Characterization of CD40+ leukocytes in flounder (Paralichthys olivaceus) and its response after Hirame novirhabdovirus infection and immunization

Xing, Jing; Tian, Hongfei; Wang, Lei; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

doi:10.1016/j.molimm.2018.11.001

Cited by 16 publications

(9 citation statements)

References 25 publications

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“…Polyclonal antibodies against flounder CD83, CD40, and MHCII, and monoclonal antibodies against flounder IgM and CD4 were previously produced in our laboratory ( 35 – 38 ), and all antibodies were diluted 1:1,000. The rabbit against OmpK antibodies were produced by Li et al.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

Liu

Xing²,

Tang³

et al. 2022

Front. Immunol.

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The CD80/86 molecule is one of the important co-stimulatory ligands and involves antigen-specific immune responses by ligating with CD28 and then delivering the required second signal to T-cell activation. In this study, a CD80/86 homolog was identified, and its expression characteristics were studied in flounder (Paralichthys olivaceus). The open reading frame (ORF) of CD80/86 is 906 bp, encoding 301 aa, and the extracellular amino acid sequence encoded two IgV- and IgC-like structural domains; fCD80/86 is highly expressed in head kidney, peripheral blood leukocytes (PBLs), and spleen, and has relatively high expression in muscle. Antibodies specific for CD80/86 were produced, and CD80/86 was colocalized with MHCII+, CD40+, and CD83+ leukocytes but not with IgM+, CD3+, or CD4+ lymphocytes. The cloned CD80/86 in flounder shares conserved structural features with its mammalian counterparts and is mainly distributed on antigen-presenting cells. Based on these data, CD80/86 as an adjuvant to enhance the immune response of DNA vaccine was investigated. A bicistronic DNA vaccine expressing both CD80/86 and the outer membrane protein (OmpK) of Vibrio anguillarum (p-OmpK-CD80/86) was successfully constructed. After immunization, p-OmpK-CD80/86 could induce the upregulation of the proportion of IgM+ and CD4+ cells in flounder, compared to the p-OmpK- or p-CD80/86-immunized group; CD28 genes were significantly induced in the p-CD80/86 and p-OmpK-CD80/86 groups. Compared to the p-OmpK group, the higher expression of CD83, MHCI, CD4, CD8, and IL-2 was detected at the injection site. The relative percent survival (RPS) produced by p-OmpK-CD80/86 is 66.11% following the V. anguillarum challenge, while the RPS of p-OmpK or p-CD80/86 is 46.30% and 5.56%, respectively. The results revealed that CD80/86 is mainly found in antigen-presenting cells, and could help elicit humoral immune responses in teleost through the CD80/86-CD28 signaling pathway involving CD4+ lymphocytes.

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Section: Methodsmentioning

confidence: 99%

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

Liu

Xing²,

Tang³

et al. 2022

Front. Immunol.

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“…The concentrations of rT-bet were determined by the Bradford method and rT-bet were used for immunization of New Zealand white rabbits according to a previous method ( 28 ). After three boosters, the serum samples were collected and purified by protein G agarose affinity chromatography (Pierce/Thermo Scientific), and then the rabbit anti-flounder rT-bet polyclonal antibodies (Abs) were obtained.…”

Section: Methodsmentioning

confidence: 99%

“…The induced recombinant Escherichia coli lysates and purified rT-bet were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue R-250. The concentrations of rT-bet were determined by the Bradford method and rT-bet were used for immunization of New Zealand white rabbits according to a previous method (28). After three boosters, the serum samples were collected and purified by protein G agarose affinity chromatography (Pierce/ Thermo Scientific), and then the rabbit anti-flounder rT-bet polyclonal antibodies (Abs) were obtained.…”

Section: Production Of Polyclonal Antibodies Against Flounder T-betmentioning

confidence: 99%

Identification and Characterization of a Master Transcription Factor of Th1 Cells, T-bet, Within Flounder (Paralichthys olivaceus)

et al. 2021

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T-bet, a T-box family member, is a transcription factor essential for the differentiation of naive CD4+ T cells into Th1 cells that are involved in both innate and adaptive immune responses. In this study, the transcription factor T-bet of flounder (Paralichthys olivaceus) was cloned and characterized, and its expression profile after infection was analyzed. T-bet+ cells were identified in flounder, and the expression and localization of T-bet in T lymphocyte subsets and B lymphocytes were investigated. Finally, the proliferation of T-bet+ cells, T lymphocyte subsets, and B lymphocytes were studied after stimulation with IFN-γ, IL-2, and IL-6, respectively, and the variations of some transcription factors and cytokines in CD4+ T lymphocyte subsets were detected. The results showed that T-bet in flounder consists of 619 aa with a conserved T-box DNA binding domain. T-bet was abundantly expressed in the spleen, head kidney, and heart, and it was significantly upregulated after infection with Vibrio anguillarum, Edwardsiella tarda, and Hirame rhabdovirus, especially in the group of Edwardsiella tarda. A polyclonal antibody against recombinant protein of T-bet was prepared, which specifically recognized the natural T-bet molecule in flounder. T-bet+ cells were found to be distributed in the lymphocytes of peripheral blood, spleen, and head kidney, with the highest proportion in spleen, and the positive signals of T-bet occurred in the cell nucleus. T-bet was also detected in the sorted CD4-1+, CD4-2+, CD8+ T lymphocytes, and IgM+ B lymphocytes. In addition, T-bet+ cells, coordinated with CD4-1+ and CD4-2+ T lymphocytes, were proliferated after stimulation with IFN-γ, IL-2, and IL-6. Especially in sorted CD4-1+ and CD4-2+ T lymphocytes, IFN-γ and IL-2 were able to upregulate the expression of T-bet, forming a positive feedback loop in Th1-type cytokine secretion. These results suggest that T-bet may act as a master transcription factor regulating flounder CD4+ T lymphocytes involved in a Th1-type immune response.

