In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) 30 and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present 31 paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total 32 of 331 mutations from a single animal, and comparing the properties of the mutation profiles of drom- 33 edary TRG and TRD variable domains. The transition preference and the significant mutation frequency 34 in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating 35 SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, 36 results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and 37 a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations 38 appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lin- 39 eages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rear- 40 rangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by 41 the fact that our structural models, computed by adopting a comparative procedure, are consistent with 42 the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated 43 by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural 44 stability of the cd subunits.

Background In most species of mammals, the TRB locus has the common feature of a library of TRBV genes positioned at the 5'- end of two in tandem aligned D-J-C gene clusters, each composed of a single TRBD gene, 6-7 TRBJ genes and one TRBC gene. An enhancer located at the 3'end of the last TRBC and a well-defined promoter situated at the 5'end of the TRBD gene and/or a undefined promoter situated at the 5'end of the TRBD2 are sufficient to generate the full recombinase accessibility at the locus. In ruminant species, the 3'end of the TRB locus is characterized by the presence of three D-J-C clusters, each constituted by a single TRBD, 5-7 TRBJ and one TRBC genes with the center cluster showing a structure combined with the clusters upstream and downstream, suggesting that a unequal crossover occurred in the duplication. An enhancer downstream the last TRBC, and a promoter at the 5'-end of each TRBD gene are also present. Results In this paper we focused our attention on the analysis of a large number of sheep TR β-chain transcripts derived from four different lymphoid tissues of three diverse sheep breed animals to certify the use and frequency of the three gene clusters in the β-chain repertoire. As the sheep TRB locus genomic organization is known, the exact interpretation of the V-D-J rearrangements was fully determined. Our results clearly demonstrate that sheep β-chain constitutes a level of variability that is substantially larger than that described in other mammalian species. This is due not only to the increase of the number of D and J genes available to the somatic recombination, but also to the presence of the trans-rearrangement process. Moreover, the functional complexity of β-chain repertoire is resolved by other mechanisms such as alternative cis- and trans-splicing and recombinational diversification that seems to affect the variety of the constant region. Conclusion All together our data demonstrate that a disparate set of molecular mechanisms operate to perform a diversified repertoire in the sheep β-chain and this could confer some special biological properties to the corresponding αβ T cells in the ruminant lineage.

The Camelidae species occupy a peculiar niche within the adaptive immune response and the camel lineage has been proposed as a fascinating model in the evolution of immune systems. In fact, the significant amount of special heavy chain-only antibodies in the serum, in addition to the tetrameric IgGs along with a repertoire, largely diversified by extensive somatic hypermutation, result in novel paratopes different from those of conventional antibodies. Moreover, for the first time in a mammalian organism, it was shown that T cell receptor evolution has been favoured in the dromedary by mutation in the productively rearranged gamma (TRG) and delta (TRD) genes, thus contributing to the repertoire diversity of γδ heterodimer. Under this scenario, in order to gain further insights into the function and evolution of the T cell receptor gamma/delta heterodimer, we investigated the genomic structure and the gene content of the TRG and TRD loci in Old World camels. The availability of recent draft genomes and new re-sequenced data of the three species, Camelus dromedarius (n = 7), Camelus bactrianus (n = 9) and Camelus ferus (n = 9) (at an average 15-fold coverage using the Illumina HiSeq2000) allowed us to determine the map of the entire TRG locus. It spans approximately 198 kb, it is flanked at its 5' end by the AMPH gene and it contains three V-J-C cassettes. Furthermore, the retrieval of relevant contigs, about 900 kb long, confirmed that TRD locus with its V, D, J and C genes, as in eutherians and birds, is clustered within the TRA locus.

The present study identifies the genomic structure and the gene content of the T cell receptor beta (TRB) locus in the Oryctolagus cuniculus whole genome assembly. The rabbit locus spans less than 600 Kb and the general genomic organization is highly conserved with respect to other mammalian species. A pool of 74 TRB variable (TRBV) genes distributed in 24 subgroups are located upstream of two in tandem-aligned D-J-C gene clusters, each composed of one TRBD, six TRBJ genes, and one TRBC gene, followed by a single TRBV gene with an inverted transcriptional orientation. All TRB genes (functional, ORF, pseudogenes) of this paper have been approved by the IMGT/WHO-IUIS nomenclature committee. Additionally, five potentially functional protease serine (PRSS) trypsinogen or trypsinogen-like genes were identified: two in tandem PRSS-like genes, followed by two PRSS genes with unique traits, lie downstream of the TRBV1 gene and one PRSS gene is located about 400 Kb away downstream of the TRBV genes. Comparative and phylogenetic analyses revealed that multiple duplication events within a few subgroups have generated the germline repertoire of the rabbit TRBV genes, which is substantially larger than those described in humans, mice, and dogs, suggesting that a strong evolutionary pressure has selected the development of a species-specific TRBV repertoire. Hence, the genomic organization of the TRB locus in the genomes appears to be the result of a balance between the maintenance of a core-number of genes essential for the immunological performances and the requirement of newly arisen genes.

