Characterization Of Fish CD4 Molecules And Further Identification Of Regulatory T Cells In Lower Vertebrate

CD4 is a crucial transmembrane glycoprotein for cellular immunity.Here we describe three CD4-like molecules in both pufferfish(Tetraodon nigroviridis) and zebrafish(Danio rerio).Although sharing an average amino acid identity of 13.8%to human CD4,they exhibit conserved molecular structures such as extracellular Ig domains with potential N-glycosylation sites and intracellular tyrosine kinase p56^lck binding sites.Immunofluorescence stain show that two major CD4 positive subsets exist in fish lymphocytes,as CD4-2^-CD4-4⁺ and CD4-2⁺CD4-4⁺.The latter population can be induced by mitogens,which is also the target of lymphocyte chemoattractant factor IL-16.It is suggested that CD4-2,rather than CD4-4,plays an essential role in fish lymphocytes.Our results will provide a deeper insight into CD4 evolution and function as well as the fish T cell immune responses.CD4⁺CD25⁺Foxp3⁺ regulatory T cells(Treg cells) are critical for the maintenance of peripheral tolerance,and the suppression of autoimmune diseases and even tumors. Although much is known about Treg cells from humans and some mammals,little is known regarding their existence or occurrence in lower vertebrates.In the present study,we describe the molecular and functional characterization of a Treg-like subset with the phenotype CD4⁺CD25⁺Foxp3⁺ from a pufferfish(Tetraodon nigroviridis) model.Functional studies show that depletion of this subset produce an enhanced mixed lymphocyte reaction(MLR) and nonspecific cytotoxic cell(NCC) activity in vitro,as well as inflammation of the intestine in vivo.Our results provide the first evidence for the existence of Treg-like regulatory mechanism underlying cellular immunity in a fish species.This strongly suggests that precise T subsets may have developed during early vertebrate evolution.The data presented here will not only be beneficial for better understanding of the origin of both Treg and Treg-mediated regulatory networks in cellular immunity,but will also present a novel fish model for the investigation of Treg-based clinical investigations and therapeutic applications.