Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish

Abstract

During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype ''IgT'' () for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout VH families, but surprisingly, the IgT isotype possesses its own exclusive set of DH and JH elements for the generation of diversity. The IgT D and J segments and constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.development ͉ immunoglobulin A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells through the process of V(D)J recombination. This process is restricted solely to gnathostomes (jawed vertebrates); no evidence for either antibody gene fragments or the VDJrecombinase machinery has been identified in agnathan fish (lampreys and hagfish) (1, 2). The specific effector function (complement fixation, recognition by phagocytic cells, and secretion in mucosal tissues) depends on the nature of the isotype constant (C) region. In mammals, there are five Ig isotypes that possess distinct effector functions for secretory immunity. Ig⌴ is the only antibody isotype found universally in gnathostomes, and until 1997, teleosts (bony fish) were thought to possess only Ig⌴; however, research on catfish (3) and, later, on Atlantic salmon (4) provided evidence for the existence of IgD in teleosts. The genomic location of the teleost delta gene (␦) immediately downstream of , coupled with modest sequence identity to mammalian ␦ and coexpression of IgM and IgD in catfish B cell lines, solidified the relationship of teleost IgD to that of mammals. Expression of teleost IgD, like that of mammals, is achieved by alternative splicing from the rearranged VDJ gene to the C ␦ genes, with the exception that the teleost IgD message retains the first exon of C 1, thus forming a chimeric antibody isotype. After the discovery of Ig heavy-chain (IgH) genes in elasmob...

Early Diversification of the TNF Superfamily in Teleosts: Genomic Characterization and Expression Analysis

“…The synteny between teleost and human CD40L loci appears to be strong, while CD40-CD40L binding and glycosylation sites are not conserved. Currently, isotype switch regions have not been found in rainbow trout (39), and it has been proposed that progenitor B cells become either of two Ig lineages (40). With this in mind, teleost CD40L may be playing a limited or alternative role to its mammalian orthologs.…”

Reconstructing immune phylogeny: new perspectives

“…Furthermore, certain characteristics of BCR genomic organization that were previously thought to be restricted to cartilaginous fish, such as cluster-type gene organization, are also present in bony fish 26 (such as catfish) and fleshy-finned fish 27 (such as lungfish). Despite the divergence of heavy-chain types that has now been recognized in cartilaginous 28 and bony [29][30][31] fish, prototypical heavy-chain CLASS-SWITCH RECOMBINATION is not seen below the phylogenetic level of amphibians 32,33 . Given the DERIVED character of the extant bony fish, the resolution of whole genomes for the pufferfish 34 , the zebrafish (see the Online links box) and other bony-fish species could offer considerable insight into the mechanisms that have driven the evolution of BCR diversification.…”

Extensive diversification of IgH subclass-encoding genes and IgM subclass switching in crocodilians

“…As IgM and IgD(W) are now commonly accepted to be the most primordial IgH classes in evolution 17,21,22 , it is reasonable to conclude that all other IgH classes evolved from them through various molecular mechanisms such as gene duplication, conversion or recombination. These processes gave rise to IgNAR and IgZ(T) after the divergence of cartilaginous fish and bony fish, respectively [12][13][14] . In comparison with fish, two additional IgH classes, IgY and IgA(X), have evolved in tetrapods, including amphibians, reptiles and birds.…”

“…Both V(D)J recombination and SHM are utilized by all of the species that have been examined, whereas gene conversion has a major role only in birds 1,8,9 . Additionally, more than 10 genes encoding different Ig classes, such as IgM, IgD (IgW), IgNAR, IgZ (IgT), IgA (IgX), IgY, IgF, IgO, IgG and IgE, have been identified in various species 3,[10][11][12][13][14][15][16][17][18][19][20] , and IgG and IgA are further diversified into a variable number of subclasses in mammalian species. Differential diversification of the Ig classes or subclasses may have arisen from a long evolutionary period of environmental selection pressure, thus conferring survival advantages.…”

“…Logically, other IgH (H: heavy-chain genes) classes should be evolutionarily derived from IgM and IgD, likely through various mechanisms such as gene duplication, gene conversion or recombination. These processes led to the origin of IgNAR in cartilaginous fish and IgZ(T) in bony fish 13,14,23 . Although both IgY and IgA(X) appeared only after the emergence of tetrapods, the former likely preceded the latter in evolution based on the evolutionary evidence that IgA (X) resulted from a genetic recombination between IgM and IgY 24,25 .…”