Genomic and comparative analysis of the T cell receptor gamma locus in two Equus species.

The genus Equus is the only extant genus of the Equidae family, which belongs to Perissodactyla, an order of mammals characterized by an odd number of toes (odd-toes ungulates). Taking advantage of the latest release of the genome assembly, we studied, for the first time in two organisms belonging to the Equus genus, the horse (Equus caballus) and the donkey (Equus asinus), the T cell receptor gamma (TRG) locus encoding the gamma chain of the γδ T cell receptor. Forty-five Variable (TRGV) genes belonging to the seven IMGT-NC validated mammalian TRGV subgroups, 25 Joining (TRGJ) and 17 Constant (TRGC) genes organized in 17 V-J-(J)-C cassettes, in tandem on about 1100 Kb, characterize the horse TRG locus, making the horse TRG locus the one with the greatest extension and with a significantly higher number of genes than the orthologous loci of the other mammalian species. A clonotype analysis of an RNA-seq transcriptomic dataset derived from spleen of an adult healthy horse, using the complete set of the horse TRGJ germline gene sequences as a probe, revealed that, in addition to the most prominent V-J rearrangements within each cassette, there is a relevant proportion of trans-cassette V-J recombination, whereby the same TRGV genes can recombine with different TRGJ genes spliced to the corresponding TRGC genes. This recombinant event strongly contributes to the diversity of the γ chain repertoire. In the donkey TRG locus, 34 TRGV, 21 TRGJ and 14 TRGC genes distributed in 14 V-J-(J)-C cassettes were found in a region of approximately 860 kb. Although the donkey's TRG is smaller than that of the horse, in Equus genus, this is still the second largest locus so far found in any mammalian species. Finally, the comparative analysis highlighted differences in size and gene content between the horse and donkey TRG loci, despite belonging to the same genus, indicating a good level of diversification within Equus. These data is in agreement with the evolutionary idea of the existence of a Equus recent common ancestor in rapid evolution, for which a mutation rate between horses and donkeys is more comparable to that between species belonging to different genera rather than to species of the same genus.

3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries.

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how γδ T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary γδ T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted γδ T cell receptor complex.

The Organization of the Pig T-Cell Receptor γ (TRG) Locus Provides Insights into the Evolutionary Patterns of the TRG Genes across Cetartiodactyla.

The domestic pig (Sus scrofa) is a species representative of the Suina, one of the four suborders within Cetartiodactyla. In this paper, we reported our analysis of the pig TRG locus in comparison with the loci of species representative of the Ruminantia, Tylopoda, and Cetacea suborders. The pig TRG genomic structure reiterates the peculiarity of the organization of Cetartiodactyla loci in TRGC "cassettes", each containing the basic V-J-J-C unit. Eighteen genes arranged in four TRGC cassettes, form the pig TRG locus. All the functional TRG genes were expressed, and the TRGV genes preferentially rearrange with the TRGJ genes within their own cassette, which correlates the diversity of the γ-chain repertoire with the number of cassettes. Among them, the TRGC5, located at the 5' end of the locus, is the only cassette that retains a marked homology with the corresponding TRGC cassettes of all the analyzed species. The preservation of the TRGC5 cassette for such a long evolutionary time presumes a highly specialized function of its genes, which could be essential for the survival of species. Therefore, the maintenance of this cassette in pigs confirms that it is the most evolutionarily ancient within Cetartiodactyla, and it has undergone a process of duplication to give rise to the other TRGC cassettes in the different artiodactyl species in a lineage-specific manner.

The T Cell Receptor (TRB) Locus in Tursiops truncatus: From Sequence to Structure of the Alpha/Beta Heterodimer in the Human/Dolphin Comparison.

The bottlenose dolphin (Tursiops truncatus) belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T. truncatus T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.

The Genomic Organisation of the TRA/TRD Locus Validates the Peculiar Characteristics of Dromedary δ-Chain Expression.

The role of γδ T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., "γδ low species") with a restricted diversity of γδ T cell receptors (TR). Conversely, artiodactyl species (i.e., "γδ high species") account for a high proportion of γδ T cells with large γ and δ chain repertoires. The genomic organisation of the TR γ (TRG) and δ (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for γ and δ chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR α (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary δ chains. Furthermore, the comparison between genomic and expressed sequences reveals that D genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary δ chain antigen binding-site.

The expansion of the TRB and TRG genes in domestic goats (Capra hircus) is characteristic of the ruminant species.