T cell receptors are heterodimers comprising alpha (TRA) and bet (TRB) or gamma (TRG) and delta (TRD) subunits, two combinations defining the two major lineages of T cells. alpha/beta T cells typically recognize peptide antigens presented on major histocompatibility complex (MHC) encoded molecules, while gamma/delta cells are either MHC restricted or resemble Ig in being able to bind free antigens. The bottlenosed dolphin is being sequenced by the Human Genome Sequencing Center (BCMHGSC) at the Baylor College of Medicine and the Broad Institute using a whole genome shotgun sequencing strategy (BioProject ID: 20367). We employed the NCBI draft genome assembly to identify the TRG and TRB loci of Tursiops truncatus. The dolphin gamma locus is the smallest and simplest of all the mammalian TRG loci as yet studied. It shows a gene cassette organization comprising two variable (V) genes, three joining (J) genes, and a single constant (C) gene. Expression analysis identified all the possible VJ rearrangements of the locus, although some transcripts are preferentially expressed. About half of the TRGV2 rearrangements originate transcripts unlikely to be functional due to stop codons in CDR3. Although a complete characterization was impossible because of gaps in the available assemblies, the dolphin beta locus is also very simple in comparison with human and ovine. We found that only four of the seven identified in the genome are TRBV expressed genes, with a significant bias toward expression of TRBV30, located immediately after the C gene in an inverted transcriptional orientation. However, there was no evidence of preferential rearrangement with regard to TRBD e TRBJ. Finally, multialignment analysis of TRGV and TRBV sequences confirmed, as expected, a close phylogenetic relationship between Tursiops truncatus and the Bovidae family of the Artiodactyla.

In mammals, T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T cell receptors. Human and mouse display a low peripheral blood γδ T cell percentage ("γδ low species") while sheep, bovine and pig accounts for a high proportion of γδ T lymphocytes ("γδ high species"). While the T cell receptor alpha (TRA) and delta (TRD) genes and the genomic organization of the TRA/TRD locus has been determined in human and mouse, this information is still poorly known in artiodactyl species, such as sheep.

We recently demonstrated for the first time in a mammalian organism (Camelus dromedarius) that somatic hypermutation (SHM) occurs in the variable domain of the productively rearranged T cell receptor gamma (TRG) and delta (TRD) genes. Here we report the mutation profile for each nucleotide, as obtained by the comparison of spleen cDNA sequences of a single animal with the corresponding germline genes. The mutations are very frequent both in TRG and TRD genes, with the significant overrepresentation at the palindromic DGYW/WRCH motifs, indicative of activation-induced cytidine deaminase (AID) targeting and at the WA/TW hotspots suggesting a possible origin due to the mismatch repair (MMR) mechanism known to take place during the second phase of SHM. The analysis of distribution of the mutations showed that CDR3, FR4 and FR1 of the TRG variable domain exhibit a statistically significant higher mutation rate than the other regions, the highest value being of 0.015/bp. There is no difference in the mutation rate between the regions of the TRD variable domain, with the exception of a CDR1 value statistically significant higher than that of FR2 in a V gene. In TRG genes, the comparison of the R/S ratios of CDRs and FRs indicated a statistically appreciable difference (chi-squared, p = 0.0009), suggesting a selection pressure acting on gamma/delta T cells. The analysis of clonal radiation in constructed lineages from cDNA TRD sequences showed that R mutations in FRs appear to be early in the lineage implying that the founder mutations do not ablate the structural integrity of the receptor. By computational analysis, we infer that the non conservative residue variations located in the FR2 of the TRD domain may indeed contribute to the further stabilization of the interaction between the two chains of the gamma/delta heterodimer.

In mammals, T cells develop along two discrete pathways characterized by expression of either the alpha beta or the gamma delta T cell receptors. Human, mouse, and dog display a low peripheral blood gamma delta T cell percentage, while sheep accounts for a high proportion of gamma delta T lymphocytes. In all these species, the genomic organization of the T cell receptor gamma (TRG) locus is well known. To gain further insight into the evolutionary significance of the gamma delta T cell lineage, the present study has defined the genomic organization of the TRG locus in rabbit (Oryctolagus cuniculus), another mammalian gamma delta high species, as deduced from the genome assembly. The rabbit TRG locus spans about 70 kb and consists of ten TRGV, two TRGJ genes, and one TRGC gene located 5' to 3' in the locus. When we compared the rabbit sequence with the human, mouse, sheep, and dog counterparts, a higher identity with human as well as sheep with respect to mouse and dog was evident, providing that in the different mammalian species, the TRG locus appears to have evolved independently without any correlation with the gamma delta condition. The complete sequence of the rabbit TRG locus described here provides also a resource for supporting functional studies especially in the context of the gamma delta T cell function.

The αβ T cells are important components of the adaptive immune system and can recognize a vast array of peptides presented by MHC molecules. The ability of these T cells to recognize the complex depends on the diversity of the αβ TR, which is generated by a recombination of specific Variable, Diversity and Joining genes for the β chain, and Variable and Joining genes for the α chain. In this study, we analysed the genomic structure and the gene content of the TRB locus in Camelus dromedarius, which is a species belonging to the Tylopoda suborder. The most noteworthy result is the presence of three in tandem TRBD-J-C clusters in the dromedary TRB locus, which is similar to clusters found in sheep, cattle and pigs and suggests a common duplication event occurred prior to the Tylopoda/Ruminantia/Suina divergence. Conversely, a significant contraction of the dromedary TRBV genes, which was previously found in the TRG and TRD loci, was observed with respect to the other artiodactyl species.