas hydrophila,the m RNA and protein levels of CD8αwere continuously and significantly increased within 24 h.In comparison,those of CD207 significantly increased within 12 h after treatment,suggesting that CD8α and CD207 play essential roles in the prophase of bacterial infection.In order to further verify the functions of CD8α and CD207,we used CD8α and CD207 polyclonal antibodies to block the antibody against the grass carp dendritic cell-mediated mixed lymphocyte reaction in vitro.The results showed that after the dendritic cells were incubated with the polyclonal antibodies of CD8α and CD207,respectively,for antibody blocking,the ability of grass carp dendritic cells to receive lipopolysaccharide(LPS)stimulation and then activate the proliferation of mixed lymphocytes was significantly decreased.Functional analysis showed that grass carp DCs could present antigen-activated lymphocytes to initiate and regulate the immune response,in which CD8α and CD207 played essential roles.2.Identification and function of grass carp Langerhans cellsThe percentage of Langerhans cells in spleen mononuclear cells was about 0.2% by flow cytometry.Giemsa stain showed that the nuclei of grass carp Langerhans cells were kidney-shaped,and the cell bodies were irregular dendritic protrusions.After sorting by flow cytometry,Langerhans cells were demonstrated by q PCR to be highly expressed in co-stimulatory molecules(CD83,CD80/86,CD40),chemokine CCR7,transcription factors(CSF1R,TGF-β,BAFF)and to have abundant Toll-like receptors(TLR1,2,3,7,8,21,22).In contrast,Langerhans cells can be activated by CD40 ligand and involved in the body’s immune response via the CD40/CD40 L pathway.Transwell migration assay revealed that Langerhans cells could be chemotactic by Poly I:C,and Langerhans cells could present antigen to stimulate mixed lymphocyte proliferation after GCRV infection significantly.q PCR showed that Langerhans cell-related immune genes were significantly up-regulated after both Poly I:C and GCRV treatment.Moreover,zymosan could dramatically enhance the phagocytic ability of Langerhans cells and up-regulate the expression of immune-related genes.