BACKGROUND: Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects their successful adaptation to diverse environmental conditions. Despite the relevance of this species, the genetic research on the goat traits is limited compared to other domestic species. Thanks to the latest goat reference genomic sequence (ARS1), which is considered to be one of the most continuous assemblies in livestock, we deduced the genomic structure of the T cell receptor beta (TRB) and gamma (TRG) loci in this ruminant species. RESULTS: Our analyses revealed that although the organization of the goat TRB locus is broadly similar to that of the other artiodactyl species, with three in-tandem D-J-C clusters located at the 3' end, a complex and extensive series of duplications have occurred in the V genes at the 5' end, leading to a marked expansion in the number of the TRBV genes. This phenomenon appears to be a feature of the ruminant lineage since similar gene expansions have also occurred in sheep and cattle. Likewise, the general organization of the goat TRG genes is typical of ruminant species studied so far, with two paralogous TRG loci, TRG1 and TRG2, located in two distinct and distant positions on the same chromosome as result of a split in the ancestral locus. Each TRG locus consists of reiterated V-J-J-C cassettes, with the goat TRG2 containing an additional cassette relative to the corresponding sheep and cattle loci. CONCLUSIONS: Taken together, these findings demonstrate that strong evolutionary pressures in the ruminant lineage have selected for the development of enlarged sets of TRB and TRG genes that contribute to a diverse T cell receptor repertoire. However, differences observed among the goat, sheep and cattle TRB and TRG genes indicate that distinct evolutionary histories, with independent expansions and/or contractions, have also affected each ruminant species.

Evolution of the T-Cell Receptor (TR) Loci in the Adaptive Immune Response: The Tale of the TRG Locus in Mammals.

T lymphocytes are the principal actors of vertebrates' cell-mediated immunity. Like B cells, they can recognize an unlimited number of foreign molecules through their antigen-specific heterodimer receptors (TRs), which consist of αß or γδ chains. The diversity of the TRs is mainly due to the unique organization of the genes encoding the α, ß, γ, and δ chains. For each chain, multi-gene families are arranged in a TR locus, and their expression is guaranteed by the somatic recombination process. A great plasticity of the gene organization within the TR loci exists among species. Marked structural differences affect the TR γ (TRG) locus. The recent sequencing of multiple whole genome provides an opportunity to examine the TR gene repertoire in a systematic and consistent fashion. In this review, we report the most recent findings on the genomic organization of TRG loci in mammalian species in order to show differences and similarities. The comparison revealed remarkable diversification of both the genomic organization and gene repertoire across species, but also unexpected evolutionary conservation, which highlights the important role of the T cells in the immune response.

The Camel Adaptive Immune Receptors Repertoire as a Singular Example of Structural and Functional Genomics.

The adaptive immune receptors repertoire is highly plastic, with its ability to produce antigen-binding molecules and select those with high affinity for their antigen. Species have developed diverse genetic and structural strategies to create their respective repertoires required for their survival in the different environments. Camelids, until now, considered as a case of evolutionary innovation because of their only heavy-chain antibodies, represent a new mammalian model particularly useful for understanding the role of diversity in the immune system function. Here, we review the structural and functional characteristics and the current status of the genomic organization of camel immunoglobulins (IG) or antibodies, α/ß and γ/δ T cell receptors (TR), and major histocompatibility complex (MHC). In camelid humoral response, in addition to the conventional antibodies, there are IG with "only-heavy-chain" (no light chain, and two identical heavy gamma chains lacking CH1 and with a VH domain designated as VHH). The unique features of these VHH offer advantages in biotechnology and for clinical applications. The TRG and TRD rearranged variable domains of Camelus dromedarius (Arabian camel) display somatic hypermutation (SHM), increasing the intrinsic structural stability in the γ/δ heterodimer and influencing the affinity maturation to a given antigen similar to immunoglobulin genes. The SHM increases the dromedary γ/δ repertoire diversity. In Camelus genus, the general structural organization of the TRB locus is similar to that of the other artiodactyl species, with a pool of TRBV genes positioned at the 5' end of three in tandem D-J-C clusters, followed by a single TRBV gene with an inverted transcriptional orientation located at the 3' end. At the difference of TRG and TRD, the diversity of the TRB variable domains is not shaped by SHM and depends from the classical combinatorial and junctional diversity. The MHC locus is located on chromosome 20 in Camelus dromedarius. Cytogenetic and comparative whole genome analyses revealed the order of the three major regions "Centromere-ClassII-ClassIII-ClassI". Unexpectedly low extent of polymorphisms and haplotypes was observed in all Old World camels despite different geographic origins.

Comparative Analysis of the TRB Locus in the Camelus Genus.

