G C A T T A C G G C A T genes

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

Rachele Antonacci 1,* , Serafina Massari 2, Giovanna Linguiti 1, Anna Caputi Jambrenghi 3 , Francesco Giannico 3 , Marie-Paule Lefranc 4 and Salvatrice Ciccarese 1

1 Department of Biology, University of Bari “Aldo Moro”, 70124 Bari, Italy; [email protected] (G.L.); [email protected] (S.C.) 2 Department of Biological and Environmental Science and Technologies, University of Salento, 73100 Lecce, Italy; [email protected] 3 Department of Agricultural and Environmental Science, University of Bari “Aldo Moro”, 70124 Bari, Italy; [email protected] (A.C.J.); [email protected] (F.G.) 4 IMGT®, the International ImMunoGeneTics Information System®, Laboratoire d’ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, UMR9002 CNRS, Université de Montpellier, CEDEX 5, 34396 Montpellier, France; [email protected] * Correspondence: [email protected]



Received: 28 April 2020; Accepted: 2 June 2020; Published: 5 June 2020


Abstract: 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 αβ or γδ chains. The diversity of the TRs is mainly due to the unique organization of the genes encoding the α, β, 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.

Keywords: T cell receptor; TRG locus; TRG genes; immunogenomics; evolution; mammals; IMGT

1. Introduction T lymphocytes play a crucial role in the immune surveillance of all jawed vertebrates. There are essentially two major subpopulations of T cells, which are classified as or according to the T-cell receptors (TRs) expressed on their membrane [1]. Each T lymphocyte expresses a unique antigen-specific TR. αβ T cells are the most abundant in the explored species, with a broad distribution in circulation and lymphoid organs. αβ T cells are mainly able to recognize peptides from degraded proteins bound to major histocompatibility (MH) proteins at the surface of antigen-presenting cells. In this context, αβ T cells comprise two subsets based on the expression of CD4 or CD8 coreceptors, which participate in the recognition of the MH-peptide antigen and in the T-cell signal transduction. CD4+ T cells recognize ligands in the context of MH class II (MH2) proteins, whereas CD8+ T cells recognize antigens via presentation by MH class I (MH1) proteins [2].

Genes 2020, 11, 624; doi:10.3390/genes11060624 www.mdpi.com/journal/genes Genes 2020, 11, 624 2 of 28

γδ T cells are an enigmatic cell population with unique features compared with αβ T cells [3]. The amount of circulating γδ T cells varies in different species. In humans and mice (“γδ-low species”), γδ T cells make up only 1–10% of circulating T lymphocytes, although they are substantially enriched in mucosa and epithelial tissues, suggesting a critical role in early immune responses. In contrast, in “γδ-high species” such as chickens, cattle, sheep, and pigs, γδ T cells constitute a major lymphocyte population in the peripheral blood (15–60%), particularly in young animals [4–8], suggesting an important role in host defense. γδ T cells are a functionally heterogeneous population, although details of their functions are becoming clearer. They recognize antigens directly and without processing, in a manner similar to immunoglobulins (IG) [9], or presented by RPI-MH1Like proteins [10], providing more flexible and not yet clear recognition of a wide range of ligands (soluble, membrane-bound, and unprocessed antigens). In line with their MH-independent antigen recognition, γδ T cells lack CD4 and CD8, and are therefore referred to as CD4 and CD8 double-negative cells. However, in ruminants, a large proportion of circulating γδ T cells express the γδ-T-cell specific WC1 proteins, which belong to the scavenger superfamily and act as pattern-recognition receptor (PRRs) and γδ-T-cell-activating coreceptors [11,12]. In all species, γδ T cells are involved in diverse and important roles in not only adaptive, but also innate immune responses, specific to each anatomical location and physiological context. Defined roles for γδ T cells are few. For example, they have been shown to play a significant function in wound healing in mouse epidermis [13]. A restricted subset of γδ T cells in human peripheral blood are involved in the recognition of phosphoantigen metabolites (PAgs), which can be produced by bacterial and eukaryotic cells as result of metabolic dysregulation [14–16]. In general, the ability of γδ T cells to recognize cell-surface molecules expressed on stressed or infected cells has direct consequences for the function of these cells in a broad range of immune settings, including antimicrobial immunity, anti-tumor immunity, and tissue homeostasis [17,18]. In ruminants, the γδ T cells recognize infectious agents in combination with WC1 proteins, making these species a valuable model for understanding the variety of ways in which these T cells respond to important pathogens [19]. The functional differences of γδ T cells among species mirror differences in the TR γ (TRG) and TR δ (TRD) chain repertoires, with a wider and more diversified repertoire in domestic species compared to humans and mice. In turn, the TRG and TRD chain repertoires depend on the gene organization of the TRG and TRD loci which, interestingly, show more variability between the different species than do the TRA and TRB loci, which encode the and chains of the T cells. The MH-independent recognition of T cells with respect to the MH restriction of T cells probably explains the plasticity of the TR and TR genomic organization.

2. The TR Chains are Encoded by Separate Multigene Families αβ and γδ heterodimers are members of the Ig superfamily, and the genes encoding for each TR chain [1] are organized in the germline DNA in a manner remarkably similar to the multigene organization of the immunoglobulin (IG) loci [2,9]. Each TR locus lies on a different chromosome region and consists of arrays of different gene types, including variable (V), diversity (D), joining (J), and constant (C) genes, each representing a multigene sub-family. Productive TRs are produced during the development of T lymphocytes in the thymus by somatic rearrangements of V and J genes in the TRA and TRG loci, and between V, D, and J genes in the TRB and TRD loci. After transcription, the resulting rearranged V-(D)-J region, encoding the variable domain of the TR chain, is spliced to the C gene, which encodes the constant domain of the receptor. The variable domain forms the antigen-binding site, while the constant domain anchors the receptor to the cell membrane and is involved in signal transduction. The resulting chain is a protein with the variable domain composed of seven distinguishable regions: three hypervariable loops or complementarity-determining regions (CDR) and four framework regions (FR). Two of the CDR loops, CDR1 and CDR2, are encoded by the V gene. The third CDR loop (CDR3) reflects the ability of the V gene to rearrange to any (D) J gene [1]. Therefore, the number of V, D, and J genes in the germline DNA, and the somatic V-(D)-J Genes 2020, 11, 624 3 of 28 rearrangement mechanism unique to the adaptive immune response, contribute to the huge diversity of the expressed TR repertoire, allowing potentially billions of different TR antigen-binding sites to be produced from a limited set of genes [2]. The gene organization in each of the four TR loci is known for many species. The genomic structure of the TRB locus has a common feature in representative species of several orders of eutherian mammals, with a pool of V (TRBV) genes, a different number from species to species, positioned upstream of tandem-aligned TRBD-J-C clusters, each composed of a single D (TRBD) gene, several J (TRBJ) genes, and one C (TRBC) gene. A single TRBV gene in inverted orientation of transcription completes each TRB locus at the 30 end. In most mammalian species, including human [20,21], mouse [22], chimpanzee and rhesus monkey [23], dog [24], rabbit [25], ferret [26], and cat [27], two TRBD-J-C clusters exist. In contrast, in the cetartiodactyl lineage [28–35], a duplication event within the 30 end of the TRB locus led to the generation of a third TRBD-J-C cluster, increasing the number of TRBD and TRBJ genes available for the somatic rearrangements. The organization of the TRA and TRD genes displays an intriguing and conserved feature, in that they are located at a single chromosomal region with the TRD locus nested within the TRA locus. In fact, this is referred to the TRA/TRD locus. Despite this complex arrangement, each TR locus exhibits specific control of its own gene assembly. The general genomic organization of the TRA/TRD region, from the 50 end to the 30 end, consists of an array of TRAV genes among which the TRDV genes are embedded. The region continues with the TRD locus, i.e., the TRDD, TRDJ, and one TRDC gene, followed by a TRDV gene in inverted orientation of transcription. At the 30 end, a cluster of TRAJ lies followed by one TRAC, which completes the TRA locus. As a consequence of the gene organization, the whole TRD locus is excised from the genomic sequence when the first TRA V-J rearrangement occurs in the TRA/TRD locus. The characterization of the TRA/TRD locus in the different mammals (https://www.imgt.org/IMGTrepertoire/: Homo sapiens, Mus musculus, Macaca mulatta, Bos taurus;[27,36–40]), has shown that in this case, the number of TRAV and TRDV genes also represents the major disparity among species. Differently from the TRB and TRA/TRD loci, the TRG locus shows great gene-organization plasticity related to the evolution of different species. Typically, the TRG genes comprise an array of multiple TRGV genes linked to TRGJ and TRGC genes organized in J-C clusters; otherwise, the TRG locus features a gene-cluster organization in V-J-C rearrangement units or cassettes. The main goal of this review was to collect all the data on the TRG locus structure of eutherian mammalian species for which the genomic organization has been characterized in detail, and to highlight differences and similarities. The Homo sapiens TRG locus is described as a paradigm, as it was the first complete locus of the adaptive immune response to be entered in databases as “genes” as well as conventional genes, leading in 1989 to the creation of IMGT and to immunoinformatics, a new science at the interface between immunogenetics and bioinformatics [2]. On the basis of the accepted phylogeny, we describe the genomic characteristic of the TRG locus in Cetartiodactyla/Carnivora lineages, with respect to Rodentia/Lagomorpha/Primata. Furthermore, for a comparative analysis, the genomic organization of the TRG locus in other vertebrate species, outgroups respective to placental mammals, is also described.

3. The Homo sapiens TRG Locus The Homo sapiens TRG locus at 7p14 spans 160 kb [1,41] (Figure1A, with permission of IMGT ® http://www.imgt.org). The orientation of the human TRG locus on the chromosome is reversed (REV) (Figure1B, with permission of IMGT ® http://www.imgt.org). It consists of 12–15 TRGV genes upstream of a duplicated J-C cluster, which comprises in the first part three TRGJ genes and the TRGC1 gene, and in the second part, two TRGJ genes and the TRGC2 gene [42–51]. The 50-most TRGV genes occupy the most centromeric position, whereas the TRBC2 gene, 30 of the locus, is the most telomeric in the TRG locus. The TRGV genes belong to six different subgroups based on the absence of cross-hybridization between them and defined, in terms of sequences, by less than 75% identity at the nucleotide level Genes 2020, 11, 624 4 of 28

Genes 2020, 11, x FOR PEER REVIEW 4 of 28 in their V regions [44]. TRGV9, expressed in 80–95% of the human peripheral T cells, is the unique membercells, is the of Subgroupunique member 2. TRGV10 of Subgroupand TRGV11 2. TRGV10, the single and membersTRGV11, ofthe subgroups single members 3 and 4, of respectively, subgroups have3 and been 4, respectively, found to be rearrangedhave been found and transcribed, to be rearranged but they and are opentranscribed, reading but frames they (ORFs) are open that reading cannot beframes expressed (ORFs) in that a gamma cannot chain, be expressed due to a splicing in a ga defectmma chain, of the pre-messengerdue to a splicing [52 ,defect53]. The of potentialthe pre- repertoiremessenger consists[52,53]. The of four potential to six repertoire functional consistsTRGV ofgenes four belonging to six functional to two TRGV subgroups, genes fivebelongingTRGJ andto two two subgroups,TRGC genes five[ 54TRGJ,55]. and The two description TRGC genes of the[54,55]. germline The description V region and of the that germline of the expressed V region rearrangedand that of V-Jthe domainexpressed and rearranged CDR lengths V-J is domain based on and the CDR IMGT lengths unique is numberingbased on the (CDR-IMGT) IMGT unique for thenumbering V domain (CDR-IMGT) [56–58] and for IMGT the V Collier domain de [56–58] Perles [and59], IMGT whereas Collier that ofde the Perles C-γ [59],domain whereas is based that onof the IMGTC-γ domain unique is numberingbased on the for IMGT the C unique domain numbering [60]. for the C domain [60].

