Annotation and classification of the bovine T cell receptor delta genes

T lymphocytes can be subdivided into at least two types based on the expression of either the αβ or γδ T cell receptor. Although both perceive antigen, they differ in the types of antigens with which they react. αβ T cell receptors react with antigenic protein peptides in the context of self major histocompatibility complex (MHC) proteins while γδ T cell receptors may react with proteins but this does not involve MHC presentation. They also react with autologous molecules on cells [1–6] as well as nonproteinaceous molecules [7]. The gene repertoires that code for the γδ T cell receptor chains and the T cell receptor gamma (TRG) and delta (TRD) gene locus organizations have been extensively described for humans and mice but to a lesser extent for the artiodactyls which includes ruminants and swine. These latter are "γδ T cell high" species because of their high levels of γδ T cells in circulation ("γδ T cell low" species exhibit much lower levels of γδ T cells in circulation). It is clear that while both αβ and γδ T cells have large and diverse T cell receptor gene repertoires [8–14], "γδ T cell high" species have a TRD gene repertoire that is much more extensive than that in the "γδ T cell low" species [15–21]. The bovine (Bos taurus) locus organization and TRD gene repertoire are the subject of this work.

T cell receptor delta and beta chains are encoded by the rearrangement of variable (V), joining (J) and diversity (D) genes making them more complex than the T cell receptor gamma and alpha chains which lack D gene products. In all mammals evaluated the genes encoding the T cell receptor beta and gamma chains are found at the T cell receptor beta (TRB) and TRG loci, respectively. The genes that encode the T cell receptor delta and alpha chains are found at a single chromosomal location with the TRD genes embedded within the T cell receptor alpha (TRA) locus. For both humans and mice these are located on chromosome 14 and encompass over 1 megabase (Mb) for the combined TRA/TRD locus [13]. The TRD locus embedded within the TRA locus spans approximately 60 kb in humans and 275 kb in mice. The loci comprise TRDV genes (two in humans and five in mice), followed by TRDD genes (three in humans and two in mice), TRDJ genes (four in humans and two in mice), a single TRDC gene and an additional TRDV gene that is located 3' of the TRDC gene in an inverted transcriptional orientation [10, 14]. In addition, five and ten functional TRAV/DV genes (that rearrange to either TRDD or TRAJ) have been identified for humans and mice, respectively, and these are found upstream of the embedded TRD locus [10, 14].

Limited evidence derived from analysis of cDNA clones from cattle, sheep and swine, as well as limited germline information for sheep, suggests that the general organization of the bovine TRA/TRD locus does not differ greatly from that of humans and mice [17, 20]. It also suggests that a much larger repertoire of TRDV genes exists for cattle, as well as for the other "γδ T cell high" species sheep and swine [15, 17, 20, 21]. Indeed, recent mapping of the bovine TRA/TRD locus identified over 100 TRDV and over 300 TRAV or TRAV/DV genes in cattle [22]. For cattle, four genes belonging to three different small subgroups (TRDV2, TRDV3 and TRDV4) have been identified that are orthologous to those same subgroups in sheep [17, 23]. In contrast, while the bovine TRDV1 genes have been found to be related to the single TRDV1 gene that occurs in humans, expansion of the TRDV1 subgroup accounts for the larger number of TRDV genes in the "γδ T cell high" species [15, 17, 20–22]. The swine and sheep TRDV1 subgroups have been estimated to contain at least 31 and 40 genes, respectively [15, 21] while the bovine TRDV1 subgroup has been reported to contain at least 37 genes [20]. However, current knowledge about ruminant and swine TRD genes is predominantly based on cDNA evidence rather than on genomic DNA. Because of this, it has been unclear whether the large number of cDNA TRDV1 sequences is the result of multiple genes or polymorphisms among animals; thus, there has been no clear way to classify and name these transcripts. By annotating the bovine genome Btau_3.1 assembly, here we demonstrated the presence of 56 TRDV genes, 52 of which belong to the TRDV1 subgroup. We also proposed TRDV1 sets to classify the bovine and ovine TRDV1 genes based on their phylogenetic grouping.