T cells can be separated into two major subsets based on the heterodimer that forms their T cell receptors. αß T cells have receptors consisting of α and ß chains, while γδ T cells are composed of γ and δ chains. αß T cells play an essential role within the adaptive immune responses against pathogens. The recent genomic characterization of the Camelus dromedarius T cell receptor ß (TRB) locus has allowed us to infer the structure of this locus from the draft genome sequences of its wild and domestic Bactrian congeners, Camelus ferus and Camelus bactrianus. The general structural organization of the wild and domestic Bactrian TRB locus is similar to that of the dromedary, with a pool of TRBV genes positioned at the 5' end of D-J-C clusters, followed by a single TRBV gene located at the 3' end with an inverted transcriptional orientation. Despite the fragmented nature of the assemblies, comparative genomics reveals the existence of a perfect co-linearity between the three Old World camel TRB genomic sequences, which enables the transfer of information from one sequence to another and the filling of gaps in the genomic sequences. A virtual camelid TRB locus is hypothesized with the presence of 33 TRBV genes distributed in 26 subgroups. Likewise, in the artiodactyl species, three in-tandem D-J-C clusters, each composed of one TRBD gene, six or seven TRBJ genes, and one TRBC gene, are placed at the 3' end of the locus. As reported in the ruminant species, a group of four functional TRY genes at the 5' end and only one gene at the 3' end, complete the camelid TRB locus. Although the gene content is similar, differences are observed in the TRBV functional repertoire, and genes that are functional in one species are pseudogenes in the other species. Hence, variations in the functional repertoire between dromedary, wild and domestic Bactrian camels, rather than differences in the gene content, may represent the molecular basis explaining the disparity in the TRB repertoire between the Camelus species. Finally, our data contribute to the knowledge about the evolutionary history of Old World camelids.

Cytochrome b marker reveals an independent lineage of Stenella coeruleoalba in the Gulf of Taranto.

Heterogeneity in geomorphological and hydrographical conditions throughout the Mediterranean Sea could be the driving factors behind the significant differences between putative sub-populations, although the existence of a large panmictic population of striped dolphin Stenella coeruleoalba (Meyen 1833) in this marine region could not be excluded. However, understanding the ecological implications of such genetic differentiation is difficult, as inferences about gene flow are usually made on evolutionary time scales and not along the ecological time frame over which most management and conservation practices are applied. In fact, as stated by the IUCN Red List, in the case of species assessed as vulnerable, the degree of genetic exchange between populations within a biogeographic region and its ecological implications represent a fascinating challenge that should be very deeply explored. This is even more significant in the Gulf of Taranto (Northern Ionian Sea, Central-eastern Mediterranean Sea), where the geomorphological and hydrographic characteristics support the hypothesis of a separated striped dolphin population genetically diverging from its original Mediterranean counterpart. To assess this hypothesis, a genetic analysis was carried out on DNA fragments of the mitochondrial cyt b gene to explore the evolutionary origin of S. coeruleoalba in the investigated area and its genetic diversity in comparison with available sequences from other Mediterranean and Atlantic populations. Results were discussed indicating ecological implications and suggesting conservation objectives. Moreover, a delphinid systematic was also suggested.

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

The α/ß T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and ß 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 ß-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.

Data characterizing the genomic structure of the T cell receptor (TRB) locus in Camelus dromedarius.

These data are presented in support of structural and evolutionary analysis of the published article entitled "The occurrence of three D-J-C clusters within the dromedary TRB locus highlights a shared evolution in Tylopoda, Ruminantia and Suina" (Antonacci et al., 2017) [1]. Here we describe the genomic structure and the gene content of the T cell receptor beta chain (TRB) locus in Camelus dromedarius. As in the other species of mammals, the general genomic organization of the dromedary TRB locus consists of a pool of TRBV genes located upstream of in tandem TRBD-J-C clusters, followed by a TRBV gene with an inverted transcriptional orientation. A peculiarity of the dromedary TRB locus structure is the presence of three TRBD-J-C clusters, which is a common feature of sheep, cattle and pig sequences.

The occurrence of three D-J-C clusters within the dromedary TRB locus highlights a shared evolution in Tylopoda, Ruminantia and Suina.

The αß 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 αß TR, which is generated by a recombination of specific Variable, Diversity and Joining genes for the ß 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.

Genomic and expression analyses of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) loci reveal a similar basic public γδ repertoire in dolphin and human.