(A)

(B)

Figure 1. Human (Homo sapiens) TRG locus representation (A) and chromosomal localization (B), reproducedFigure 1. Human with permission (Homo sapiens) from IMGTTRG ®locus(http: representation//www.imgt.org (A).) Inand (A chromosomal), colors are according localization to IMGT (B), ® colorreproduced menu with for genes permission (https: from//www.imgt.org IMGT (http://www.imgt.org)./IMGTScientificChart In/ (RepresentationRulesA), colors are according/colormenu. to IMGT php#LOCUScolor ). The boxes representingmenu the genes are not to scale. Exonsfor are not shown. A double arrowgenes (https://www.imgt.org/IMGTScientificChart/RepresentationRules/colormenu.php#LOCUS). The indicates insertion/deletion polymorphisms. The Amphiphysin (AMPH) (IMGT 50 borne) was identified boxes representing the genes are not to scale. Exons are not shown. A double arrow indicates 16 kb upstream of TRGV1 (ORF), the most 50 gene in the locus, and the Related to steroidogenic acute insertion/deletion polymorphisms. The Amphiphysin (AMPH) (IMGT 5′ borne) was identified 16 kb regulatory protein D3-N-terminal like (STARD3NL) (30 IMGT borne) was identified 9.4 kb downstream of upstream of TRGV1 (ORF), the most 5′ gene in the locus, and the Related to steroidogenic acute regulatory TRGC2 (F), the most 30 gene in the locus. In (B), a vertical red line indicates the localization of the TRG locusprotein at D3-N-terminal 7p14. A blue like arrow (STARD3NL indicates) the (3′ orientationIMGT borne) 5 was3 identifiedof the locus, 9.4 kb and downstream the gene group of TRGC2 order 0 → 0 in(F), the the locus. most The 3′ gene blue in arrow the locus. is proportional In (B), a vertical to the size red of line the indicates locus, indicated the localization in kilobases of the (kb). TRG The locus total numberat 7p14. ofA genesblue arrow in the indicates locus is shown the orientation in parentheses. 5′ → 3′ of the locus, and the gene group order in the locus. The blue arrow is proportional to the size of the locus, indicated in kilobases (kb). The total number of genes in the locus is shown in parentheses.

Polymorphisms in the number of TRGV genes and in the exon number of the TRGC2 gene have been described in different populations. Variation of the number of TRGV subgroup genes (from 7

Genes 2020, 11, 624 5 of 28

Polymorphisms in the number of TRGV genes and in the exon number of the TRGC2 gene have been described in different populations. Variation of the number of TRGV subgroup genes (from 7 to 10) has been observed [61,62]. These allele polymorphisms, which result from the deletion of V4 and V5, or from the insertion of an additional gene, V3P, between V3 and V4, can be detected via restriction-fragment length polymorphism (RFLP). The two TRGC genes, which are 16 kb apart, resulted, with their associated TRGJ genes, from a recent duplication in the locus. However, there are structural differences. TRGJP1, TRGJ1, and TRGC1 cross-hybridize to TRGJP2, TRGJ, and TRGC2, respectively, whereas the TRGJP has no equivalent in the TRGJP2-J2-C2 cluster. The TRGC genes encode the constant domain (C-γ) of 110 amino acids (AA), the connecting region (CO), the transmembrane region (TM), and the cytoplasmic region (CY). The TRGC1 has three exons and encodes a C-region of 173 AA, whereas the TRGC2 gene has four or five exons, owing to the duplication or triplication of a region that includes Exon 2 (EX2, EX2T and/or EX2R) and encodes a C-region of 189 or 205 AA, respectively [1]. This allelic polymorphism of the TRGC2 with duplication (C2(2 )) or triplication × (C2(3 )) of Exon 2 can be identified via RFLP [63]. Exon 2 of the TRGC1 gene has a cysteine involved × in the interchain disulfide bridge, whereas the cysteine is not conserved in Exon 2 of the human TRGC2 gene. Enhancer and silencer sequences have been characterized 6.5 kb downstream of the TRGC2 gene [64]. The total number of TRG genes per haploid genome in human is 19 to 22, of which 11 to 13 are functional.

