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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...

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.

Cluster of differentiation (CD) antigens are cell surface molecules expressed on leukocytes and other cells associated with the immune system. Antibodies that react with CD antigens are known to be one of the most essential tools for identifying leukocyte subpopulations. T lymphocytes, as an important population of leukocytes, play essential roles in the adaptive immune system. Many of the CD antigens expressed on T lymphocytes are used as surface markers for T lymphocyte classification, including CD3, CD4 and CD8 molecules. In this review, we summarize the recent advances in the identification of CD molecules on T lymphocytes in teleosts, with emphasis on the functions of CD markers in the classification of T lymphocyte subsets. We notice that genes encoding CD3, co-receptors CD4 and CD8 have been cloned in several fish species and antibodies have been developed to study protein expression in morphological and functional contexts. T lymphocytes can be divided into CD4+ and CD8+ cells discriminated by the expression of CD4 and CD8 molecules in teleost, which are functionally similar to mammalian helper T cells (Th) and cytotoxic T cells (Tc), respectively. Further studies are still needed on the particular characteristics of teleost T cell repertoires and adaptive responses, and results will facilitate the health management and development of vaccines for fish.

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