Characteristics of CD4-1 gene and its immune responses against Aeromonas veronii infection by activating NF-κB signaling in Qihe crucian carp Carassius auratus.

CD4-1 found in bony fish contains four extracellular immunoglobulin (Ig)-like domains similar to that of mammalian CD4, which is crucial for the activation of CD4+ helper T-cell. However, there is limited knowledge regarding the molecular markers, immune functions and regulation mechanism of CD4-1 in teleosts due to their vast diversity. In this study, we cloned and characterized two isoforms of Qihe crucian carp CD4-1, designated as CaCD4-1.1 and CaCD4-1.2. We further explored their expression responses upon stimulation with Aeromonas veronii, and the regulation of their immune responses against A. veronii by NF-κB. The ORF of CaCD4-1.1 and CaCD4-1.2 cDNA encoded 477 and 466 amino acids, respectively. Both proteins contained seven conserved cysteine residues in the extracellular domain, and a CCC motif in their cytoplasm, respectively. However, CaCD4-1.1 exhibited a relatively limited similarity with CaCD4-1.2 in the ectodomain. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the mRNA expression of CaCD4-1.1 and CaCD4-1.2 exhibited differential constitutive expression across all examined tissues. Furthermore, the expression level of CD4-1.2 was higher than that of CD4-1.1 in the gills, head kidney, and spleen of Qihe crucian carp subjected to A. veronii challenge, while it was lower in the trunk kidney. Inhibition of NF-κB activity resulted in a decrease in the expression levels of CD4-1.1 and CD4-1.2 mRNA in the gill, while inducing an increase in expression levels in the spleen, in accordance with the observed ultrastructural changes in both organs. Interestingly, the impact of NF-κB on the mRNA expression level of CD4-1.1 appears to be stronger than that of CD4-1.2. Our results suggest that CaCD4-1.1 and CaCD4-1.2 could be expressed on T cells and antigen-sampling cells that exhibit similar characteristics to mammalian M cells, respectively, and differentially regulated by NF-κB in adaptive immune responses against bacterial infection. This research contributes to a better understanding of the crucial role of CD4-1 in the immune response of Qihe crucian carp and provide novel insights for the prevention and treatment of fish diseases in aquaculture.

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii.

In this study, we established a murine cell line that expresses ginbuna crucian carp (ginbuna) CD4-2 and used it to develop an anti-CD4-2 monoclonal antibody (mAb). An established mAb, named D5, showed good reactivities to BALB/c 3T3 cells expressing CD4-2 and a lymphocyte population in the ginbuna leukocytes. Gene expression analysis showed that D5+ cells express CD4-2 and TCRß genes but not CD4-1 and IgM genes, meanwhile May Grunwald-Giemsa staining of sorted D5+ cells had the typical morphology of lymphocytes. Two-color immunofluorescence analysis with anti-CD4-1 mAb (6D1) and anti-CD4-2 mAb (D5) by flow cytometry revealed that the percentages of CD4-1 single positive (SP) and CD4-2 SP lymphocytes were comparatively higher than CD4-1/CD4-2 double positive (CD4 DP) lymphocytes in all tissues examined in ginbuna. The highest percentage of CD4-2 SP cells (∼40%) was found in the thymus, while the head-kidney exhibited the highest percentages of CD4-1 SP (∼30%) and CD4 DP (∼5%) cells. These findings indicated that ginbuna CD4+ lymphocyte population consists of two major subpopulations (CD4-1 SP and CD4-2 SP) and a minor subset (CD4 DP).

Production of monoclonal antibody against grouper (Epinephelus coioides) CD4-1 and the distribution of CD4-1+ cells.

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1+ cells in the various tissues from grouper. Tissue distribution analyses revealed that the grouper CD4-1+ cells were expressed in all tissues tested in the healthy grouper, with greater localization in the thymus, head kidney, and spleen tissues. In addition, we tested the changes in the proportion of CD4-1+ cells in the thymus, head kidney, and the gills of grouper post the infection by C. irritans. Our data suggest that the CD4-1 mAb produced against grouper in the current study can be used as a tool to characterize CD4-1+ cells and to investigate the functions of the grouper CD4-1+ cells in the host response against pathogens infection.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Involvement of CD4-1 T cells in the cellular immune response of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and nervous necrosis virus (NNV) infection.

The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.