4. The “Gene Cluster” Organization of the TRG Locus Is Predominant in the Outgroups The first paper on the chicken (Gallus gallus), reported the screening of a splenic cDNA library and Northern blot analysis of the thymus and spleen. Results identified three multimember TRGV subgroups, three TRGJ genes, and a single constant TRGC gene in the TRG locus [65]. At that time, the genomic organization of the TRG loci was known only for mice [66–68] and humans [41,42,45], and a TRGV gene expansion was proposed to explain the association between the high frequency of γδ T cells in chickens as well as in cattle, sheep, and pigs [4–7], respective to the low frequency of γδ T cells in mice and humans. In a very recent paper, the Gallus gallus TR genomic organization was obtained by using Illumina and single-molecule real-time sequencing technology to re-sequence genomic regions of chicken TR loci based on 10 mapped bacterial artificial chromosome clones. The chicken TRG locus has been mapped to Chromosome 2 and it spans only 82 kb; DNA-dependent protein kinase catalytic subunit (PRKDC) and leucine-rich repeat flightless-interacting protein 2 (LRRFIP2) genes are found immediately flanking the 50 and 30 end of the TRG locus (Table1, Supplementary Figure S1A) [69]. The chicken TRG locus organization recalls the cluster scheme. It consists of a single J-C (three TRGJ–one TRGC) gene cluster equipped with 37 upstream TRGV genes that are divided into 11 subgroups (Table1, Supplementary Figure S1A). This expansion of TRGV genes is slightly at odds with their usage in the peripheral blood, where only 15 have been found to be expressed [69]. The single TRGC gene is made up of three exons. Recently, the genomic organization of the TRG locus and the germline and expressed repertoire of TRG genes in the White Peking duck were determined. In this avian, the TRG locus consists of 13 TRGV genes classified into six subgroups upstream of a single J-C (five TRGJ–one TRGC) gene cluster (Table1, Supplementary Figure S1B) [ 70]. The total number of variables is certainly lower than that found in chickens, although this number may not be the correct one. In fact, the 50 end of the locus lacks the PRKDC flanking gene, and this is an indication of the genomic incompleteness of the locus itself. In the sandbar shark (Carcharhinus plumbeus), a cartilaginous fish, the TRG locus consists of a single J-C (three TRGJ–one TRGC) gene cluster preceded by five TRGV genes (Table1, Supplementary Figure S1C) [71]. Approximately equal numbers of clones have been found containing TRGV1 (18), TRGV2 (19), TRGV3 (12), and TRGV4 (17), indicating that there is no bias in the rearrangement of these V genes; however, the author found only four clones expressing TRGV5, suggesting that there may be significantly less rearrangement of this most 50 distal V gene. Similarly, no bias was apparent in the expression of the three TRGJ genes. However, expression analysis conducted on spleen Genes 2020, 11, 624 6 of 28 tissue from a single shark revealed the presence of a high degree of nucleotide mutations in the V region of cDNA sequences with respect to parental genomic sequences. Somatic hypermutation (SHM) in the sandbar shark TRG V region is the explanation for these data. The SHM process has shark-specific characteristics, such as the presence of tandem mutations [72]. Genes 2020, 11, x FOR PEER REVIEW 6 of 28 Genes 2020, 11, x FOR PEER REVIEW 6 of 28 GenesGenes 20202020,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 2828 Table 1. Genomic organization and gene content of the TRG locus in the outgroups. Table 1. GenomicGenes 2020 organization, 11, x FOR PEER REVIEW and gene content of the TRG locus in the outgroups.6 of 28 Table 1. Genomic organization and gene content of the TRG locus in the outgroups. Genes 2020, 11, xTable FOR PEER 1. Genomic REVIEW organization and gene content of the TRG locus in the outgroups. 6 of 28 Table 1. Genomic organization and gene content of the TRG locus in the outgroups. Table 1. Genomic organization and gene content of theChromosomal TRG locus in the outgroups. Reference TRGV genes TRGJ genes TRGC genes Chromosomal Miniature locus TRGV GenesTable TRGJ1. Genomic Genes organization TRGC and gene Genes content of theChromosomal TRG locus in the outgroups.Miniature Locus ReferencesReference TRGV genes TRGJ genes TRGC genes LocalizationChromosomalChromosomallocalization Miniature locus sReferenceReference TRGVTRGVTRGV1 genesgenes (6) TRGJ TRGJ genesgenes TRGC TRGC genesgenes Chromosomallocalization Miniature locus sReference localization s TRGV1 (6) TRGVTRGV2TRGV1 genes (7)(6) TRGJ genes TRGC genes Chromosomallocalization Miniature locus sReference TRGV genes TRGJ genes TRGC genes localization Miniature locus s TRGV3TRGV2TRGV1TRGV1 (2)(7)(6)(6) TRGV2 (7) TRGV1 (6) localization s TRGV4TRGV3TRGV2TRGV2 (5)(2)(7)(7) TRGV3 (2) TRGV2TRGV1 (7)(6) TRGV5TRGV4TRGV3TRGV3 (2)(5)(2) TRGJ1 (1) one locus TRGV3TRGV2 (2)(7) Liu et al., TRGV4 (5) TRGV6TRGV4TRGV4 (4)(5)(5) TRGJ2 (1) one locus Chicken TRGV5 (2) TRGJ1 (1) TRGC one locus TRGV5 (2) TRGV4TRGV5TRGV3TRGV5TRGJ1 (5)(2)(2) (1) TRGJ1TRGJ1 (1)(1) oneone locus locus Liu et al., Chicken TRGV7TRGV6 (4) TRGJ3TRGJ2 (1) TRGC one locus LiuLiu2020 etet al.,al., TRGV5TRGV6TRGV4TRGV6 (2)(4)(5)(4) TRGJ1TRGJ2TRGJ2 (1)(1) Chrom. 2 Chicken TRGV6ChickenChicken (4) TRGV8TRGV7TRGJ2 (2)(4) (1) TRGJ3 (1) TRGCTRGCTRGC one locus LiuLiu2020 et al., et al., [69]; TRGV6TRGV5TRGV7 (4)(2)(4) TRGJ2TRGJ1TRGJ3 (1)(1) TRGV7Chicken (4) TRGV9TRGV8TRGV7TRGJ3 (3)(2)(4) (1) TRGJ3 (1) TRGC Chrom.Chrom. 2 Gallus gallus Liu20202020 et al., TRGV7TRGV6 (4)(4) TRGJ3TRGJ2 (1)(1) Chrom.Chrom. 22 BIRDS Chicken TRGV10TRGV9TRGV8TRGV8 (3)(2)(2)(1) TRGC Gallus gallus 2020 TRGV8 (2) TRGV8TRGV7 (2)(4) TRGJ3 (1) Chrom. 2 GallusGallus gallusgallus 2020 BIRDS TRGV11TRGV10TRGV9TRGV9 (3)(3)(1) Gallus gallus TRGV9 (3) TRGV9TRGV8 (3)(2) Chrom. 2 Gallus gallus BIRDS BIRDSBIRDS TRGV11TRGV10TRGV10 (1)(1) BIRDSTRGV10 (1) TRGV10TRGV11TRGV11TRGV1TRGV9 (5) (3) (1)(1) Gallus gallus TRGJ1 (1) BIRDS TRGV11TRGV10TRGV2TRGV1 (2)(5) (1)(1) TRGV11 (1) TRGJ2TRGJ1 (1) one locus TRGV11TRGV3TRGV2TRGV1TRGV1 (3)(2)(5) (5)(1) Yang et TRGJ3TRGJ1TRGJ1 (1)(1) one locus Duck TRGV1TRGV2TRGV2 (5)(2)(2) TRGJ2 (1) TRGC one locus TRGV1 (5) TRGV4TRGV3 (1)(3) TRGJ1TRGJ2TRGJ2 (1)(1) one locus Yang et Duck TRGV2TRGV3TRGV1TRGV3TRGJ1 (2)(3)(5)(3) (1) TRGJ4TRGJ3 (1) TRGC al.,YangYang 2017 etet TRGV2 (2) TRGV5TRGV4 (1) TRGJ2TRGJ1TRGJ3 (1)(1) one locus DuckDuck TRGV3TRGV2 (3)(2) TRGJ5TRGJ4TRGJ3 (1) TRGCTRGC no assigned al.,Yang 2017 et TRGV6TRGV5TRGV4TRGV4TRGJ2 (1)(1) (1) oneone locus locus TRGV3Duck (3) TRGJ3TRGJ4TRGJ2TRGJ4 (1)(1) TRGC no assigned Anas platyrynchos al.,al., 20172017 TRGV4TRGV5TRGV3TRGV5 (1)(3)(1) TRGJ5 (1) Yang et Duck TRGV6TRGJ3 (1) (1) TRGJ4TRGJ5TRGJ3TRGJ5 (1)(1) TRGC nono assignedassigned Anas platyrynchos Yangal., 2017 et al., [70]; TRGV4Duck (1) TRGV5TRGV4TRGV6 (1)(1) TRGC TRGV6TRGJ4 (1) (1) TRGJ5TRGJ4 (1)(1) nono assigned assigned AnasAnas platyrynchosplatyrynchos al., 2017 TRGV6TRGV5 (1)(1) TRGV5 (1) TRGJ5 (1) no assigned Anas platyrynchos TRGV6TRGJ5 (1) (1) AnasAnas platyrynchos platyrynchos TRGV6 (1) TRGV1 (1) TRGV2TRGV1 (1) TRGJ1 (1) one locus TRGV1 (1) Chen et TRGV3TRGV2TRGV1TRGV1 (1)(1) TRGJ2TRGJ1 (1) one locus Shark TRGV1 (1) TRGC oneone locuslocus Chen et TRGV2 (1) TRGV4TRGV3TRGV2TRGV2TRGJ1 (1)(1) (1) TRGJ3TRGJ2TRGJ1TRGJ1 (1)(1) one locus Shark TRGC one locus al.,ChenChen 2009 etet Shark TRGV3 (1) TRGV2TRGV3TRGV1TRGV3TRGJ2 (1)(1) (1) TRGJ1TRGJ2TRGJ2 (1)(1) TRGC no assigned Chen et al., [71]; SharkShark TRGV5TRGV4 (1) TRGJ3 (1) TRGCTRGC one locus al.,Chen 2009 et TRGV3TRGV4TRGV2TRGV4 (1)(1) TRGJ2TRGJ3TRGJ1TRGJ3 (1)(1) al., 2009 TRGV4Shark (1) TRGV5TRGJ3 (1) (1) TRGC nono assigned assigned al.,Chen 2009 et FISHES TRGV4TRGV3 (1)(1) TRGJ3TRGJ2 (1)(1) nono assignedassigned Carcharhinus plumbeus Shark TRGV5TRGV5 (1)(1) TRGC Carcharhinus plumbeus al., 2009 FISHESTRGV5 (1) TRGV5TRGV4 (1)(1) TRGJ3 (1) no assigned Carcharhinus plumbeus al., 2009 CarcharhinusCarcharhinus plumbeusplumbeus FISHESFISHES TRGV5 (1) no assigned FISHES Carcharhinus plumbeus FISHES TRGJ1 (1) TRGC1 (1) FISHES TRGJ1 (1) TRGC1 (1) Carcharhinus plumbeus TRGJ2 (1) TRGJ2TRGJ1 (1)TRGC2 TRGC2TRGC1 (1) (1) twotwo loci loci Atlantic TRGV1Atlantic (4) TRGV1 (4) Yazawa et TRGJ3TRGJ1TRGJ1 (1)(1) TRGC3TRGC1TRGC1 (1)(1) two loci TRGJ3 (1) TRGJ2 (1)TRGC3 TRGC2 (1) (1) two loci Yazawa et al., [73]; salmon TRGV2Atlantic (3) TRGV2TRGV1 (3)(4) TRGJ1TRGJ2TRGJ2 (1)(1) TRGC1TRGC2TRGC2 (1)(1) two loci Yazawa et AtlanticsalmonAtlantic TRGV1TRGV1TRGJ4 (4)(4) (1) TRGJ4TRGJ3 (1)TRGC4 TRGC4TRGC3 (1) (1) no assignedtwo loci YazawaYazawaal., 2008 etet TRGV2 (3) TRGJ2TRGJ3TRGJ1TRGJ3 (1)(1) TRGC2TRGC3TRGC1TRGC3 (1)(1) Atlanticsalmon TRGV1TRGV2 (4)(3) TRGJ5TRGJ4 (1) TRGC5TRGC4 (1) no assigned Yazawaal., 2008 et TRGV2TRGJ5 (3) (1) TRGJ3TRGJ2 (1)(1)TRGC5 TRGC3TRGC2 (1) (1)(1) two loci salmonsalmon TRGJ5TRGJ4TRGJ4 (1)(1) TRGC5TRGC4TRGC4 (1)(1) no assigned al.,al., 20082008 Atlantic TRGV2TRGV1 (3)(4) Salmo salar Yazawa et salmon TRGJ4TRGJ5TRGJ3TRGJ5 (1)(1) TRGC4TRGC5TRGC3TRGC5 (1)(1) nono assignedassigned Salmo salar al., 2008 TRGV2 (3) salmon TRGV1 (1) TRGJ5TRGJ4 (1)(1) TRGC5TRGC4 (1)(1) no assigned Salmo salar al., 2008 TRGV1 (1) TRGJ1 (1) TRGV2TRGV1TRGJ1 (1) (1) TRGJ5 (1) TRGC5 (1) no assigned SalmoSalmo salarsalar TRGV2 (1) TRGV1TRGV1 (1)(1) TRGJ2TRGJ1 (1) Salmo salar TRGV3TRGV2TRGJ2 (1) (1) TRGJ1TRGJ1 (1)(1) TRGV3 (1) TRGV1TRGV2TRGV2 (1)(1) TRGJ3TRGJ2 (1) Salmo salar TRGV4TRGV3TRGJ3 (1) (1) TRGJ1TRGJ2TRGJ2 (1)(1) one locus TRGV2TRGV1TRGV3 (1)(1) TRGJ4TRGJ3 (1) TRGV4 (1) TRGV5TRGV4TRGV3 (6)(1) TRGJ2TRGJ1 (1)(1) Wang et TRGJ4 (1) TRGJ5TRGJ3TRGJ3 (1)(1) oneone locus locus REPTILES Alligator TRGV3TRGV4TRGV2TRGV4 (1)(1) TRGJ4 (1) TRGC one locus TRGV5 (6) TRGV6TRGV5 (3)(6) TRGJ3TRGJ4TRGJ2TRGJ4 (1)(1) one locus Wang et REPTILES Alligator REPTILES Alligator TRGV4TRGV5TRGV3TRGV5TRGJ5 (1)(6)(1)(6) (1) TRGJ6TRGJ5 (1) TRGCTRGC WangWangal., 2018 et et al., [74]; TRGV7TRGV6 (1)(3) TRGJ4TRGJ3TRGJ5 (1)(1) one locus REPTILESREPTILESTRGV6 AlligatorAlligator (3) TRGV5TRGV4 (6)(1) TRGJ7TRGJ6TRGJ5 (1) TRGCTRGC no assigned Wangal., 2018 et TRGV8TRGV7TRGV6TRGV6TRGJ6 (2)(1)(3)(3) (1) no assignedone locus REPTILES Alligator TRGJ5TRGJ6TRGJ4TRGJ6 (1)(1) TRGC no assigned al.,al., 20182018 TRGV7 (1) TRGV6TRGV7TRGV5TRGV7 (3)(1)(6)(1) TRGJ8TRGJ7 (1) Wang et REPTILES Alligator TRGV9TRGV8TRGJ7 (1)(2) (1) TRGJ6TRGJ7TRGJ5TRGJ7 (1)(1) TRGC nono assignedassigned Alligator sinensis al., 2018 TRGV8 (2) TRGV7TRGV8TRGV6TRGV8 (1)(2)(3)(2) TRGJ9TRGJ8 (1) Alligator sinensis TRGV10TRGV9TRGJ8 (1)(1) (1) TRGJ7TRGJ8TRGJ6TRGJ8 (1)(1) no assigned Alligator sinensis al., 2018 TRGV9 (1) TRGV8TRGV9TRGV7TRGV9 (2)(1)(1) TRGJ9 (1) TRGV10 (1) TRGJ8TRGJ9TRGJ7TRGJ9 (1)(1) no assigned AlligatorAlligator sinensissinensis TRGV10TRGV9TRGV8TRGJ9 (1)(2) (1) (1) TRGJ1 (1) TRGV10 (1) TRGV10 (1) TRGJ9TRGJ8 (1)(1) Alligator sinensis TRGV10TRGV9 (1)(1) TRGJ2TRGJ1 (1) TRGV1 (5) TRGJ9 (1) Alligator sinensis TRGV10TRGJ1 (1) (1) TRGJ3TRGJ2TRGJ1TRGJ1 (1)(1) one locus TRGV2TRGV1 (1)(5) TRGJ1 (1) Parra et TRGJ4TRGJ2TRGJ2 (1)(1) one locus MARSUPIALS Opossum TRGV1TRGV1TRGJ2 (5)(5) (1) TRGJ3 (1) TRGC one locus TRGV1 (5) TRGV3TRGV2 (2)(1) TRGJ2TRGJ3TRGJ1TRGJ3 (1)(1) one locus Parra et MARSUPIALS Opossum TRGV1TRGV2TRGV2 (5)(1)(1) TRGJ5TRGJ4 (1) TRGC al.,ParraParra 2008 etet TRGV4TRGV3TRGJ3 (1)(2) (1) TRGJ3TRGJ2TRGJ4 (1)(1) oneone locus locus MARSUPIALSTRGV2 Opossum (1) TRGV2TRGV1 (1)(5) TRGJ6TRGJ5TRGJ4 (1) TRGCTRGC Chrom. 6q al.,Parra 2008 et TRGV4TRGV3TRGV3 (1)(2)(2) one locus MARSUPIALS OpossumMARSUPIALS Opossum TRGJ4 (1) TRGJ4TRGJ5TRGJ3TRGJ5 (1)(1) TRGCTRGC Chrom. 6q Parraal.,al., 20082008 et al., [75]; TRGV3 (2) TRGV3TRGV4TRGV2TRGV4 (2)(1)(1) TRGJ7TRGJ6 (1) Parra et TRGJ5TRGJ4TRGJ6 (1)(1) Chrom.Chrom. 6q6q Monodelphis domestica al., 2008 MARSUPIALS Opossum TRGV4TRGV3TRGJ5 (1)(2) (1) TRGJ7TRGJ6 (1) TRGC Chrom. 6q TRGV4 (1) TRGJ6TRGJ5TRGJ7 (1)(1) Chrom. 6q al., 2008 TRGV4Legend:TRGJ6 (1) (1)The numbersTRGJ7 (1) in brackets indicate the number of the genes. Monodelphis domestica TRGJ7TRGJ6 (1)(1) Chrom. 6q MonodelphisMonodelphis domestica Legend:TRGJ7 (1)The numbers in brackets indicate the number of the genes. Legend:Legend: TheThe numbersnumbersTRGJ7 (1) inin bracketsbrackets inindicatedicate thethe numbernumber ofof thethe genes.genes. Monodelphis domestica In the Atlantic salmonLegend: (TheSalmo numbers salar), intwo brackets different indicate TRG the loci number were identifiedof the genes. [73]. Monodelphis Unlike domesticathe other Legend:outgroupIn the Thespecies, Atlantic numbers the salmonLegend: salmon (inTheSalmo TRG bracketsnumbers salar loci), inaretwo brackets indicate organi different inzeddicate TRG thein cassettes,the loci number number were each identifiedofof the containing thegenes. [73]. genes. Unlike the basic the V-J-Cother InIn thethe AtlanticAtlantic salmonsalmon ((SalmoSalmo salarsalar),), twotwo differentdifferent TRGTRG lociloci werewere identifiedidentified [73].[73]. UnlikeUnlike thethe otherother outgroup species, the salmon TRG loci are organized in cassettes, each containing the basic V-J-C outgroupoutgroupIn the species, species,Atlantic thethe salmon salmonsalmon (Salmo TRGTRG salar lociloci), aretwoare organiorganidifferentzedzed TRG inin cassettes,cassettes, loci were each eachidentified containingcontaining [73]. Unlike thethe basicbasic the V-J-CotherV-J-C In the Atlantic salmon outgroup (InSalmo the species, Atlantic salar the salmon salmon), two(Salmo TRG disalar lociff), erent aretwo organi different TRGzed TRG in cassettes, loci loci were were each identified containing identified [73]. Unlikethe basic [the73 V-J-Cother]. Unlike the other outgroup species, the salmon TRG loci are organized in cassettes, each containing the basic V-J-C outgroup species, the salmon TRG loci are organized in cassettes, each containing the basic V-J-C unit.

