Molecular Cloning, Functional Characterization Of Th Cell Related Molecule And Preliminary Identification Of Th Cell In Fish

This article focused on the molecular cloning, functional characterization of helper T cell related molecule and preliminary identification of helper T cell in Danio rerio and Tetraodon nigroviridis.In part one, we firstly found that the antibody production to thymus-dependent (TD) antigen also need the help of T cell in fish with the immunosuppressive agent specifically targets T cells (cyclosporin A) which indicates that Th-like-mediated cellular cooperation between T and B cells may have developed in the adaptive immunity as early as fish did during vertebrate evolution. To provide further evidence, we also performed phenotypic identification with THPOK, the specific transcription factor of help T cell.In part two, we successfully identified and cloned CD154 and CD40 homologs which mediated the cooperation of helper T cell and B cell in TD immune response in mammals from the zebrafish. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by TD antigen. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and enhanced by soluble CD154 or CD154-encoding plasmid in vivo. TD antigen-induced CD154 expression was inhibited by cyclosporin A suggesting that CD154 functionally associate with T cells. Double immunofiuorescence staining showed that CD40 and membrane IgM (mIgM) co-localized in B cells. CD154-CD40 binding assays showed CD154 specifically binds to CD40 at homodimeric form. The results of this part provide the first evidence for the existence of the functional CD154-CD40-mediated T-B cell cooperation underlying adaptive immunity in a fish species.In part three, we cloned and analyzed the TIM gene famlily which can be used to distinguish Th1 and Th2 subsets in mammals for the first time. The identification of the TIM gene family will contribute to a better understanding for the evolutionary history and function of them, but also lay a foundation for further research to identify the Th1 and Th2 subsets with them in fish.