Production and characterization of monoclonal antibodies against grass carp CD4-1 and CD4-2

CD4 T helper cells are an important group of cells in the immune system of vertebrates and express CD4 receptor on the cell surface.In mammals,the CD4 receptor is encoded by a single copy gene,whilst in fish,two copies of cd4 genes,namely cd4-1 and cd4-2,are found.In this study,the ectodomains of grass carp(Ctenopharyngodon idella,Ci)CD4-1 and CD4-2 were expressed in the E.coli cells and used to generate monoclonal antibodies in mice.Western blotting,confocal microscopy and flow cytometry were performed to characterize the monoclonal antibodies.It has been shown that the CiCD4-1 and CiCD4-2 monoclonal antibodies had good specificity to react with the recombinant ectodomains of CiCD4-1 and CiCD4-2 expressed in the CHO-S cells and the native CD4 molecules of grass carp.The CiCD4-1 monoclonal antibody did not recognize CiCD4-2 and verse versa.In addition,the CD4-1 and CD4-2 monoclonal antibodies specifically recognized the CD4-1 and CD4-2 receptors expressed in the HEK293 cells and native molecules of fish cells.Further,the percentages of lymphocytes in immune tissues of healthy fish were analyzed by flow cytometry.It was found that 17.6%of lymphocytes were CD4-1^(+)cells and 22.5%were CD4-2^(+)in the head kidney.In the spleen,13.1%of lymphocytes were CD4-1^(+)and 18.6%were CD4-2^(+)while 7.3%of blood lymphocytes were CD4-1^(+)cells and 8.8%were CD4-2^(+)cells.The availability of the CD4-1 and CD4-2 monoclonal antibodies provides antibody-based tools for further elucidation of the functions of CD4 T cells in grass carp.

Distinct expression profiles and overlapping functions of IL-4/13A and IL-4/ 13B in grass carp (Ctenopharyngodon idella)

In mammals,interleukin(IL)-4 and IL-13 are important cytokines involved in the immune response of the host defence against pathogens and play critical roles in the polarization of monocytes/macrophages(MO/Mϕs).However,in teleost fish,the role of IL-4/13 homologues in the polarization of MO/Mϕremains unknown.In this paper,we identified a second isoform of IL-4/13(CiIL-4/13A)from grass carp(Ctenopharyngodon idella)in addition to the IL-4/13 homologue previously reported and comparatively studied their expression profiles and functions.The two CiIL-4/13 transcripts were constitutively expressed in tissues but displayed distinct expression patterns.Infection with Flavobacterium columnare or grass carp reovirus II(GCRV-Ⅱ)resulted in the alteration of expression levels of both IL-4/13 isoforms.Furthermore,in the primary head kidney cells,the recombinant CiIL-4/13A and B could up-regulate the expression of IL-10,inducible nitric oxide synthase(iNOS)and M2 macrophage marker gene(KLF4,kruppel like factor 4),whilst down-regulate the expression of IL-1βand M1 macrophage marker gene(MARCO,macrophage receptor with collagenous structure).The results suggest that the CiIL-4/13A and B play similar roles in regulating inflammation and differentiation of MO/Mϕs in grass carp.