The first locus spans 260 kb and contains five tandem-repeated cassettes, each of which consists of one to four TRGV, one TRGJ, and one TRGC genes. The only exception is the TRGJ3-C3, which is missing the 50 end V genes. A total of 10 TRGV (7 functional genes belonging to two subgroups and 3 pseudogenes), 5 TRGJ, and 5 TRGC genes were found in Locus 1 (Table1, Supplementary Figure S1D). Within Locus 2, a single V-J-C cassette was found, consisting of one gene for each type. Genomic comparison between the two TRG loci indicated that the duplication of these loci was not derived from whole-genome duplication in salmonids, but rather that it might be the result of a separate partial duplication event [73]. The six TRGC genes are all potentially functional and present a different structure. The TRGC2, TRGC3, and TRGC5 genes are organized into three exons (EX1, EX2, and EX3), while the TRGC1 gene has four exons, including two EX2 (EX2A and EX2B). The TRGC4 and TRGC6 genes have only two exons (EX1 and EX3), and are missing Exon 2 (EX2), which codes for the connecting region. Expression Genes 2020, 11, 624 7 of 28 study within Locus 1 has revealed that the V genes located within each cassette preferentially recombine with the J genes in their own cassette. However, several cDNA clones show unique rearrangements of V genes that are rearranged by skipping over cassettes [73]. This form of rearrangement may be a mechanism for generating more potential diversity for antigen recognition. The genomic organization of the TRG locus in Alligator sinensis was recently defined based on the analysis of BAC clones [74]. The TRG locus of Alligator sinensis spans 115 kb and consists of a J-C cluster (nine TRGJ–one TRGC) (Table1, Supplementary Figure S1E) preceded by 18 TRGV genes belonging to 10 subgroups. Two of these 18 TRGV genes are pseudogenes, as they contain in-frame stop codons. The TRGC gene presents the three-exon basic structure. Amphiphysin (AMPH) is found at the 50 end of the locus, and Related to steroidogenic acute regulatory protein D3-N-terminal like (STARD3NL or MLN64 in Supplementary Figure S1E) with an inverted orientation is found at the 30 end of the locus. The organization of the conventional TRG locus in the opossum Monodelphis domestica is highly conserved, with a similar complexity to that of eutherians (placental mammals) (Table1, Supplementary Figure S1F). Only a single TRG locus has been identified, and this corresponds to the locus mapped previously to Chromosome 6q [76]. AMPH and the STARD3NL are found at the 50 and 30 end of the TRG locus, respectively. From the 50-most TRGV gene to the 30 untranslated region (UTR) of the single TRGC gene, the opossum TRG locus spans only approximately 90 kb. There are nine TRGV genes present in the opossum and these are divided into four subgroups [75]. All TRGV genes appear to be functional, and have been found to be expressed in the thymus. In the J-C-cluster (seven TRGJ–one TRGC), the opossum TRGC is encoded by three exons. Exon 1, which encodes the C-γ domain, contains a single N-glycosylation site at position IMGT N101 [60]. An unusual characteristic described in marsupials is the absence of the second cysteine (2nd-CYS), C104, required for the formation of the intrachain disulfide bond in Exon 2 [60]. All non-eutherian mammals (monotremes and marsupials) lack 2nd-CYS C104, a loss apparently due to independent mutations in marsupials and monotremes [77].

5. The TRG Locus in Cetartiodactyla and Carnivora: The “Gene Cassette” Model

5.1. Genomic Organization of Ovine, Bovine, Camel, and Dolphin TRG Loci In this review of the evolution of the TRG locus in mammals, the unexpected finding described by Massari et al. [78] is certainly relevant. In the paper, the authors reported the cytological mapping of two sheep phage λ genomic clones containing two distinct TRGC sequences. FISH experiments on sheep metaphases highlighted the presence of two TRG paralogous loci separated by at least five chromosomal bands on Chromosome 4 (Figure2). One locus, named TRG1, is located on 4q3.1 within a region of homology with the human Chromosome 7p14, where the TRG locus maps [79]. The other locus, TRG2, mapping on 4q15–22, is not included in the region of synteny with respect to humans, thus appearing to be peculiar to sheep. This finding represented the first case in mammals in which a TR locus was not found in a single chromosomal region. In humans, only one case of extensive interchromosomal duplication has been reported. It regards the TRB locus, where several TRBV genes moved from the main TRB locus on Chromosome 7q34 to Chromosome 9p21, causing the emergence of an orphan locus [80,81]. Conversely, expression assays [82–84] have shown that both sheep TRG loci are functional. The subsequent availability of BAC clones containing TRG1 and TRG2 locus sequences [85] made it possible to extend FISH experiments to goat, cattle, and river buffalo. These studies located TRG2 loci on the homologous chromosome band of cattle and goat 4q22 and river buffalo 8q17 [86], showing that the presence of the paralogous TRG2 locus is typical of Bovidae. Genes 2020, 11, 624 8 of 28 Genes 2020, 11, x FOR PEER REVIEW 8 of 28

Figure 2. Sheep (Ovis aries) TRG locus representation, reproduced with permission from IMGT® Figure 2. Sheep (Ovis aries) TRG locus representation, reproduced with permission from IMGT® (http://www.imgt.org). The TRG genes are organized in two loci on Chromosome 4. Colors are (http://www.imgt.org). The TRG genes are organized in two loci on Chromosome 4. Colors are according according to IMGT color menu for genes to IMGT color menu for genes (https://www.imgt.org/IMGTScientificChart/RepresentationRules/ (https://www.imgt.org/IMGTScientificChart/RepresentationRules/colormenu.php#LOCUS). The colormenu.php#LOCUS). The boxes representing the genes are not to scale. Exons are not shown. boxes representing the genes are not to scale. Exons are not shown. Arrows indicate the TRGC Arrows indicate the TRGC cassettes and the transcriptional orientation of their genes. cassettes and the transcriptional orientation of their genes. The genomic structure of both TRG loci in sheep (Figure2) highlights the peculiarity of the organizationOne locus, ofnamed these TRG1, loci, consisting is located in aon set 4q3.1 of six within (three fora region each locus) of homology closely related with the “cassettes”, human eachChromosome containing 7p14, the basicwhere structure the TRG V–J–J–C locus maps unit [79]. arranged The other in the locus, same TRG2, transcriptional mapping orientation on 4q15–22, [84 is]. Allnot theincluded J-J-C regions in the areregion delimited of synteny at their with 5’ end respect by promoters to humans, for thus germline appearing transcription to be peculiar containing to sheep. STAT This finding represented the first case in mammals in which a TR locus was not found in a single motifs, which control the local recombinational accessibility [87], and at their 30 end by enhancer-like elements,chromosomal which region. govern In the humans, general only recombinational one case of extensive accessibility interchromosomal [88,89]. Preliminary duplication expression has studies been havereported. shown It regards that each theTRGV TRB locus,gene preferentially where several rearranges TRBV genes with moved the TRGJ fromgenes the main of its TRB cassette locus and, on afterChromosome transcription, 7q34 the to V-J Chromosome region is spliced 9p21, to thecausing relevant theC inemergence mature transcripts of an orphan [82,83 ].locus The isolation[80,81]. ofConversely, five TRG1 expression and two TRG2 assays BAC [82–84] clones, have their shown subcloning that both in plasmidsheep TRG vectors, loci are and functional. the sequencing of theirThe inserts subsequent allowed theavailability authors to of obtain BAC contiguousclones cont genomicaining TRG1 sequences and TRG2 spanning locus 158.8 sequences kb for TRG1 [85] andmade 95.0 it possible kb for TRG2 to extend [89]. In FISH Table experiments2 and Figure 2to, thegoat overall, cattle, organization and river buffal of theo. ovine These TRG1 studies and located TRG2 lociTRG2 is shown.loci on the The homologous entire TRG1 locuschromosome encompasses band threeof cattle cassettes, and goat TRGC5, 4q22 TRGC3,and river and buffalo TRGC4, 8q17 named [86], accordingshowing that to the the constant presence genes of the [82 paralo,83] andgous following TRG2 locus the recommendations is typical of Bovidae. of the IMGT Nomenclature Committee.The genomic Similarly, structure the entire of both TRG2 TRG locus, loci consistingin sheep (Figure of TRGC1, 2) highlights TRGC2, andthe peculiarity TRGC6 cassettes, of the isorganization schematically of these represented. loci, consisting Comparative in a set genomics of six (three and for evolutionary each locus) analysesclosely related of the “cassettes”, sheep TRG sequenceseach containing support the thebasic idea structure that the V–J–J–C TRGC5 unit cassette arranged resembles in the same the ancestral transcriptional one, which orientation underwent [84]. theAll the first J-J-C duplicative regions eventare delimited that led at to their the birth 5' end of by the promoters two TRG locifor germline [89]. Overall, transcription six TRGV containinggenes are locatedSTAT motifs, in the TRGC5which cassettecontrol andthe threelocal inrecombin the TRGC3,ational while accessibility all the other [87], cassettes and at consists their 3 of′ aend single by gene.enhancer-like The sheep elements,TRGV genes which belong govern to eleventhe general subgroups, recombinational defined by accessibility a percentage [88,89]. of identity Preliminary less than 75%expression for the studies V-region have at the shown nucleotide that each level TRGV [44]. gene All sheep preferentially V subgroups rearranges consist with of only the one TRGJ member genes gene,of its cassette except the and, TRGV3 after andtranscription, the TRGV5 the subgroups V-J region which is spliced have twoto the genes relevant each. C Only in mature two TRGV transcriptsgenes and[82,83]. three TheTRGJ isolationgenes of are five pseudogenes. TRG1 and two TRG2 BAC clones, their subcloning in plasmid vectors, and theWith sequencing regard to the of TRGCtheir insertsgenes, allallowed sheep genesthe authors are functional to obtain and, contiguous studied in genomic detail, show sequences a great structuralspanning diversity158.8 kb [for90]. TRG1 Exon 1and (EX1) 95.0 is similarkb for inTR allG2 the [89].TRGC In genesTable and2 and encodes Figure the 2, C-theγ domain, overall whichorganization contains of thethe twoovine conserved TRG1 an 1st-CYSd TRG2 23loci and is shown. 2nd-CYS The 104 entire required TRG1 for locus the intra-chain encompasses disulfide three andcassettes, N-glycosylation TRGC5, TRGC3, sites N-X-S and /TRT atGC4, positions named IMGT according N13, N83 to the (TRGC4 cons),tant and genes N84.1 [82,83] (TRGC5 andand followingTRGC6), the recommendations of the IMGT Nomenclature Committee. Similarly, the entire TRG2 locus, consisting of TRGC1, TRGC2, and TRGC6 cassettes, is schematically represented. Comparative

