Characterization of TCRand CD8head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α.

Abstract

T lymphocytes play an important role in cellular and adaptive immunity in vertebrates. The mechanisms of the fish immune system are little studied because of the lack of population-specific antibodies. This study examined the expression of two T lymphocyte markers, TCRα (PoTCRα) and CD8α (PoCD8α) in the Japanese flounder (Paralichthys olivaceus). The expression of PoTCRα and PoCD8α was mainly detected in immune/mucosal tissues. Recombinant PoTCRα and PoCD8α were expressed in pET32a and pET259, respectively. Then, rabbit anti-PoTCRα serum and rat anti-PoCD8α serum were prepared. Using serum, the characteristics of TCR+ and CD8+ head kidney leucocytes (HKLs) were investigated. The results of laser scanning confocal microscopy (LSCM) demonstrated that TCRα and CD8α were transmembrane proteins localized on the cell surface. The populations of CD8α, CD8α, TCRα, and TCRαwere sorted by flow cytometry (FCM) and analysed using qRT-PCR. The results demonstrated that all TCRα/TCRαor CD8α/CD8αHKLs expressed IFN-γ. The CD4-1 and IgM transcripts were detected only in TCRαand CD8αcells. Furthermore, HKL mitogenesis was induced with concanavalin A (ConA) stimulation. Taken together, the results from LSCM and FCM analyses showed that mammalian and P. olivaceus TCRand CD8leucocytes share basic characteristics.