Characterizations of CD4-1, CD4-2 and CD8β T cell subpopulations in peripheral blood leucocytes, spleen and head kidney of Japanese flounder (Paralichthys olivaceus)

Publication date: May 2017
Source:Molecular Immunology, Volume 85
Author(s): Jing Xing, Junjie Ma, Xiaoqian Tang, Xiuzhen Sheng, Wenbin Zhan
In the previous study, antibodies against CD3 molecule have been produced and were used in labeling T cells in Japanese flounder (Paralichthys olivaceus). In this paper, CD4⁺ and CD8⁺ lymphocytes subpopulations in peripheral blood leucocytes (PBL), spleen and head kidney of flounder were investigated. The flounder CD4-1, CD4-2 and CD8β recombinant proteins and their antibodies (Abs) were produced, then the cross-reactivity of the Abs to CD4-1, CD4-2 and CD8β was detected by Western blotting, respectively, and the reactions of Abs to PBL were analyzed by immunofluorescence staining (IFS) and flow cytometry (FCM). CD4-1⁺/CD3⁺, CD4-2⁺/CD3⁺, and CD8β⁺/CD3⁺ lymphocytes in PBL, spleen and head kidney were observed by double IFS, then their proportions were analyzed using two-color FCM, respectively. Further, CD4-1/CD8β, CD4-2/CD8β, or CD4-1/CD4-2 lymphocytes were analyzed using double-IFS and two-color FCM. Finally, CD4-1⁺, CD4-2⁺, and CD8β⁺ lymphocytes in spleen and head kidney were observed by immunohistochemistry. The results showed that the Abs were specific for CD4-1, CD4-2 and CD8β molecules, respectively. The proportions of CD4-1⁺/CD3⁺, CD4-2⁺/CD3⁺, and CD8β⁺/CD3⁺ lymphocytes were 6.7±2.0%, 8.6±2.8%, 2.1±1.3% in PBL; 13.6±3.6%, 15.6±5.2%, 2.8±1.4% in spleen; 20.0±4.6%, 20.5±4.6%, 3.2±1.5% in head kidney, respectively. Most CD4⁺ and CD8⁺ cell subpopulations belonged to CD3⁺ cells; there were no cross-reactivity between CD4⁺ and CD8⁺ cells. CD4-1⁺/CD4-2⁻, CD4-1⁻/CD4-2⁺, and CD4-1⁺/CD4-2⁺ cells presented different proportions in PBL, spleen and head kidney, among them, CD4-1⁺/CD4-2⁺ cell is the majority of CD4T cell subpopulation.



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