Genes 2020, 11, 624 9 of 28 according to the IMGT unique numbering for C-DOMAIN [60]. The connecting region (CO), which contains a single conserved cysteine that forms the interchain disulfide bond with the TRDC chain, differs in size between the sheep TRGC genes. The size differences result from a different number of exons encoding the CO: one exon (EX2A) for the TRGC1 and TRGC5 genes, two exons (EX2A and EX2C) for the TRGC3 gene, and three exons (EX2A, EX2B, and EX2C) for the TRGC2, TRGC4, and TRGC6 genes. Furthermore, the EX2A exon of all TRGC genes (excluding the TRGC5 gene) contains a TTE(K)P(S)P motif in single copy or, for TRGC2, in triplicate. Finally, all TRGC genes have an almost identical Exon 3 (EX3) encoding the last part of the connecting region, the transmembrane region, and the cytoplasmic region (https://www.imgt.org/IMGTrepertoire/Proteins/protein/sheep/TRG/TRGC/Sh_TRGCallgenes.html). The gene organization of the bovine TRG loci has been also determined [91,92]. Similarly to the ovine organization, the structure of the two bovine TRG loci consists of corresponding tandem-repeated V-J-J-C cassettes at each locus (Tables2 and3, Supplementary Figure S2A). To facilitate comparative studies between ruminants, and on the basis of their high sequence identity [89], gene names identical to those of sheep were assigned to the bovine TRGC cassettes by the IMGT Nomenclature Committee [93]. However, differences in the genomic organization with respect to the sheep loci can be observed, as summarized in Table3. The bovine TRG1 locus is 178 kb long and includes an extra cassette (TRGC7) which, however, appears not to be functional because the TRGC7 gene is a pseudogene and the TRGJ genes are absent [92]. Moreover, the incomplete nature of the bovine TRGC5 cassette (Supplementary Figure S2A) may justify the absence of the TRGV11 gene and the presence of two more TRGJ5 genes with respect to sheep (Table3), while gene duplications involving the bovine TRGV8 and TRGV9 gene subgroups have occurred (Table2, Supplementary Figure S2A and Table S2B). Conversely, the bovine TRG2 locus spans 103 kb and it appears to be more similar to the corresponding ovine locus except for the duplication of the TRGV6 gene. The bovine TRGC genes present a structural diversity similar to that of the sheep genes (Table3). In particular, the TRGC5 has the classic three exons (EX1, EX2A, and EX3); TRGC3 and the TRGC7 pseudogene have four exons (EX1, EX2A, EX2C, and EX3); all the others have five exons (EX1, EX2A, EX2B, EX2C, and EX3). A TT(A)EPP motif has been found in TRGC4, TRGC2, and TRGC1 in single, duplicate, or quadruplicate form, respectively (https://www.imgt.org/IMGTrepertoire/ Proteins/protein/Btaurus/Bt_TRCallgenes.html). In this species, expression analyses have also shown that V-J rearrangements among genes are favored within the same TRG cassette [91,92,94,95]. Comparative genomics in ruminants (Ovis aries and Bos taurus) highlights the fact that requirements related to immunoprotective functions, including the first defensive barrier in the epithelia of the digestive tract, are likely to have induced a sort of functional genome variability within TRG loci. As a consequence, the large number of TRGV genes, due to the reiterated duplications of TRG gene cassettes within the locus, increase the number of rearrangement events, which in turn produced transcripts with highly diversified variable domains [89]. Only recently, the identification of the 50 and 30 boundary genes (defined as IMGT 50 and 30 bornes), AMPH and STARD3NL, allowed the complete TRG locus in all its parts to be established in dromedary species [96]. The dromedary TRG locus is single. Starting from the first TRGV11 gene at its 50 end, and ending with TRGC2 gene at its 30 end, it spans about 105 kb and, similarly to each of the two TRG loci in sheep, it is organized into three V-J-J-C cassettes [97]. The distance between the STARD3NL gene and the last exon of the TRGC2 gene is 8725 bp and in this portion no genes are present. The cassettes are classified as TRGC1, TRGC2, and TRGC5 [96] (Table2, Supplementary Figure S2B). The ancestral nature of the TRGC5 cassette is highlighted by the high structural correspondence and the close phylogenetic relationship with the sheep cassette genes, with the exception of the presence of only one TRGV3 gene and the functionality of the TRGV11 and TRGV10 genes in the dromedary cassette. As in sheep and bovine genomes, the TRGC5 gene consists of three exons (EX1, EX2, and EX3). There are three TRGJ genes in the dromedary, as in Bovidae. Finally, the extensive collinearity shared between the entire dromedary TRGC5 cassette sequence and the corresponding cassette of the ovine TRG1 locus further confirmed the “ancient cassette” characteristic of the latter [96]. Genes 2020, 11, 624 10 of 28

Only one TRGV gene is present in the other dromedary TRGC cassettes, whereas, the TRGC1 and TRGC2 genes, encoded by five exons, consist of three EX2, as observed in the sheep TRGC2, TRGC4, and TRGC6 genes [97]. The total number of dromedary TRGV germline genes is certainly lower compared to that of sheep and cattle, and the TRG chain diversity due to the potential gene rearrangements is therefore more limited. However, cDNA sequencing clearly revealed that besides the combinatorial diversity and the introduction of N region diversity typical of all known IG and TR genes, a further mechanism enhances the TRG diversity in Camelus dromedarius. In line with previous reports [97], more recent studies [35,98] have provided direct evidence that somatic hypermutation (SHM) heavily contributes to the expansion of the γδ TR repertoire even in the absence of functional reiterated genome duplications. The frequency of mutations observed in the V-γ domain was comparable with that found in targeted genes in AID-induced T lymphomas [99], rearranged shark TRGV [71], and dromedary TRDV regions [100]. Previously, somatic hypermutation had been completely demonstrated only for IG genes in the B cells of higher vertebrates in order to produce antibodies with higher affinity. In contrast, in the dromedary as well as in sharks, the purpose of the somatic mutations in V-γ domains is to generate a more diverse repertoire of receptors. Finally, the dolphin TRG locus is the smallest and simplest of all mammalian loci studied to date [38]. It spans only 48 kb and its genes are arranged in a pattern comprising two TRGV belonging to two distinct subgroups, three TRGJ genes and a single TRGC gene (Table2, Supplementary Figure S2C). A closer inspection of the shared dolphin and sheep genes revealed that the dolphin TRGC gene possesses a single small Exon 2 (EX2) similar to the sheep TRGC5 EX2. The dot-plot matrix of dolphin TRG and sheep TRG1 loci genomic comparison displays a remarkable consistency of the identity diagonals from the sheep TRGV11-1 gene to the TRGC5 gene, with a remarkable compactness of the three J genes [38]. The overall organization of the dolphin TRG locus is reminiscent of the typical single-cassette structure of artiodactyls [89], with a small number of genes. However, an evolutionary correlation can be also found between the two dolphin TRGV genes and the human TRGV9 and TRGV11 genes, as well as between the dolphin J-J-J-C region and the human J-J-J-C1 region [38]. Dolphin TRG-chain expression analysis from the blood and skin of unrelated subjects demonstrated that the two TRGV and three TRGJ genes were used in every possible combination, although a bias towards some transcripts (TRGV1-TRGJ2 and TRGV2-TRGJ3) was noted. Furthermore, about half of the transcripts using TRGV2 were unproductive due to the presence of stop codons in CDR3. The percentage values of the productive/unproductive rearrangements were similar for both cDNA and genomic clones, and in the same and different individuals, in contrast to what is usually observed (the percentage of unproductive rearrangements being lower in cDNA, due to nonsense-mediated decay of RNA). The authors argued that the occurrence of clonotypes shared by different individuals living both in marine and in artificial marine “habitats”, and previously described as “convergent recombination” [101], could be in fact strictly related to the biased V-J rearrangement events. A comparable preferential usage of the TRGJP (localized between TRGJP1 and TRGJ1) and the predominant expression of TRGV9-JP-C1 chain paired with a TRDV2-D-J-C in humans may be related to promoter characteristics [102]; thus, the same observation in both dolphins and humans supports an accurate determination of the TRGJ gene usage. As a consequence, the high frequency of TRGV1-J2/TRDV1-D1-J4 productive rearrangements in dolphins may represent a situation of oligoclonality comparable to that found in human TRGV9-JP/TRDV2-D-J T cells [103].

5.2. Genomic Organization of Canine and Feline TRG Loci Table2 and Supplementary Figure S2D show the overall organization of the dog TRG locus, which encompasses eight cassettes, named according to the constant genes. All TRG cassettes lie in the same transcriptional orientation, are closely spaced, and contain the basic recombinational unit V-J-J-C, except for the last cassette, J-J-C, which lacks the V gene and occupies the 30 end of the locus. Genes 2020, 11, 624 11 of 28