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“…The mass spectrometry analysis experiments were performed by Sangon Biotech (Shanghai, China). Indirect immunofluorescence staining and flow cytometry were also performed as previously described 53 , in which the rabbit anti-CD3ɛ polyclonal antibody 54 (diluted 1:1,000 in PBS) was mixed with mouse anti-CD4-1 or CD4-2 monoclonal antibody (diluted 1:1,000 in PBS) as the primary antibody. The indirect immunofluorescence staining results were observed under a fluorescence microscope (Olympus DP70, Tokyo, Japan), and the results of flow cytometry were analyzed on an Accuri C6 flow cytometer (BD, USA).…”

Section: Methodsmentioning

confidence: 99%

Kinetics of T lymphocyte subsets and B lymphocytes in response to immunostimulants in flounder (Paralichthys olivaceus): implications for CD4+ T lymphocyte differentiation

Xing

Tian

Tang

et al. 2020

Sci Rep

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CD4 + T lymphocytes play crucial roles in the adaptive immune system. CD4, as the most effective marker to delineate the T-helper subsets, was identified in many fish species. Two CD4 homologs, CD4-1 and CD4-2, have been reported in flounder (Paralichthys olivaceus). In this study, monoclonal antibodies (mAbs) against CD4-1 and CD4-2 of flounder were produced, CD4 + T lymphocytes were isolated and identified, and the variations in CD4 + and CD8 + t lymphocytes and igM + B lymphocytes after Poly I:C, PMA or β-glucan stimulation were investigated. Then, the expression of transcription factors and cytokines in sorted CD4 + T lymphocytes was analyzed. The results showed that the mAbs were specific to flounder CD4-1 + and CD4-2 + T cells. CD4-1 + and CD4-2 + cells responded to all three stimulants, while CD8 + T lymphocytes only give a strong response to Poly I:C, and the percentages of igM + B lymphocytes showed a tendency to increase. After stimulation, the expression of transcription factors and cytokines of Th1, Th2 and Th17 cells varied in CD4 + T cells. These results will provide crucial foundations for the differentiation and function of teleost CD4 + t lymphocytes. CD4 + T cells, also known as T helper (Th) cells, play essential roles in the function of the immune system 1,2. They display extensive functional and phenotypic diversity in response to pathogens and promote cancer surveillance and tolerance to "self " antigens and environmental allergens 2. These functions are performed through specific antigen (Ag) recognition by CD4 + T cells and subsequent secretion of effector and regulatory cytokines 3. Upon Ag encounter, the T cell co-receptor CD4 present on T helper cells binds to the peptide/MHC (pMHC) class II molecules on antigen-presenting cells and transduces activation signals 4,5. Activated CD4 + Th cells can further differentiate into a variety of effector Th cell subsets, such as Th1, Th2, Th17 cells and regulatory T (Treg) cells 6 , and Th1 and Th2 cells were discovered to be the dominant populations of Th cells 7. The differentiation of Th cells towards a Th1 profile occurs in the presence of IL-12 and IFN-γ and is controlled by the master transcription factor T-bet 6. These Th1 cells secrete effector cytokines such as IFN-γ, TNF-a and IL-2 that stimulate macrophages and cytotoxic T lymphocytes (CTLs), which play important roles in cellular immunity against intracellular pathogens 6. Th2 cells, which are promoted by IL-4 and induction of GATA-3, produce cytokines IL-4, IL-5 and IL-13 that stimulate B cells to secrete different antibody isotypes and thus control extracellular infections 6,8,9. Th17 cells produce cytokines, including IL-17, IL-22, and IL-26, and they express the master transcription factor RORγt. They have a role in host defense against extracellular bacteria, fungi, inflammation and autoimmune diseases 10. Treg cells, which are regulated through Foxp3, have an essential role in regulating Th1-, Th2-, and Th17-type responses and are responsible for peripheral tolerance 11. Addition...

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Characterization of CD40+ leukocytes in flounder (Paralichthys olivaceus) and its response after Hirame novirhabdovirus infection and immunization

Cited by 16 publications

References 25 publications

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

Identification and Characterization of a Master Transcription Factor of Th1 Cells, T-bet, Within Flounder (Paralichthys olivaceus)

Kinetics of T lymphocyte subsets and B lymphocytes in response to immunostimulants in flounder (Paralichthys olivaceus): implications for CD4+ T lymphocyte differentiation

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