Development of a monoclonal antibody against the CD3ε of olive flounder ( Paralichthys olivaceus ) and its application in evaluating immune response related to CD3ε

Jung, Jin Tae; Lee, Jung Seok; Kim, Young Rim; Im, Se Pyeong; Kim, Si Won; Lazarte, Jassy Mary S.; Kim, Jae-Sung; Thompson, Kim D.; Suh, Jong Pyo; Jung, Tae Sung

doi:10.1016/j.fsi.2017.04.016

Cited by 22 publications

(22 citation statements)

References 37 publications

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“…However, studies on the origin and primordial roles of helper T cell subsets in teleosts have been hindered by the lack of appropriate tools [1,16]. In previous studies, we produced mAbs against CD3ε and CD4-1 lymphocytes of olive flounder and confirmed their specificity and identity through Western blotting, flow cytometry and immunofluorescence staining [1,14]. In order to investigate the functions of CD4-2-positive T cells in teleosts, a monoclonal antibody against CD4-2 lymphocytes from olive flounder was produced in the present study.…”

Section: Discussionmentioning

confidence: 87%

“…Leukocyte populations isolated from tissues contain not only lymphocytes but also granulocytes, monocytes and thrombocytes, with a similar size to lymphocytes under the microscope and in flow cytometry analysis [17,26]. Through previous studies, we revealed that the mAbs we produced specifically detect lymphocytes and did not target other cells of a similar size, using Giemsa staining and flow cytometry analysis [1,14]. The small cells recognized by mAb (3C8) were believed to be lymphocytes, because their dot plot profile in the flow cytometry was characteristic of lymphocytes [2,16,27,28], and very low populations of monocytes, macrophages and thrombocytes were present in the kidney and spleen [17,29,30].…”

Section: Discussionmentioning

confidence: 93%

“…To show that the reactivity of mAb 3C8 was against only CD4-2 cells of olive flounder, and not against the CD4-1 molecule, we screened recombinant CD3ε, CD4-1 and CD-2 proteins and T cells with the specific mAbs we produced in previous studies against CD3ε and CD4-1 [1,14]. Anti-CD3ε mAb (4B2) and anti-CD4-1 mAb (10F8) detected 63 ng to 500 ng of recombinant CD3ε and CD4-1 protein, respectively.…”

Section: Two-color Flow Cytometry To Confirm That the Staining Of Mabmentioning

confidence: 99%

“…Monoclonal antibodies (mAbs) against cell surface markers are one of the most frequently used tools for characterizing mammalian leukocytes, and these are also used for a variety of research and therapeutic applications [1,14]. Although the number of studies characterizing components of the immune response of teleosts is increasing, studies investigating T cell populations and their function are hindered by the lack of specific mAbs for characterizing target T cells [11,[14][15][16]. Many of the mAbs that have been developed against cell markers of teleost leukocytes so far focus on B cells, granulocytes and thrombocytes.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

Jung

Chun

Lee

et al. 2020

IJMS

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The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 93%

Section: Two-color Flow Cytometry To Confirm That the Staining Of Mabmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

Jung

Chun

Lee

et al. 2020

IJMS

Self Cite

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“…The microplate wells were firstly coated with 100 µL of purified Cµ 1 protein as an antigen and incubated overnight at 4 • C. The wells were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST; pH 7.4) and blocked with 100 µL of BlockPRO™ Blocking Buffer (Visual Protein, Taipei City, Taiwan) for 1 hour at 37 • C. During the washing period, the 100 µL of serial dilutions of test fish sera from 1:100 to 1:10,240 were preincubated with 100 µL of enzyme-labelled mAb IgMHCµ 1 primary antibodies for 1 hour and then added mixture to the wells and incubated for 1 hour at 37 • C. The cELISA began when any of the unbound enzyme-labelled mAb IgMHCµ 1 in the fish serum in the preincubation step binds to the coated recombinant Cµ 1 antigen. After washing, the microplate wells were incubated with 100 µL of secondary HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St Louis, USA) diluted 1:12000 in PBS (pH 7.4) for 1 hour at 37 • C. After the last washing, 100 µL of TMB One Component HRP Microwell Substrate (Surmodics IVD, Inc., Eden Prairie, MN, USA) was added to each well, and the plates were incubated for 30 minutes at room temperature in the dark [45,59]. Then, the reaction was stopped with TMB Stop Solution (Surmodics IVD, Inc.).…”

Section: Indirect Elisa Analysismentioning

confidence: 99%

Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish

2020

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Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cμ1 of the IgMH of catfish (IgMHCμ1 mAb) was developed in a rabbit model using sequence information from bighead catfish (Clarias macrocephalus). The IgMHCμ1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish (Clarias gariepinus) and their hybrid (C. macrocephalus × C. gariepinus), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ1 mAb successfully recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0–39.9%, 45.6–53.2%, and 58.7–60.0%, respectively. Furthermore, the IgMHCμ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish.

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Glutamine Metabolism Underlies the Functional Similarity of T Cells between Nile Tilapia and Tetrapod

Wei

Jiao

et al. 2023

Advanced Science

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As the lowest organisms possessing T cells, fish are instrumental for understanding T cell evolution and immune defense in early vertebrates. This study established in Nile tilapia models suggests that T cells play a critical role in resisting Edwardsiella piscicida infection via cytotoxicity and are essential for IgM+ B cell response. CD3 and CD28 monoclonal antibody crosslinking reveals that full activation of tilapia T cells requires the first and secondary signals, while Ca2+–NFAT, MAPK/ERK, NF‐κB, and mTORC1 pathways and IgM+ B cells collectively regulate T cell activation. Thus, despite the large evolutionary distance, tilapia and mammals such as mice and humans exhibit similar T cell functions. Furthermore, it is speculated that transcriptional networks and metabolic reprogramming, especially c‐Myc‐mediated glutamine metabolism triggered by mTORC1 and MAPK/ERK pathways, underlie the functional similarity of T cells between tilapia and mammals. Notably, tilapia, frogs, chickens, and mice utilize the same mechanisms to facilitate glutaminolysis‐regulated T cell responses, and restoration of the glutaminolysis pathway using tilapia components rescues the immunodeficiency of human Jurkat T cells. Thus, this study provides a comprehensive picture of T cell immunity in tilapia, sheds novel perspectives for understanding T cell evolution, and offers potential avenues for intervening in human immunodeficiency.

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Development of a monoclonal antibody against the CD3ε of olive flounder ( Paralichthys olivaceus ) and its application in evaluating immune response related to CD3ε

Cited by 22 publications

References 37 publications

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish

Glutamine Metabolism Underlies the Functional Similarity of T Cells between Nile Tilapia and Tetrapod

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