The limit dividing the eight cassettes from each other is approximately given by a space ranging from 10 to 18 kb (from the last exon of each C gene to the L-PART1 of the V gene of the downstream cassette), except for one of about 35 kb between Cassettes 6 and 7. The AMPH and STARD3NL genes flank, respectively, the 50 and 30 ends of the TRG locus which spans 460 kb. There are 16 TRGV genes assigned to seven subgroups. Four subgroups (TRGV2, TRGV3, TRGV5, and TRGV7) are multimembers with three or four genes; the other subgroups have only one gene member. The germline configuration and the exon–intron organization of the eight TRGC genes (TRGC1–TRGC8) has been well analyzed [104]. Six of them (TRGC2 to TRGC5,TRGC7, and TRGC8) are functional, whereas TRGC1 is an open reading frame (ORF) and TRGC6 is a pseudogene. The first exon (EX1) encodes the C-γ domain, which comprises 110 amino acids or is slightly shorter (105 aa for TRGC1, 109 aa for TRGC2 and TRGC4). The first part of the connecting region is encoded by one or two exons (EX2A and/or EX2B), a situation reminiscent of the artiodactyl TRGC genes. Thus, the canine TRGC2, TRGC3, and TRGC4 genes have both EX2A and EX2B exons, whereas the TRGC1, TRGC6, TRGC7, and TRGC8 genes have only a single EX2B exon, and TRGC5 has a single EX2A exon. The remaining part of the connecting region (CO), the transmembrane region, and the cytoplasmic region are encoded by EX3 (https://www.imgt.org/IMGTrepertoire/Proteins/protein/dog/TRG/TRGC/Cf_TRGCallgenes.html). The reiterated cassette duplication in the canine TRG locus resulted in a total of 40 genes, with 21 of them functional and 19 pseudogenes or ORFs. On the other hand, the low ratio of functional genes to the total number of canine TRG genes (23/40), suggests that there is no correlation between the extensive duplications of the cassettes and a need for new functional genes. In contrast to the bovine and ovine TRG loci, the extensive duplication of the TRG cassettes does not seem to match a real need of the adaptive immune response. The Felis catus TRG locus spans approximately 260 kb in the pericentromeric region of Chromosome A2. The 50 IMGT borne is AMPH and the 30 IMGT borne is STARD3NL (Table2, Supplementary Figure S2E). The TRG locus contains 30 genes. There are 12 TRGV genes (six functional and six pseudogenes) assigned to six subgroups, 12 TRGJ genes (four functional, two ORFs, and six pseudogenes), and 6 TRGC genes (four functional and two pseudogenes) arranged into five complete and one incomplete V-J-(J)-C cassettes [27]. The feline TRGV genes belong to six subgroups, two of which have four members (TRGV2 with four functional genes, TRGV5 with one functional and three pseudogenes) and the four others with a single member each; TRGV7 is the only functional one, TRGV6 is a pseudogene owing to two stop codons in the V region, and TRGVA and TRGVB are degenerate pseudogenes. The canine TRGV1 and TRGV3 subgroup orthologs are absent in the cat genome [104]. The 12 feline TRGJ genes were designated based on the cassette they belong to. There are four functional TRGJ genes, two ORFs, and six pseudogenes. Each TRGC region is encoded by three exons (EX1, EX2A or EX2B, and EX3) and all are functional except for TRGC5 and TRGC6 due to frameshifts in EX1 and EX3, respectively [27]. The feline TRG locus most closely resembles that of the dog, which has 8 V-J-(J)-C cassettes [http://www.imgt.org/ IMGTrepertoire/LocusGenes]. The fact that Cassettes 4 and 5 are in an inverted orientation in the cat, despite a high homology to dog V genes, suggests that the inversion likely occurred after speciation. Expression data have shown that the genomic cassette organization of the cat TRG genes may favor physical V and J proximity in the rearrangement, and that the greater effectiveness of this physical proximity in pursuing a strong gene expression depends on the functionality of the constant gene with which it is associated [27]. Genes 2020, 11, x FOR PEER REVIEW 10 of 28 Genes 2020, 11, x FOR PEER REVIEW 10 of 28 Table 2. Genomic organization and gene content of the TRG locus in Cetartiodactyla and Carnivora. Genes 2020, 11, 624 Genes 2020, 11, x FOR PEER REVIEW 10 of 28 12 of 28 Table 2. Genomic organization and gene content of the TRG locus in Cetartiodactyla and Carnivora.

TRGV TRGJ TRGC Chromosomal Miniature Table 2. Genomic organization and gene content of the TRG locus in Cetartiodactyla and Carnivora. References

TRGVgenes genesTRGJ TRGCgenes Chromosomallocalization Miniaturelocus References Table 2. Genomic organization and gene contentTRGVgenes of theTRGJgenes TRG locusTRGCgenes in CetartiodactylaChromosomallocalization and Carnivora.Miniaturelocus TRGV1 (1) References TRGV2genes (1) genes genes localization locus Massari et TRGV Genes TRGJ GenesTRGV1TRGV2 (1) TRGC Genes Chromosomal Localization Miniature LocusMassari References et TRGV3 (2) al., 1998; TRGV1 (1) TRGV2 (1) TRGJ1 (2) TRGC1 (1) Massari et TRGV1TRGV4 (1) Miccoli et TRGV2 (1) TRGV3 (2) TRGJ2 (2) TRGC2 (1) Two loci al., 1998; TRGV2TRGV5 (1)(2) TRGJ1 (2) TRGC1 (1) Massarial., 2003; et TRGV4 (1) TRGJ3 (2)* TRGC3 (1) Miccoli et TRGV3 (2) Ovine TRGV3TRGV6 (2)(1) TRGJ2 (2) TRGC2 (1) Two loci al.,Vaccarelli 1998; TRGJ1 (2) Ovine TRGV5TRGV6TRGC1 (2)(1) (1) TRGJ1TRGJ4 (2)(2)* TRGC1TRGC4 (1) Chrom. 4q31 al.,Vaccarelli 2003; TRGV4 (1) TRGV4TRGV7 (1) TRGJ3TRGJ4 (2)* TRGC3TRGC4 (1) Chrom. 4q31 Miccoliet et TRGJ2 (2) TRGV6TRGV7TRGC2 (1) (1) TRGJ2TRGJ5 (2)(3)° TRGC2TRGC5 (1)Two lociChrom.Two loci 4q22 Vaccarelliet TRGV5 (2) Ovine TRGV5TRGV8 (2)(1) TRGJ4TRGJ5 (2)*(3)° TRGC4TRGC5 (1) Chrom. 4q314q22 al.,Massari 2003;2005; et al., [78]; TRGJ3 (2) * TRGV7TRGV8TRGC3 (1) (1) TRGJ3TRGJ6 (2)*(2) TRGC3TRGC6 (1) etal., 2005; Ovine TRGV6 (1) TRGV6TRGV9 (1) TRGJ5TRGJ6 (3)°(2) TRGC5TRGC6 (1) Chrom. 4q22 VaccarelliMiccoli et al., [85]; TRGJ4 (2) * Ovine TRGV8TRGV9TRGC4 (1) (1) TRGJ4 (2)* TRGC4 Chrom.(1) 4q31Chrom. 4q31 Ovis aries al.,Vaccarelli 2005; TRGV7 (1) TRGV7TRGV10 (1) (1P) TRGJ6 (2) TRGC6 (1) Ovis aries etVaccarelli al., 2008 et al., [84,89]; TRGJ5 (3) ◦ TRGV9TRGV10TRGC5 (1) (1P) (1) TRGJ5 (3)° TRGC5 Chrom.(1) 4q22Chrom. 4q22 Vaccarelliet al., 2008 TRGV8 (1) TRGV8TRGV11 (1) (1P) Ovis aries al., 2005; TRGJ6 (2) TRGV10TRGV11TRGC6 (1P) (1) TRGJ6 (2) TRGC6 (1) et al., 2008 TRGV9 (1) TRGV9 (1) Vaccarelli TRGV11 (1P) OvisOvis aries TRGV10 (1P) TRGV10 (1P) et al., 2008 TRGV11 (1P) TRGV11TRGV1 (1) (1P) TRGV2 (1) TRGV1 (1) TRGV1 (1) TRGC1 (1) TRGV3 (2) TRGJ1 (2) TRGV2 (1) TRGV2 (1) TRGC2 (1) Antonacci TRGV1TRGV4TRGC1 (1) (1) TRGJ2 (2) TRGC1 (1) Two loci TRGV3 (2) TRGJ1 (2) TRGV3 (2) TRGJ1 (2) TRGC3 (1) et TRGV2TRGV5(1+1O)TRGC2 (1) (1) TRGJ3 (1) TRGC2 (1) Antonacci TRGV4 (1) TRGJ2 (2) Bovine TRGV4 (1) TRGJ2 (2) TRGC1TRGC4 (1)Two loci Two loci al., 2007; Bovine TRGV3TRGV6TRGC3 (2) (1) TRGJ1TRGJ4 (2) TRGC3TRGC4 (1) Chrom. 4q31 etal., 2007; TRGV5(1+1O) TRGJ3 (1) TRGV5(1+1O)TRGV6 (2) TRGJ3TRGJ4 (1)(2) TRGC2TRGC5 (1) Chrom. 4q31 AntonacciConradAntonacci et et al., [86]; CETARTIODACTYLA TRGV7 (1) TRGJ5 (1) TRGC5 (1) Chrom. 4q1.5-2.2 Conrad et Bovine CETARTIODACTYLA Bovine TRGV4TRGV7TRGC4 (1) (1) TRGJ2TRGJ5 (2)(1) TRGC6TRGC4 (1) Chrom.Two 4q1.5-2.2loci al., 20072007; TRGV6 (2) TRGJ4 (2) TRGV8TRGV6 (4)(2) TRGJ6TRGJ4 (1)(2) TRGC3TRGC6 Chrom.(1) 4q31Chrom. 4q31 etal.,Conrad 2007 et al., [92]; CETARTIODACTYLA TRGV5(1+1O)TRGV8TRGC5 (4) (1) TRGJ3TRGJ6 (1) TRGC7TRGC5 (1P)(1) Conrad et TRGV7 (1) CETARTIODACTYLATRGJ5 (1) Bovine TRGV7 (1) TRGJ5 (1) TRGC4TRGC7Chrom. (1)(1P) 4q1.5-2.2Chrom. 4q1.5-2.2 al., 2007; TRGV6TRGV9TRGC6 (2) (1) TRGJ4 (2) TRGC6 (1) Chrom. 4q31 al., 2007 TRGV8 (4) TRGJ6 (1) TRGV8 (4) TRGJ6 (1) TRGC5 (1) Bos taurus Conrad et CETARTIODACTYLA TRGV7TRGV10TRGC7 (1) (1) (1P) TRGJ5 (1) TRGC7 (1P) Chrom. 4q1.5-2.2 TRGV9 (2) TRGV9TRGV10 (2) (1) TRGC6 (1) al., 2007 TRGV8 (4) TRGJ6 (1) BosBos taurustaurus TRGV10 (1) TRGC7 (1P) TRGV10 (1) TRGV9 (2) TRGV1 (1) Bos taurus TRGV1 (1) TRGV10TRGV1 (1) (1) TRGV2 (1) TRGV2 (1) TRGV2 (1) One locus Vaccarelli TRGV3TRGV1 (1) TRGJ1 (2)* TRGC1 (1) One locus Vaccarelli TRGV3 (1) TRGJ1 (2) * TRGV2TRGV3TRGC1 (1) (1) TRGJ1 (2)* TRGC1 (1)One locus et al., 2012; TRGV4 (1O) TRGJ2 (2)* TRGC2 (1) etVaccarelli al., 2012; et al., [97]; Camel TRGV1TRGV4 (1)(1O) TRGJ2 (2)* TRGC2 (1) Chrom.One locus7 q11.12 AntonacciVaccarelli Camel TRGV4 (1O) TRGJ2 (2) * Camel TRGV3TRGC2 (1) (1) TRGJ1 (2)* TRGC1 (1) Chrom. 7 q11.12 Antonacci TRGV2TRGV7 (1) TRGJ5 (3)° TRGC5 (1) etAntonacci al., 2012; et al., [96]; TRGV7 (1) TRGJ5 (3) ◦ TRGV4TRGC5 (1O) (1) TRGJ2 (2)* TRGC2Chrom. (1) 7 q11.12One locus Vaccarelliet al., 2020 Camel TRGV3TRGV10 (1) (1) TRGJ1 (2)* TRGC1 (1) Chrom. 7 q11.12 Antonacciet al., 2020 TRGV10 (1) TRGV7TRGV10 (1) (1) TRGJ5 (3)° TRGC5 (1) CamelusCamelus dromedariusdromedarius et al., 2012; TRGV4TRGV11 (1O) (1) TRGJ2 (2)* TRGC2 (1) et al., 2020 TRGV11 (1) Camel TRGV10TRGV11 (1) Chrom. 7 q11.12 Antonacci TRGV7 (1) TRGJ5 (3)° TRGC5 (1) Camelus dromedarius TRGV11 (1) et al., 2020 TRGV10 (1) Camelus dromedarius TRGV11 (1) TRGJ1 (1) TRGJ1 (1) One locusOne locus TRGV1 (1) Dolphi TRGV1 (1) TRGJ1 (1) One locus Linguiti et Dolphin TRGJ2 (1) Dolphi TRGV1TRGC (1) TRGJ2 (1) TRGC LinguitiLinguiti et et al., [38]; TRGV2 (1) TRGV2 (1) TRGJ1TRGJ2 (1) TRGC One locus al., 2016 TRGJ3 (1) nDolphi TRGV1TRGV2 (1) TRGJ3 (1) no assignedno assigned Linguitial., 2016 et n TRGJ2TRGJ3 (1) TRGC no assigned TRGV2 (1) TRGJ1 (1) One locus al., 2016 Dolphin TRGV1 (1) TRGJ3 (1) no assigned Linguiti et TRGJ2 (1) TRGC TursiopsTursiops truncatus TRGV2 (1) Tursiops truncatus al., 2016 n TRGJ3 (1) no assigned Tursiops truncatus Tursiops truncatus TRGJ1 (2)* TRGC1 (1O) TRGV1(1P) TRGJ2 (2)* TRGC2 (1) TRGV2(4) TRGJ1 (2)* TRGC1 (1O) TRGV1(1P) TRGJ3 (2)* TRGC3 (1) Massari et TRGV3 (3P) TRGJ2 (2)* TRGC2 (1) One locus TRGV2(4) TRGJ1TRGJ4 (2)* TRGC1TRGC4 (1O)(1) al., 2009; CARNIVORA Canine TRGV1(1P)TRGV4 (1O) TRGJ3 (2)* TRGC3 (1) Massari et CARNIVORA Canine TRGV3TRGV4 (3P)(1O) TRGJ2TRGJ5 (2)*(2) TRGC2TRGC5 (1) One locus Martin et TRGV2(4)TRGV5(3)§ TRGJ4TRGJ5 (2)*(2) TRGC4TRGC5 (1) Chrom. 18 al.,Martin 2009; et TRGV4TRGV5(3)§ (1O) TRGJ3TRGJ6 (2)*(2) TRGC3TRGC6 (1)(1P) Chrom. 18 Massarial., 2003 et CARNIVORA Canine TRGV3TRGV6(1P) (3P) TRGJ5TRGJ6 (2) TRGC5TRGC6 (1)(1P) One locus Martinal., 2003 et TRGV5(3)§TRGV6(1P) TRGJ4TRGJ7 (2)* TRGC4TRGC7 (1) Chrom. 18 al., 2009; TRGV4TRGV7(3)# (1O) TRGJ6TRGJ7 (2)(2)* TRGC6TRGC7 (1P)(1) al., 2003 CARNIVORA Canine TRGV6(1P)TRGV7(3)# TRGJ5TRGJ8 (2)(2)* TRGC5TRGC8 (1) Martin et TRGV5(3)§ TRGJ7TRGJ8 (2)* TRGC7TRGC8 (1) Chrom. 18 Canis lupus familiaris TRGV7(3)# TRGJ6 (2) TRGC6 (1P) al., 2003 TRGV6(1P) TRGJ8 (2)* TRGC8 (1) TRGJ7 (2)* TRGC7 (1) Canis lupus familiaris TRGV7(3)# TRGJ8 (2)* TRGC8 (1) Canis lupus familiaris

