A second TCRδ locus in Galliformes uses antibody-like V domains: insight into the evolution of TCRδ and TCRμ genes in tetrapods

Abstract

Analyses of the available avian genomes revealed the presence of a second TCRδ locus in the Galliformes. This second TCRδ locus is nonsyntenic to the conventional TCRα/δ and is unusual in that the V genes are more related to IgH V genes (VH) than to TCR V genes. The second TCRδ is not found in another avian lineage, the passerine zebra finch. Rather the finch's conventional TCRα/δ locus contains VH genes that are expressed with the conventional Cδ gene, similar to what has been found in amphibians. A comparison between Galliformes and Passeriformes genomic organization suggests an origin of the second TCRδ in the former lineage involving gene duplication. Expression of these atypical TCRδ transcripts with a VH domain paired with Cδ was found in lymphoid tissues of both avian lineages. The configuration of the second TCRδ in chicken and turkey is reminiscent of the TCRδ duplication that is present in nonplacental mammals and provides insight into the origin of the uniquely mammalian TCRμ locus.

Figure 1

Zebra finch TCRα/δ locus. V, D and J gene segments and C regions were numbered based on their position on the locus (5′ to 3′) and were color coded as follow: V, red; D, orange; J, green; Cδ, dark blue; Cα, light blue. The VHδ gene segment is indicated in yellow. Genes with conserved synteny in tetrapods are shaded light grey. Transcriptional orientation is shown with the direction of the arrow in each segment.

Figure 2

Phylogenetic tree of the avian VHδ compared with VH genes from birds, mammals and amphibians. Avian VHδ genes are in bold and bracketed. The tree shown was generated using the Minimum evolution distance method. Similar results were achieved using Neighbor Joining. Bootstrap values are based on 1000 replicate samples. The last three digits of the accession number are indicated for those sequences taken from GenBank. The three VH clans are indicated with brackets on the right and their bootstrap support is shown in bold. A distance bar is shown below the tree.

Figure 3

Predicted amino acid alignment of atypical avian TCRδ genes contain residues conserved in conventional Ig domains. Translations were based on nucleotide sequence from the genomic assemblies, except for duck that is cDNA. A. Predicted amino acid alignment of frameworks (FR) FR1 to FR3 regions of the VHδ from four different avian species. FR and CDR are indicated at the top of the alignment. Dashes indicate identity with the first sequence. Dots indicate gaps. Cysteine residues involved in intrachain disulfide bond and extra cysteine residues are highlighted in black with white letters. B. Alignment of Ig-C domain of conventional and atypical TCRδ. Human and mouse sequences are shown as references on top of the alignment. Dashes in the mouse sequence indicate identity to the human sequence. Dashes in all avian Cδ sequences indicate identity with the chicken Cδ1 sequence. Gaps are indicated with dots. Cysteine residues are indicated as in A. Potential glycosylation sites are highlighted in light gray.

Figure 4

Junctional diversity in avian atypical TCRδ CDR3 regions from different avian species. GenBank accession numbers are shown on the left of each sequence. P and N nucleotides are highlighted in black and grey, respectively. V-D-J germline sequences are at the top of the figure and indicated in bold. Gaps are shown with dots. Dashes indicate identity with the sequence at the top of each alignment for V and J gene segments. A. Adult zebra finch unique thymus cDNA sequences. D and J gene segments are indicated above the sequences. B. Unique V-D-J recombination sequences obtained from day 4 and day 21 chicken thymuses. Germline sequences are shown as described above. C. The 3′ end of the germ-line VHδ, the complete germ-line D, and complete germ-line J genes (un-recombined) from the chicken turkey genome assembles compared with the corresponding regions from the duck cDNA sequence (AF415216). D. Comparison of Dδ and Jδ germline gene segments from chicken, turkey, zebra finch (Dδ2 and Jδ2) and duck cDNA sequence. Conserved motif in the Jδ is highlighted in grey. Similar sequences between Dδ and Jδ gene segments are indicated in bold or underlined.

Figure 5

Representation of the zebra finch TCRα/δ locus and of a second TCRδ locus in chicken and turkey. V, D and J gene segments and C regions were color coded as in Fig. 1.

Figure 6

Phylogenetic tree of TCR Cδ and Cμ regions. The tree was constructed using the Ig-C domains and analyzed using the minimum evolution distance method. Bootstrap values are indicated per 1000 replicates. Avian sequences are shown in bold. A distance bar is shown below the tree.

Figure 7

Simplified representation of the TCRα/δ locus, atypical TCR and atypical isoforms in distinct vertebrate lineages. Genes are color coded as indicated at the bottom. Extended red and yellow rectangles in the locus maps represent the presence of several gene segments. Vδ* represent Vδ genes in cartilaginous fish that have lost the sequence encoding the leader peptide and that are only used as supportive Vδ on NAR-TCR.