Overview of the Germline and Expressed Repertoires of the TRB Genes in Sus scrofa

Abstract

The α/β T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and β chains are encoded by different genes localized on two distinct chromosomal loci: TRA and TRB. The present study employed the recent release of the swine genome assembly to define the genomic organization of the TRB locus. According to the sequencing data, the pig TRB locus spans approximately 400 kb of genomic DNA and consists of 38 TRBV genes belonging to 24 subgroups located upstream of three in tandem TRBD-J-C clusters, which are followed by a TRBV gene in an inverted transcriptional orientation. Comparative analysis confirms that the general organization of the TRB locus is similar among mammalian species, but the number of germline TRBV genes varies greatly even between species belonging to the same order, determining the diversity and specificity of the immune response. However, sequence analysis of the TRB locus also suggests the presence of blocks of conserved homology in the genomic region across mammals. Furthermore, by analysing a public cDNA collection, we identified the usage pattern of the TRBV, TRBD, and TRBJ genes in the adult pig TRB repertoire, and we noted that the expressed TRBV repertoire seems to be broader and more diverse than the germline repertoire, in line with the presence of a high level of TRBV gene polymorphisms. Because the nucleotide differences seems to be principally concentrated in the CDR2 region, it is reasonable to presume that most T cell β-chain diversity can be related to polymorphisms in pig MH molecules. Domestic pigs represent a valuable animal model as they are even more anatomically, genetically and physiologically similar to humans than are mice. Therefore, present knowledge on the genomic organization of the pig TRB locus allows the collection of increased information on the basic aspects of the porcine immune system and contributes to filling the gaps left by rodent models.

Keywords: IMGT; T cell receptor; TRB locus; TRB repertoire; domestic pig.

Figure 1

Schematic representation of the genomic organization of the pig TRB locus deduced from the genome assembly Sscrofa11.1. The diagram shows the position of all the related and unrelated TRB genes according to nomenclature. The boxes representing the genes are not to scale. The exons are not shown. The arrows indicate the transcriptional orientation of the genes.

Figure 2

The NJ tree inferred from the pig, human, dog and dromedary TRBV gene sequences. The evolutionary analysis was conducted in MEGA7 (20). The optimal tree with the sum of branch length = 14.02591240 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (24). The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer phylogenetic trees. The evolutionary distances were computed using the p-distance method (22) and are in the units of the number of base differences per site. The analysis involved 124 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 163 positions in the final dataset. The pig TRBV subgroup classification is performed according to the clustering with the orthologous mammalian TRBV subgroups. The different colors highlight the distribution of the phylogenetic groups. The gene functionality according to IMGT rules (F: functional, ORF: open reading frame, P: pseudogene) is indicated. The IMGT 6-letter for species (Homsap, Camdro, Susscr) and 9-letter for subspecies (Canlupfam) standardized abbreviation for taxon is used.

Figure 3

The IMGT Protein display of the pig TRBV genes. Only functional genes and in-frame pseudogenes are shown. The description of the strands and loops and of the FR-IMGT and CDR-IMGT is according to the IMGT unique numbering for V-REGION (25). The five conserved amino acids of the V-DOMAIN (1st-CYS 23, except for the TRBV6 pseudogene, CONSERVED-TRP 41, hydrophobic AA 89, 2nd-CYS 104 and J-PHE 118) are indicated in bold. The amino acid length of the CDR-IMGT AA is also indicated in square brackets.

Figure 4

Nucleotide and deduced amino acid sequences of the pig TRBD (A), TRBJ (B) and TRDC (C) genes. The consensus sequence of the heptamer and nonamer is provided at the top of the figure and is underlined. The numbering adopted for the gene classification is reported on the left of each gene. In (A), the inferred amino acid sequence of the TRBD genes in the three coding frames are reported. In (B), the donor splice site for each TRBJ is shown. The canonical FGXG amino acid motifs are underlined. In (C), IMGT Protein display of the pig TRBC gene as derived from the alignment by Clustal W with the human, sheep and dromedary Cβ proteins. The descriptions of the strands and loops were collected according to the IMGT unique numbering for the C-DOMAIN (26).

Figure 5

Dot-plot of the pig TRB locus sequence against itself. (A) With the exception of the main diagonal, the dots and diagonal lines indicate internal homology units in the sequence. The red boxes show the two TRBV regions underwent to duplication events and the internal homology of the D-J-C cluster portion. The gray arrow points to the homology region enlarged in (B). (B) The pattern of parallel lines indicates the duplicated region of about 28 kb containing the TRBV2 and TRBV4 genes.

Figure 6

Evolutionary relationship of the TRBV4 (A), TRBV5 (B) and TRBV7 (C) genes. The evolutionary history was inferred using the Neighbor-Joining method (21). The percentage of replicate trees in which the associated genes clustered together in the bootstrap test (100 replicates) are shown next to the branches (24). The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic trees. The evolutionary distances were computed using the p-distance method (22) and are in the units of the number of base differences per site. The analysis involved 33 (in A), 35 (in B) and 41 (in C) nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 333 (A,B) and 348 (C) positions in the final dataset. The evolutionary analyses were conducted in MEGA7 (20).

Figure 7

The IMGT Protein display of the pig TRBV4 (A) and TRBV7 (B) germline and cDNA genes. The gene groups represent the phylogenetic clusters of Figure 6. The TRBV cDNA genes are indicated by the Accession number.The description of the strands and loops and of the FR-IMGT and CDR-IMGT is according to the IMGT unique numbering for the V-REGION (25). The five conserved amino acids of the V-DOMAIN (1st-CYS 23, except for the TRBV6 pseudogene, CONSERVED-TRP 41, hydrophobic AA 89, 2nd-CYS 104 and J-PHE 118) are indicated in bold.