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" (tau) 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 V(H) families, but surprisingly, the IgT isotype possesses its own exclusive set of D(H) and J(H) elements for the generation of diversity. The IgT D and J segments and tau 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.

Fig. 1.

Comparative analysis of IgT in teleost fish. (A) Amino acid alignment of the rainbow trout (Onmy) IgT and zebrafish (Dare) IgZ C domains. C regions (C_H) are based on cDNA and germ-line sequences for rainbow trout. Cysteine and tryptophan residues typical of the Ig fold are in boldface type. Potential N-linked glycosylation sites are underlined, and residues differing between the IgT duplicates are shown immediately under the IgT.1 sequence. * denotes identity, and – indicates gaps. S, T, and Y residues found in the transmembrane region typical of associating with B cell coreceptors CD79A/B are in boldface type. (B) CDR3 junctions for three IgT VDJ rearrangements.

Fig. 2.

Phylogenetic relationships for individual vertebrate IgH C domains; individual Ig C domains were aligned by using clustalw, and trees were generated by using the neighbor-joining method, with genetic distances obtained by Poisson correction (scale bar below the tree). (A)C_H1 domain. (B)C_H3 domain. GenBank accession nos. follow. IgA: duck, U27222; chicken, S40610; human, BAC85198; and rabbit, X82116. IgE: rat, AAA41365; sheep, M84356; and horse, U15150. IgG: rabbit, K00752; sheep, X69797; and mouse, J00453. IgY: axolotl, X69492; and Xenopus, X15114. NAR and IgW: nurse shark NAR, U18701 and sandbar shark IgW, U40560. IgX: Xenopus, X13779. IgM: skate, M35185; shark, Y00840; Xenopus, J03631; Atlantic salmon, S48652; catfish, M27230; trout, AY870256; and zebrafish, CAI11475. IgT/Z: rainbow trout, AY870265; Atlantic salmon, TC29571; and zebrafish, AY643750.

Fig. 3.

Genomic organization of the rainbow trout and zebrafish IgH loci. Two major sequence contigs (107 and 14 kbp) resulted for the trout IgH.1 locus, which have been merged into a single contig within the δ gene. The three trout V_H genes are members of the V_H2, -8, and -5 families, respectively (18). The zebrafish locus (≈75 kbp) is based on ensembl supercontigs ctg14038, ctg1404, and ctg14057 and BAC sequences BX649502 and BX510335. ψ, pseudogenes; R, repetitive regions; →, transcriptional orientation.

Fig. 4.

Tissue-specific expression of IgM, IgD, and IgT in naïve rainbow trout. (A) Northern blot analysis (12 μg) by using RNA from 1-yr-old rainbow trout (Clear Springs strain). Th, thymus; Pn, pronephros; Mn, mesonephros; Sp, spleen; Lv, liver; Int, intestine; Mu, muscle; H, heart; Ts, testis. OSU-142 (OSU) and Hot Creek (HC) spleen samples. (B) RT-PCR analysis of IgM and IgT in select tissues. Hrt, heart; Ms, mesonephros; E1, day-12 embryo; E2, day-22 embryo; YS, yolk sac fry; Pbl, peripheral blood lymphocytes.