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

Abstract

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...

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

“…As important immune cells, T/B lymphocytes play an irreplaceable role in cellular and humoral immunity in fish. In our previous study, the monoclonal antibodies against flounder IgM, CD4-1, CD4-2, and polyclonal antibodies against flounder CD8β molecule were produced, respectively, T and B lymphocytes were identified and characterized in flounder ( 29 – 31 ). In this study, T-bet was found to be expressed in sorted IgM + B lymphocytes, and also in CD4-1 + , CD4-2 + , and CD8 + T lymphocytes.…”

“…However, very few functional studies have been carried out on fish CD4 + cells due to a lack of tools. Recently, monoclonal antibodies against CD4 molecules were generated for ginbuna crucian carp, rainbow trout, and flounder ( 31 , 53 , 54 ). CD4 + cells have been identified using monoclonal antibodies, and the function of teleost CD4 + cells was demonstrated to be similar to that of mammalian Th cells.…”

“…The lymphocytes isolated from head kidney of flounder were incubated with the monoclonal antibodies against CD4-1, CD4-2, IgM or mouse polyclonal antibodies anti-CD8b, respectively (29)(30)(31). After the lymphocytes were incubated with the secondary antibody (Alexa Fluor 488-conjugated goat antimouse IgG, 1:1000 diluted in PBS), CD4-1 + , CD4-2 + , CD8 + T lymphocytes, and IgM + B lymphocytes were isolated by flow cytometry (BD FACSAria).…”

“…High purity of CD4-1 + T cells, CD4-2 + T cells, CD8 + T cells, and IgM + B cells was successfully sorted from head kidney lymphocytes using the previously developed monoclonal and polyclonal antibodies (29)(30)(31). Before sorting, T lymphocyte subsets and B lymphocytes contained different proportions in the head kidney lymphocytes, and the purity of the sorted cells could be 98.5% or more as seen in the flow cytometry results after sorting (Figure 6).…”

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus)

“…AB082957.1 andAB643633.1) were translated using Expasy (https://web.expasy.org/translate/). The signal peptide, transmembrane domain and immunoglobulin-like domains were predicted using Signal P 4.1 (http://www.cbs.dtu.dk/ services/SignalP-4.1/), TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and SMART software (http://smart.embl-heidelberg.de/), respectively [37]. The secondary structures and the physico-chemical characteristics of amino acids, including antigenicity, hydrophobicity, charge density and flexibility were analyzed using DNAStar Protean program.…”