Genes 2020, 11, x FOR PEER REVIEW 10 of 28

Table 2. Genomic organization and gene content of the TRG locus in Cetartiodactyla and Carnivora.

TRGV TRGJ TRGC Chromosomal Miniature References genes genes genes localization locus

TRGV1 (1) TRGV2 (1) Massari et TRGV3 (2) al., 1998; TRGJ1 (2) TRGC1 (1) TRGV4 (1) Miccoli et TRGJ2 (2) TRGC2 (1) Two loci TRGV5 (2) al., 2003; TRGJ3 (2)* TRGC3 (1) TRGV6 (1) Vaccarelli Ovine TRGJ4 (2)* TRGC4 (1) Chrom. 4q31 TRGV7 (1) et TRGJ5 (3)° TRGC5 (1) Chrom. 4q22 TRGV8 (1) al., 2005; TRGJ6 (2) TRGC6 (1) TRGV9 (1) Vaccarelli Ovis aries TRGV10 (1P) et al., 2008 TRGV11 (1P)

TRGV1 (1) TRGV2 (1) TRGC1 (1) TRGV3 (2) TRGJ1 (2) TRGC2 (1) Antonacci TRGV4 (1) TRGJ2 (2) Two loci TRGC3 (1) et TRGV5(1+1O) TRGJ3 (1) TRGC4 (1) al., 2007; Bovine TRGV6 (2) TRGJ4 (2) Chrom. 4q31 TRGC5 (1) Conrad et CETARTIODACTYLA TRGV7 (1) TRGJ5 (1) Chrom. 4q1.5-2.2 TRGC6 (1) al., 2007 TRGV8 (4) TRGJ6 (1) TRGC7 (1P) TRGV9 (2) Bos taurus TRGV10 (1)

TRGV1 (1) TRGV2 (1) One locus Vaccarelli TRGV3 (1) TRGJ1 (2)* TRGC1 (1) et al., 2012; TRGV4 (1O) TRGJ2 (2)* TRGC2 (1) Camel Chrom. 7 q11.12 Antonacci TRGV7 (1) TRGJ5 (3)° TRGC5 (1) et al., 2020 TRGV10 (1) Camelus dromedarius TRGV11 (1)

Genes 2020, 11, 624 13 of 28 TRGJ1 (1) One locus Dolphi TRGV1 (1) Linguiti et TRGJ2 (1) TRGC TRGV2 (1) al., 2016 n TRGJ3 (1) no assigned

Table 2. Cont. Tursiops truncatus

TRGV Genes TRGJ Genes TRGC Genes Chromosomal Localization Miniature Locus References TRGJ1 (2) * TRGC1 (1O) TRGJ1 (2)* TRGC1 (1O) TRGV1(1P) TRGV1(1P) TRGJ2 (2) * TRGC2 (1) TRGJ2 (2)* TRGC2 (1) TRGV2(4) TRGV2(4) TRGJ3 (2) * TRGC3 (1) TRGJ3 (2)* TRGC3 (1) Massari et TRGV3 (3P) TRGV3 (3P) One locusOne locus TRGJ4 (2) * TRGC4 (1) TRGJ4 (2)* TRGC4 (1) al.,Massari 2009; et al., [104]; Canine TRGV4 (1O) TRGV4 (1O) CARNIVORATRGJ5 (2) Canine TRGC5 (1) TRGJ5 (2) TRGC5 (1) MartinMartin et et al., [39]; TRGV5(3) § TRGV5(3)§ Chrom. 18Chrom. 18 TRGJ6 (2) TRGC6 (1P) TRGJ6 (2) TRGC6 (1P) al., 2003 TRGV6(1P) TRGV6(1P) TRGJ7 (2) * TRGC7 (1) TRGJ7 (2)* TRGC7 (1) TRGV7(3) # Genes 2020, 11, x FOR PEERTRGV7(3)# REVIEW 11 of 28 TRGJ8 (2) * TRGC8 (1) TRGJ8 (2)* TRGC8 (1) CanisCanis lupuslupus familiaris CARNIVORA

TRGV2(4) TRGJ1 (2) * TRGV2(4)TRGC1 (1) TRGJ1 (2)* TRGC1 (1) TRGV5(4) $ TRGJ2 (3) ˆ TRGV5(4)$TRGC2 (1) TRGJ2 (3)^ TRGC2 (1) Radtanakat One locusOne locus TRGVA(1P) TRGJ3 (2) * TRGVA(1P)TRGC3 (1) TRGJ3 (2)* TRGC3 (1) i= Feline Radtanakatikanon et al., [27]; TRGVB(1P) TRGJ4 (1P) Feline TRGVB(1P)TRGC4 (1) TRGJ4 (1P) TRGC4 (1) kanon et Chrom. A2Chrom. A2 TRGV6(1P) TRGJ5 (2) * TRGV6(1P)TRGC5 (1P) TRGJ5 (2)* TRGC5 (1P) al.,2020 TRGV7(1) TRGJ6 (2) TRGV7(1)TRGC6 (1P) TRGJ6 (2) TRGC6 (1P)

FelisFelis catus Legend: TRGV: §(1F+2P); #(2F+1P); $(1F+3P); TRGJ: *(1F+1P); °(2F+1P); ^(1F+2P); P = pseudogene, O Legend: TRGV: § (1F+2P); # (2F+1P); $ (1F+3P); TRGJ: * (1F+1P); ◦ (2F+1P); ˆ (1F+2P); P = pseudogene, O = ORF. The numbers in brackets indicate the number of the genes. = ORF. The numbers in brackets indicate the number of the genes.

Table 3. Cassette comparison between Ovis aries and Bos taurus.

Table 3. CassetteTRG1 comparison between Ovis aries and Bos taurus. Ovis aries 4q3.1 Bos taurus 4q3.1 locus TRG1 locus OvisCassettes aries 4q3.1IMGT gene Nb of CDR-IMGT Bos taurusNb4q3.1 of CDR-IMGT IMGT gene names Cassettes names IMGT gene names Nbnames of alleles names CDR-IMGT alleles lengths lengths IMGT gene names Nb of allelesalleles CDR-IMGTlengths lengths TRGC5 TRGV11-1 TRGC51 P TRGV11-1 [8.6.4] 1 P [8.6.4] TRGV3-1 2 FTRGV3-1 [8.7.4] 2 F [8.7.4]TRGV3-1 TRGV3-1 1 F, 11 (F)F, 1 (F) [8.7.4] [8.7.4] TRGV3-2 1 FTRGV3-2 [8.7.5] 1 F [8.7.5]TRGV3-2 TRGV3-2 1 F, 11 (F)F, 1 (F) [8.7.4] [8.7.4] TRGV7 1 FTRGV7 [8.6.6] 1 F [8.6.6]TRGV7-1 TRGV7-1 1 F, 11 (F)F, 1 (F) [8.6.6] [8.6.6] TRGV10-1 1PTRGV10-1 [9.7.4] 1P [9.7.4]TRGV10-1 TRGV10-1 1 F1 F [9.7.5] [9.7.5] TRGV4 2 FTRGV4 [8.4.5] 2 F [8.4.5]TRGV4-1 TRGV4-1 1 (F)1 (F) [8.4.x] [8.4.x] TRGJ5-1 1 FTRGJ5-1 - 1 F - TRGJ5-1 TRGJ5-1 1 F1 F - - TRGJ5-2 1 PTRGJ5-2 - 1 P - TRGJ5-3 1 FTRGJ5-3 - 1 F - TRGC5 (EX2A) 1 (F) - TRGC5 (EX2A) 1 F, 2 (F) - TRGC5 (EX2A) 1 (F) - TRGC5 (EX2A) 1 F, 2 (F) - (TRGC7) TRGV8-1 1 F, 1 F [3.8.4] TRGV8-3 1 F, (1 F) [3.8.5] (TRGC7) TRGV8-1 1 F, 1 F [3.8.4] TRGV8-3 1 F, (1 F) [3.8.5] TRGV8-4 1 F [3.8.5] TGRV8-1 TRGV8-4 1 F1 F [3.8.5] [3.8.5] TRGV8-2 TGRV8-1 1 F, (1 F)1 F [3.8.5] [3.8.5] [6.8.5] TRGV8-2 1 F, (1 F) [3.8.5][6.8.5] TRGV2-1 2 F, 1 (F) [6.8.5]TRGV2-1 1 F [6.8.5] TRGV2-1 del81,82,832 F, 1 (F) TRGV2-1 1 F del81,82,83 del81,82,83 del81,82,83 TRGC7 (EX2A,2C) 1 P - TRGC3 TRGV9-1 1 F, 1 (F) [6.8.4] TRGV9-1 1 F [6.8.5] TRGV9-2 1 F [6.8.5] TRGJ3-1 1 F - TRGJ3-1 1 F, 1 (F) - TRGJ3-2 1 P - TRGC3 1 F, 1 (F) - TRGC3 (EX2A,2C) 1 F, 1 (F) - (EX2A,2C) TRGC4 TRGV1 (L41) 2 F [5.8.5] TRGV1-1 (L41) 1 F, 2 (F) [5.8.5] TRGJ4-1 1 P - TRGJ4-1 1 F - TRGJ4-2 1 F - TRGJ4-2 1 F - TRGC4 TRGC4 1 (F) - 1 F, 1 (F) - (EX2A,2B,2C) (EX2A) TRG2 Ovis aries 4q2.1 Bos taurus 4q1.5-2.2 locus IMGT gene Nb of Bos taurus Nb of Cassettes CDR-IMGT CDR-IMGT names alleles alleles [5.8.5] [5.8.5] TRGC1 TRGV5-1 (L41) 2 F TRGV5-1 (L41) 1 F, 4 (F) del5-8 del5-8 TRGJ1-1 1 F - TRGJ1-1 1 F - TRGJ1-2 1 F - TRGJ1-2 1 F, 2 (F) - TRGC1 TRGC1 (EX2A) 1 F - 1 F, 3 (F) - (EX2A,2B,2C) [5.8.5] TRGC2 TRGV6-1 1 F, 1 (F) del16