The existence of multiple D and J genes also contributes to diversity of possible T cell receptor delta chain amino acid sequences since a particular TRDV gene is expected to be able to recombine with any D and J gene. The region of the protein derived from the V-D-J gene rearrangement is known as the complementarity determining region 3 (CDR3), while the CDR1 and CDR2 loops are germline-encoded by the TRDV gene. The V-D-J gene rearrangement involves recognition of the recombination signal (RS) sequences for subsequent DNA cleavage by the enzymes RAG1 and RAG2 [24]. T cell receptor delta chain sequence diversity is augmented by junctional flexibility and the addition of N and P nucleotides. RS flank the V, D and J genes and are composed of highly conserved heptamer and nonamer sequences separated by either a 12 base pair (bp) spacer (located 5' of TRDD and TRDJ genes) or a 23 bp spacer (located 3' of TRDV and TRDD genes). Since the conserved heptamer and nonamer sequences and spacer lengths have been found to be important for efficient recombination [25] they were also evaluated here. Finally, recent evidence demonstrated that diversity of T cell receptor delta chain sequences is amplified by the occurrence of multiple TRDD genes within a single CDR3 [20, 26]. Thus we also evaluated TRDD gene usage by analyzing cDNA sequences and report those findings here.

In order to evaluate and characterize the bovine T cell receptor delta gene repertoire and their genomic organization we annotated the TRD genes in the Btau_3.1 assembly. Here we describe the existence of two TRDV2 genes, as well as a single gene each for TRDV3 and TRDV4. We confirmed the presence of three TRDJ and five TRDD genes [20] and evaluated TRD junctional diversity incorporating these genes. Furthermore, we described the existence of 52 TRDV1 genes (46 of them functional and six of them ORF), evaluated them for their sequence characteristics and further classified them into eleven sets based on phylogenetic analysis. As for other mammals, the bovine TRD locus was found embedded within the TRA locus and thus TRDV1 genes were distributed amongst the TRAV genes. Indeed, there were a number of genes for which assignment to the TRAV or TRDV group was ambiguous. It is important to note that the presence of TRAV/DV genes is certain and has been reported [17] but the ability of TRDV or TRAV genes (as defined based on sequence characteristics) to rearrange with either TRDD or TRAJ must be determined experimentally and will be evaluated in future studies. Evaluation of the genes contained within the TRA/TRD locus was reported [22] following the initial submission of this work with overall findings similar to those discussed here.

The question remains as to whether or not the findings reported here represent the complete TRD gene repertoire because of the gaps in the Btau_3.1 assembly. The presence of additional TRDV1 genes is almost certain because of extensive gaps in this region although it is unlikely that additional TRDD or TRDJ genes are present in the genome since that region is fully scaffolded and has been carefully evaluated for the presence of additional genes. That region also contains TRDV4 and its analysis is likely definitive. In addition, previous cDNA evidence supports the existence of only two TRDV2 genes [17] and is consistent with our findings. The existence of two TRDV3 genes has been reported [20] but is not supported by our findings which may be due to gaps in the genome assembly. Even with gaps, locus organization can be assisted by comparison to other species. Indeed, all mammals studied have genes that are closely related to human TRDV1 with some being distributed among the TRAV genes. The bovine TRDV4 gene is located downstream of the single TRDC gene in inverse orientation, as found for the human TRDV3 and mouse TRDV5 genes. It is notable that there is significant sequence conservation among the orthologous bovine TRDV4, human TRDV3, mouse TRDV5 and ovine TRDV4 genes as well as their entire RS. This suggests that they derive from a common ancestral gene that appeared before the separation of these species. By comparison, there are no human or mouse genes that are equivalent to bovine TRDV2 or TRDV3. The enormous expansion of the TRDV1 repertoire that has been observed for cattle, sheep and pig [15, 21] is striking when compared with the single TRDV1 gene found in human (and its two orthologs in mouse) and thus does not assist us in predicting the extent of the possible missing information.

TRDV1 gene subgroup expansion is also striking when compared with other TRDV gene subgroups which, even in artiodactyls, are found to contain only one or two members. While the number of TRGV genes found in each species does not vary considerably [31, 35, 36], expansion of the TRDV1 genes in bovine, sheep and pig is consistent with them being "γδ T cell high" species, implying that this increase in antigen receptor diversity permits them to be stimulated by and to respond to more types of antigens. The presence of fewer TRDV genes in "γδ T cell low" species, such as humans and mice, suggests that these species evolved so that γδ T cells play a less important role in their immune function. Sequence conservation among bovine TRDV1 RS (including the 23-bp spacer sequences), supports the concept that the large TRDV1 subgroup arose from multiple duplication events. Many bovine TRDV1 genes share high percent identities, indicating that in some cases this subgroup is not as diverse as the number of individual genes might suggest. For example, some TRDV1 sequences differed by as few as four (TRDV1m and TRDV1s), three (TRDV1v and TRDV1as) or even one (TRDV1aa and TRDV1bn) nucleotide in their coding regions even though all were determined to be distinct genes based on analysis of the flanking genomic sequences. However, it cannot be ruled out that some of these gene models represent the same gene but do not appear as such due to sequencing and/or assembly anomalies. The question of why numerous and highly similar TRDV1 genes have been maintained in the bovine genome is compelling and studies to evaluate their functions will be pursued.