BACKGROUND: The bottlenose dolphin (Tursiops truncatus) is a mammal that belongs to the Cetartiodactyla and have lived in marine ecosystems for nearly 60 millions years. Despite its popularity, our knowledge about its adaptive immunity and evolution is very limited. Furthermore, nothing is known about the genomics and evolution of dolphin antigen receptor immunity. RESULTS: Here we report a evolutionary and expression study of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) genes. We have identified in silico the TRG and TRA/TRD genes and analyzed the relevant mature transcripts in blood and in skin from four subjects. The dolphin TRG locus is the smallest and simplest of all mammalian loci as yet studied. It shows a genomic organization comprising two variable (V1 and V2), three joining (J1, J2 and J3) and a single constant (C), genes. Despite the fragmented nature of the genome assemblies, we deduced the TRA/TRD locus organization, with the recent TRDV1 subgroup genes duplications, as it is expected in artiodactyls. Expression analysis from blood of a subject allowed us to assign unambiguously eight TRAV genes to those annotated in the genomic sequence and to twelve new genes, belonging to five different subgroups. All transcripts were productive and no relevant biases towards TRAV-J rearrangements are observed. Blood and skin from four unrelated subjects expression data provide evidence for an unusual ratio of productive/unproductive transcripts which arise from the TRG V-J gene rearrangement and for a "public" gamma delta TR repertoire. The productive cDNA sequences, shared both in the same and in different individuals, include biases of the TRGV1 and TRGJ2 genes. The high frequency of TRGV1-J2/TRDV1- D1-J4 productive rearrangements in dolphins may represent an interesting oligo-clonal population comparable to that found in human with the TRGV9- JP/TRDV2-D-J T cells and in primates. CONCLUSIONS: Although the features of the TRG and TRA/TRD loci organization reflect those of the so far examined artiodactyls, genomic results highlight in dolphin an unusually simple TRG locus. The cDNA analysis reveal productive TRA/TRD transcripts and unusual ratios of productive/unproductive TRG transcripts. Comparing multiple different individuals, evidence is found for a "public" gamma delta TCR repertoire thus suggesting that in dolphins as in human the gamma delta TCR repertoire is accompanied by selection for public gamma chain.

Sheep (Ovis aries) T cell receptor alpha (TRA) and delta (TRD) genes and genomic organization of the TRA/TRD locus.

BACKGROUND: In mammals, T cells develop along two discrete pathways characterized by expression of either the αß 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. RESULTS: The analysis of the current Ovis aries whole genome assembly, Oar_v3.1, revealed that, as in the other mammalian species, the sheep TRD locus is nested within the TRA locus. In the most 5' part the TRA/TRD locus contains TRAV genes which are intermingled with TRDV genes, then TRD genes which include seven TRDD, four TRDJ genes, one TRDC and a single TRDV gene with an inverted transcriptional orientation, and finally in the most 3' part, the TRA locus is completed by 61 TRAJ genes and one TRAC gene. Comparative sequence and analysis and annotation led to the identification of 66 TRAV genes assigned to 34 TRAV subgroups and 25 TRDV genes belonging to the TRDV1 subgroup, while one gene was found for each TRDV2, TRDV3 and TRDV4 subgroups. Multiple duplication events within several TRAV subgroups have generated the sheep TRAV germline repertoire, which is substantially larger than the human one. A significant proportion of these TRAV gene duplications seems to have occurred simultaneously with the amplification of the TRDV1 subgroup genes. This dynamic of expansion has also generated novel multigene subgroups, which are species-specific. Ovis aries TRA and TRD genes identified in this study were assigned IMGT definitive or temporary names and were approved by the IMGT/WHO-IUIS nomenclature committee. The completeness of the genome assembly in the 3' part of the locus has allowed us to interpret rearranged CDR3 of cDNA from both TRA and TRD chain repertoires. The involvement of one up to four TRDD genes into a single transcript makes the potential sheep TRD chain much larger than any known TR chain repertoire. CONCLUSIONS: The sheep genome, as the bovine genome, contains a large and diverse repertoire of TRA and TRD genes when compared to the "γδ T cell low" species genomes. The composition and length of the rearranged CDR3 in TRD V-delta domains influence the three-dimensional configuration of the antigen-combining site thus suggesting that in ruminants, γδ T cells play a more important and specific role in immune recognition.

Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains.

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

Genomic characteristics of the T cell receptor (TRB) locus in the rabbit (Oryctolagus cuniculus) revealed by comparative and phylogenetic analyses.

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.

Generation of diversity by somatic mutation in the Camelus dromedarius T-cell receptor gamma variable domains.

In jawed vertebrates the V-(D)-J rearrangement is the main mechanism generating limitless variations of antigen-specific receptors, immunoglobulins (IGs), and T-cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T-cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V-J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.

Structural and comparative analysis of the T cell receptor gamma (TRG) locus in Oryctolagus cuniculus.

In mammals, T cells develop along two discrete pathways characterized by expression of either the αß or the γδT cell receptors. Human, mouse, and dog display a low peripheral blood γδ T cell percentage, while sheep accounts for a high proportion of γδ 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 γδ T cell lineage, the present study has defined the genomic organization of the TRG locus in rabbit (Oryctolagus cuniculus), another mammalian γδ 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 γδ 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 γδ T cell function.