Annotation and classification of the bovine T cell receptor delta genes

Abstract

Background: gammadelta T cells differ from alphabeta T cells with regard to the types of antigen with which their T cell receptors interact; gammadelta T cell antigens are not necessarily peptides nor are they presented on MHC. Cattle are considered a "gammadelta T cell high" species indicating they have an increased proportion of gammadelta T cells in circulation relative to that in "gammadelta T cell low" species such as humans and mice. Prior to the onset of the studies described here, there was limited information regarding the genes that code for the T cell receptor delta chains of this gammadelta T cell high species.

Results: By annotating the bovine (Bos taurus) genome Btau_3.1 assembly the presence of 56 distinct T cell receptor delta (TRD) variable (V) genes were found, 52 of which belong to the TRDV1 subgroup and were co-mingled with the T cell receptor alpha variable (TRAV) genes. In addition, two genes belonging to the TRDV2 subgroup and single TRDV3 and TRDV4 genes were found. We confirmed the presence of five diversity (D) genes, three junctional (J) genes and a single constant (C) gene and describe the organization of the TRD locus. The TRDV4 gene is found downstream of the C gene and in an inverted orientation of transcription, consistent with its orthologs in humans and mice. cDNA evidence was assessed to validate expression of the variable genes and showed that one to five D genes could be incorporated into a single transcript. Finally, we grouped the bovine and ovine TRDV1 genes into sets based on their relatedness.

Conclusions: The bovine genome contains a large and diverse repertoire of TRD genes when compared to the genomes of "gammadelta T cell low" species. This suggests that in cattle gammadelta T cells play a more important role in immune function since they would be predicted to bind a greater variety of antigens.

Figure 1

Schematic representation of TRD gene exon-intron structures. (A) Representative structures of TRDV genes (based on genomic sequence for TRDV1au, GLEAN_15708), which contain two exons, and (B) the single TRDC gene (GLEAN_19705), which contains three exons, are shown. Scale is shown in base pair increments beneath the schematic. V-RS, recombination signal sequence of a V gene.

Figure 2

Schematic representation of the bovine (Bos taurus) TRD locus organization. (A) 52 unique TRDV1 genes were identified and are all predicted to be functional (or ORF) and found on chromosome 10, although many of them were unplaced in the Btau_3.1 assembly. Multiple TRDV genes were identified in two or three gene prediction models, as indicated, and labels correspond to those used in Table 1. However, evidence suggests that in each case only a single gene is represented. Genomic organization is shown in those cases when three or more TRDV genes were found within a single scaffolded region. TRA genes are not shown although in many cases occur among the TRDV genes. In addition to some TRDV1 genes, the schematic includes TRDV2-1 on ChrUn.158 (no other TRDV genes are included in the schematic). (B) Genomic organization of the five TRDD genes, 3 TRDJ genes and the single TRDC gene is shown, along with TRDV4 and TRAC. Gene designations, orientations and Bovine Genome Scaffold identifications are as indicated. The determination of TRDV gene orientation was based on the assembly of the Bovine Genome Scaffolds, it is possible that some scaffolds were assembled in an incorrect orientation individually and/or in relation to other scaffolds. Diagrams are shown to scale with base pair increments beneath the schematics. With the exception of TRAC, TRA genes are not shown. TRDV2-2 and TRDV3 are not shown because those genes were identified on scaffolds lacking additional TRDV genes.

Figure 3

TRDV gene sequences. (A) TRDV deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with JalView. Analysis included all non-redundant genomic sequences (see Table 1 for GLEAN identification numbers). IMGT unique numbering for V-DOMAIN [29,32] is indicated above the alignment and conserved cysteines are indicated below the alignment. (B) IMGT Collier de Perles [33] are shown for TRDV1 (based on TRDV1a, GLEAN_22158), TRDV2-1 (no glean identification), TRDV3-1 (no glean identification) and TRDV4 (GLEAN_19724) and were determined using IMGT/V-QUEST [32]. The CDR-IMGT lengths are indicated.

Figure 4

Phylogenetic tree of bovine TRDV genes. The Neighbor-Joining method [47] was used to classify TRDV genes using non-redundant bovine (bo) genomic TRDV sequences identified here and the previously classified ovine (ov), murine (mo) and human (hu) TRDV sequences (described in Methods). Bovine TRAV (GenBank accession number BC148926) was included in the analysis and was used to root the tree. The optimal tree with the sum of branch length = 5.37540770 is shown. Complete deletion to eliminate gaps was performed and the final dataset included a total of 207 positions. Eleven phylogenetic sets are indicated along with the percentage interior branch test value based on 1000 replicates for each set.

Figure 5

Comparison of TRDV gene recombination signal sequences. Sequence logos for recombination signal (RS) sequences are shown for (A) bovine and ovine TRDV1 genes (52 and 12 sequences, respectively). (B) bovine TRDV4 and its ovine, human and murine orthologs (TRDV4, TRDV3 and TRDV5, respectively; four sequences) and (C) remaining human, murine and bovine TRDV genes (human TRDV1, TRDV2; murine TRDV1, TRDV2-1, TRDV2-2, TRDV4; bovine TRDV2-1, TRDV2-2, TRDV3-1; nine sequences). Heptamer and nonamer sequences and spacer lengths are indicated. Logos were generated using WebLogo3 [45].

Figure 6

TRDJ gene sequence alignments and phylogenetic tree. Alignments of TRDJ gene (A) nucleotide and (B) deduced amino acid sequences, using bovine (bo) genomic TRDJ sequences identified here and previously classified ovine (ov), swine (sw), human (hu) and murine (mo) TRDJ sequences (described in Methods) are shown with identities indicated by a dot (.) and gaps indicated by a dash (-). The Neighbor-Joining method [47] was used to infer evolutionary history of the above mentioned sequences. The optimal tree (C) with the sum of branch length = 2.43145392 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [48]. The Maximum Composite Likelihood method [49] was used to compute evolutionary distances. Complete deletion to eliminate gaps was performed and the final dataset included a total of 42 positions.

Figure 7

TRD rearranged CDR3 sequences and TRDD gene usage. TRD rearranged CDR3 nucleotide sequences, and their corresponding deduced amino acid sequences, derived from cDNA from peripheral blood mononuclear cells, were aligned to demonstrate TRDD usage. Germline TRDD sequences are shown above representative TRDV1, TRDV2, TRDV3 and TRDV4 sequences containing between one and five TRDD genes. TRDD genes are shaded and gene usage was determined by the presence of at least six nucleotides of a particular TRDD gene. Sufficient cDNA sequence was analyzed to determine TRDV gene usage; however, the sequences shown here have been truncated after the 2nd-CYS 104 and at the beginning of the TRDJ gene.