Based on phylogenetic sequence analysis and gene features, we subdivided the bovine TRDV1 genes into eleven sets. Each set corresponds to intraspecies gene duplications of ancestral genes that were present before the separation between bovine and ovine species. We also assigned bovine cDNA sequences to germline genes identified in this work (see Table 5). Of 55 previously identified TRDV1 cDNA sequences evaluated, 20 could not be assigned to a germline gene, possibly because of gaps in that region of the genome assembly. Of the 52 TRDV1 genes identified in this study, cDNA evidence existed for only 16. It is possible that, because a majority of the previously identified TRDV1 cDNA sequences were derived from peripheral blood mononuclear cells [16–20], the remaining TRDV1 genes that lack cDNA verification might be expressed in other tissues that have not been evaluated. It is also possible that other factors, such as age or immunological experience, might impact the expression of particular genes and thus prevented verification of those genes. Future studies will focus on specifically amplifying individual TRDV1 transcripts in order to verify that they are functional and to determine whether they are expressed in an age and/or tissue dependent manner.

The potential importance of the role of the T cell receptor delta rearranged CDR3 is evident instudies evaluating the binding of the T10 and T22 antigens [26, 37, 38] by murine γδ T cells since antigen recognition by the CDR3 was found to be autonomous, that is, not dependent on the TRDV gene associated with it [39]. It could be reasoned that, because the genomes of humans and mice lack the extensive TRDV repertoirethat has been observed in artiodactyls, thiswould be compensated for by diversity of the genes that rearrange to form the CDR3 (i.e. TRDD and TRDJ genes). Moreover, because many bovine TRDV1 amino acid sequences are very similar, structurally it might be expected that their ligand binding regionsincluding CDR3 would also need to be as diverse as that in humans and mice. Humans have three TRDD and four TRDJ genes while mice have two TRDD and two TRDJ genes. With the exception of the human TRDJ genes, these numbers all fall short of the five TRDD and three TRDJ genes identified in cattle. We demonstrated here that in cattle the TRD CDR3 contained combinations that include between one and five TRDD genes and would range in length from nine to 37 amino acids. With the exception of TRDD4, the germline bovine TRDD genes predominantly encode glycine and the hydrophobic residues valine and tryptophan, with the neutral residues threonine and tyrosine being encoded to a lesser extent (see Additional File 1). This is reminiscent of findings for the equine VH CDR3 loops in which a higher proportion of glycine and lower proportion of cysteine content was identified when sequences were compared with those from humans, sheep and pigs [40]. As was suggested for equine VH CDR3, this indicates that bovine TRDV CDR3 loops have increased flexibility and are therefore better suited to recognize a large number of antigenic conformations. Considering this, it is unlikely that CDR3 diversity in mice and humans compensates for their less diverse TRDV repertoire and, in fact, it seems that cattle have more scope for antigen binding on all levels.

If the highly diverse CDR3 is indeed the main structural component involved in antigen recognition, then the TRDV gene products might be most important for interactions with co-receptors or antigen presenting cells, whatever their nature may be. While the interactions between γδ T cell receptor and the antigens that they bind are still largely unknown, it has been demonstrated that for αβ T cell receptor the CDR1 loops (which are germline encoded) are involved with binding peptide as well as MHC. The CDR2 loops (also germline encoded) bind only MHC. The TRDV CDR1 in cattle exhibited diverse lengths and were generallyfound to be five to ten amino acids long as for mouse and human TRDV, TRBV and TRAV CDR1. Thisdiverse bovine TRDV CDR1 repertoire may contribute to antigen binding in contrast to what has been reported for murine γδ T cell recognition of T22 as described above. It was found that bovine TRDV1 CDR2 were either only three amino acids long or absent completely (e.g., TRDV1f, TRDV1ae, TRDV1ar and TRDV1o) in contrast to TRBV CDR2 lengths of five to seven amino acids in mammals. It is yet to be determined whether the TRDV1 genes lacking CDR2 are functional, however, if they are found to be so this lackof, or abbreviation of, the CDR2 inbovine TRDV1 genes is logical since γδ T cells are not MHC-restricted.

Based on annotations of the bovine genome we identified the TRD genes including 56 TRDV genes, five TRDD genes, three TRDJ genes and the single TRDC gene and described their organization within the TRD locus. Furthermore, we report that the TRDV1 subgroup contains 52 genes, indicating that this subgroup underwent expansion as has been found for sheep and swine. Also, because this large gene subgroup has been maintained in the bovine genome this indicates a unique role for these genes in γδ T cell biology.