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CD154-CD40 Interactions Are Essential for Thymus-Dependent Antibody Production in Zebrafish: Insights into the Origin of Costimulatory Pathway in Helper T Cell-Regulated Adaptive Immunity in Early Vertebrates¹

College of Life Sciences, Zhejiang University, Hangzhou, People’s Republic of China; and Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Hangzhou, People’s Republic of China

The CD154-CD40-mediated costimulatory pathway is critical for T-B cell cooperation in thymus-dependent (TD) immune response in mammals. However, little is known about its existence and occurrence in lower vertebrates. Here, we report on the identification and functional characterization of CD154 and CD40 homologs from the zebrafish (Danio rerio) model. Zebrafish CD154 is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, whose tertiary structure is a sandwich containing two stacked sheets with "jelly roll" topology, just as the human TNF members do. The zebrafish CD40 is a type I membrane-bound protein with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The consensus TNFR-associated factor (TRAF)2- and TRAF6-binding motifs in mammalian CD40 are found in the cytoplasmic tail of zebrafish CD40, which indicates similar signal transduction mechanisms to higher vertebrates. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by thymus-dependent Ag. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and it was enhanced by soluble CD154 or CD154-encoding plasmid in vivo. Thymus-dependent Ag-induced CD154 expression was inhibited by cyclosporin A, suggesting that CD154 functionally associates with T cells. Double immunofluorescence staining showed that CD40 and membrane IgM colocalized in B cells. CD154-CD40 binding assays showed that CD154 specifically binds to CD40 at homodimeric form. Our results provide the first evidence for the existence of the functional CD154-CD40-mediated costimulatory pathway and helper T cell regulatory mechanism underlying adaptive immunity in a fish species.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Basic Research Program of China (973; 2006CB101805), Hi-Tech Research and Development Program of China (863; 2008AA09Z409), the National Natural Science Foundation of China (30871936, 30571423), and the Science and Technology Foundation of Zhejiang Province (2006C12038, 2006C23045, 2006C12005).

² Address correspondence and reprint requests to Li-Xin Xiang and Dr. Jian-Zhong Shao, College of Life Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, People’s Republic of China. E-mail addresses: xianglx{at}zju.edu.cn and shaojz{at}zju.edu.cn

³ Abbreviations used in this paper: TD, thymus dependent; BCA, bicinchoninic acid; CRD, cysteine-rich domain; CsA, cyclosporin A; EST, expressed sequence tag; KLH, keyhole limpet hemocyanin; THD, TNF homology domain; TI, thymus independent; TMB, tetramethylbenzidine; TRAF, TNFR-associated factor; UTR, untranslated region; zCD154, zebrafish CD154; zCD40, zebrafish CD40.