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

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.