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 Vertebrates

Abstract

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.

An evolutionarily conserved program of B-cell development and activation in zebrafish

“…These results explain why IgM secretion is not detected until 4 weeks after intraperitoneal immunization of zebrafish or trout. 35,36 We found increases in B cell numbers in the kidney, spleen, gut, and PEX after intraperitoneal immunization, although the most dramatic increases in antigen-binding B cells were in the spleen and gut. Correspondingly, these sites were reported to be likely sites of immune responses to bacterial infections in zebrafish.…”

Essential Roles of TIM-1 and TIM-4 Homologs in Adaptive Humoral Immunity in a Zebrafish Model

“…The coding sequences for the extracellular region of DrTIM-1 (DrTIM1ex) or DrTIM-4 (DrTIM-4ex) and the open reading frame (ORF) of sDrTIM-4 were subcloned into pET28a and pET41b (Invitrogen) to construct prokaryotic expression vectors (pET28a-DrTIM-1ex, pET28a-DrTIM-4ex, pET41b-DrTIM-4ex, and pET41b-sDrTIM-4) with His-tags and GST-tags (29). The ORFs of DrTIM-1 and DrTIM-4 were inserted into pEGFP-N1 (Clontech), pCMV-Tag2B (Stratagene), and pcDNA6/myc-His B (Invitrogen) to construct eukaryotic expression vectors (pEGFP-DrTIM-1, pEGFP-DrTIM-4, pCMV-DrTIM-1, and pcDNA6-DrTIM-4) with enhanced GFP, flag-tag, and myc-tag, respectively (42).…”

“…The PCR products were purified from agarose gel (1.2%) by using a gel extraction kit (Qiagen), inserted into the pGEM-T EASY vector (Promega), and then transformed into competent Escherichia coli TOP 10 (Invitrogen). The positive plasmid DNA was purified following the Miniprep protocol (Omega Bio-tek) and sequenced on an ABI 3730 sequencer (Invitrogen) as previously described (29).…”

Use of Microarray Technology to Improve DNA Vaccines in Fish Aquaculture - The Rhabdoviral Model

“…Nevertheless, the use of cds as vaccine adjuvants has been described for the cd154 gene in zebrafish (Gong et al, 2009); the reasoning being that co-expression of cds with antigen in the same cell might accelerate specific immune responses. Thus, specific antibody responses obtained using cd154 and the pMCV1.4 plasmid coding for the G gene of VHSV (Ruiz et al, 2008) were increased 3-4-fold with respect to the plasmid alone (Gong et al, 2009). …”