Genes 2020, 11, 624 14 of 28

Table 3. Cont.

TRG1 locus Ovis aries 4q3.1 Bos taurus 4q3.1 Cassettes names IMGT gene names Nb of alleles CDR-IMGT lengths IMGT gene names Nb of alleles CDR-IMGT lengths TRGC7 (EX2A,2C) 1 P - TRGC3 TRGV9-1 1 F, 1 (F) [6.8.4] TRGV9-1 1 F [6.8.5] TRGV9-2 1 F [6.8.5] TRGJ3-1 1 F - TRGJ3-1 1 F, 1 (F) - TRGJ3-2 1 P - TRGC3 (EX2A,2C) 1 F, 1 (F) - TRGC3 (EX2A,2C) 1 F, 1 (F) - TRGC4 TRGV1 (L41) 2 F [5.8.5] TRGV1-1 (L41) 1 F, 2 (F) [5.8.5] TRGJ4-1 1 P - TRGJ4-1 1 F - TRGJ4-2 1 F - TRGJ4-2 1 F - TRGC4 (EX2A,2B,2C) 1 (F) - TRGC4 (EX2A) 1 F, 1 (F) - TRG2 locus Ovis aries 4q2.1 Bos taurus 4q1.5-2.2 Cassettes IMGT gene names Nb of alleles CDR-IMGT Bos taurus Nb of alleles CDR-IMGT [5.8.5] [5.8.5] TRGC1 TRGV5-1 (L41) 2 F TRGV5-1 (L41) 1 F, 4 (F) del5-8 del5-8 TRGJ1-1 1 F - TRGJ1-1 1 F - TRGJ1-2 1 F - TRGJ1-2 1 F, 2 (F) - TRGC1 TRGC1 (EX2A) 1 F - 1 F, 3 (F) - (EX2A,2B,2C) [5.8.5] TRGC2 TRGV6-1 1 F, 1 (F) del16 [5.8.5] [5.8.5] TRGV5-2 2 F TRGV5-2* 10 (F) del5-8 del5-8 TRGJ2-1 1 F - TRGJ2-1* 1 F, 2 (F) - TRGJ2-2 1 F - TRGJ2-2 1 F, 2 (F) - TRGC2 (EX2A,2B,2C) 1 F, 1 (F) - TRGC2 (EX2A,2B,2C) 1 F, 4 (F) - [5.8.5] [5.8.5] TRGC6 TRGV6-1 1 F, 1 (F) TRGV6-2* 1 F del16 del16 TRGJ6-1 1 F - TRGJ6-1* 1 F - TRGJ6-2 1 F - - TRGC6 (EX2A,2B,2C) - TRGC6 (EX2A,2B,2C) 1 F, 1 (F) - Highlighted (in yellow) are sequence features found in both Ovis aries and Bos taurus TRG sequences (‘del’ followed by numbers indicate gap (unoccupied) positions according to the IMGT unique numbering for V-DOMAIN [58], ‘L41’ indicates that the CONSERVED-TRP 41 is replaced by a leucine (L) in the V-REGION. ‘EX2A’, ‘EX2A,2C’ and ‘EX2A,2B,2C’ indicate exon designation for TRGC gene with 1, 2 or 3 Exons 2, respectively. Genes 2020, 11, 624 15 of 28

6. The TRG Locus in Rodentia, Lagomorpha, and Primata The Mus musculus TRG is located in a single position on Chromosome 13A2 and it occupies a region of about 200 kb, typically delimited by the AMPH and STARD3NL genes at the 50 and 30 ends, respectively (Table4, Supplementary Figure S3A) [ 66,68,105–107]. The arrangement of the TRG genes resembles the Artiodactyla and Carnivora organization into cassettes, even if the number both of cassettes and of total genes are lower. Overall, the mouse locus comprises seven TRGV genes belonging to five subgroups, four TRGJ and four TRGC functional genes organized into four classical V-J-C cassettes. The first cassette, TRGC1, is the most extensive at 41 kb where, proceeding from 50 to 30, the TRGV7, TRGV4, TRGV6, and TRGV5 subgroup genes (one gene for each subgroup) are located. The TRGC3, TRGC2, and TRGC4 cassettes follow, all consisting of one TRGV and each belonging to a diverse subgroup, followed by one TRGJ and one TRGC gene. However, the TRGC3 cassette is not functional because of the TRGC3, while the entire TRGC2 cassette is inverted in the locus with respect to the other three cassettes. Enhancer elements that control the general accessibility of the region have been identified at the 30 end of all V–J–C cassettes [108]. The mouse TRGC1, TRGC2, and TRGC3 chains have a short hinge domain while the TRGC4 chain is polymorphic with a large hinge region [67]. Analysis of the genomic organization of the mouse TRGC genes revealed that they are characterized by three exons (EX1, EX2, and EX3). However, the TRGC4 gene can be constituted by one exon (EX2A) or, as in dog or artiodactyl TRGC genes [90,104], by two small exons (EX2A + EX2B), respectively 18 and 15 amino acids long, encoding the first part of the polymorphic hinge region of the TRGC4 chain (https://www.imgt.org/IMGTrepertoire/Proteins/ protein/mouse/Mu_TRallgenes.html). If the dolphin TRG represents the smallest and simplest locus within the mammalian superorder Laurasiatheria (Cetartiodactyla/Perissodactyla/Carnivora), the rabbit (Oryctolagus cuniculusis) TRG can be considered the smallest and simplest locus identified to date within the Eurarchontoglires (Primate/Lagomorpha/Rodentia). In fact, the rabbit TRG locus spans about 70 kb and contains 11 TRGV genes upstream of 2 TRGJ genes and 1 TRGC gene (Table4, Supplementary Figure S3B) [ 109]. Hence, the TRG gene arrangement in the rabbit is comparable to the cluster organization described earlier in most of the outgroups. A possible enhancer element has been identified about 8 kb downstream of the last exon of the TRGC gene. The AMPH gene is 13 kb upstream of the first TRGV gene, while STARD3NL is 1 kb downstream of the enhancer-like region, in an inverted transcriptional orientation. The TRGV genes (eight functional and three ORFs) are classified in four subgroups: the TRGV1 subgroup comprises eight members, and the TRGV2, TRGV3, and TRGV4 subgroups only have one member each. A high level of nucleotide identity between rabbit and human TRGV1 subgroup genes, both located in the 50 part of the respective locus, has been found. The only TRGC gene consists of four exons, with two exons (EX2A and EX2B) that encode for the first part of the connecting region. Genes 2020, 11, x FOR PEER REVIEW 16 of 28 Genes 2020, 11, x FOR PEER REVIEW 16 of 28

Table 4. Genomic organization and gene content of the TRG locus in Rodentia, Lagomorpha and Table 4. Genomic organization and gene content of the TRG locus in Rodentia, Lagomorpha and Primata. Genes Primata.2020, 11, x FOR PEER REVIEW 16 of 28

TRGV TRGJ TRGC Chromosomal Miniature Table 4. Genomic organizationTRGV andTRGJ gene contentTRGC of the TRGChromosomal locus in Rodentia, LagomorphaMiniature and References genes genes genes localization locus References Primata. genes genes genes localization locus Genes 2020, 11, x FOR PEER REVIEW 16 of 28 Hayday et al., TRGV TRGJ TRGC Chromosomal Miniature Hayday et al., 1985; TRGV1 References1985; Genes 2020, 11, 624 Table 4. Genomic organizationTRGV1genes andgenes gene contentgenes of the TRGlocalization locus in Rodentia, Lagomorphalocus and Garman et al., 16 of 28 TRGV2 TRGC1 Garman et al., Primata. TRGV2 TRGJ1 TRGC1 1986; TRGV3 TRGJ1 TRGC2 One locus 1986; TRGV3 TRGJ2 TRGC2 One locus TrauneckerHayday et al., et RODENTIA Mouse TRGV4 TRGJ2 TRGC3 Traunecker et TRGV4 TRGJ3 TRGC3 al.,1985; 1986; RODENTIA Mouse TRGV5TRGV1TRGV TRGJ3TRGJ (P)TRGC ChromosomalChrom. 13A2 Miniature al., 1986; TRGV5 TRGJ4 (P) Chrom. 13A2 ReferencesPelkonenGarman etet al.,al., Table 4. Genomic organization and gene contentTRGV6TRGV2 of the TRGTRGJ4 locus inTRGC4TRGC1 Rodentia, Lagomorpha and Primata. Pelkonen et al., TRGV6genes TRGJ1genes TRGC4genes localization locus 1987;1986; TRGV7TRGV3 TRGC2 One locus Mus musculus 1987; TRGV7 TRGJ2 Mus musculus VernooijTraunecker et al., et TRGV Genes TRGJ Genes TRGCTRGV4 Genes ChromosomalTRGC3 Localization Miniature LocusVernooij References et al., RODENTIA Mouse TRGJ3 Haydayal.,1993; 1986; et al., TRGV5 (P) Chrom. 13A2 1993;1985; TRGV1 TRGV1 TRGJ4 Pelkonen et al., TRGV6 TRGC4 Garman et al., TRGV2 TRGV2 TRGC1 1987;Hayday et al., [66]; TRGJ1 TRGC1TRGV7 TRGJ1 Mus musculus 1986; TRGV3 TRGV3 TRGC2One locus One locus VernooijGarman et al., et al., [105]; TRGJ2 TRGC2 TRGJ2 Traunecker et RODENTIA Mouse TRGV4 TRGV4 TRGC3 1993;Traunecker et al., [106]; RODENTIATRGJ3 Mouse TRGC3 (P) TRGJ3 al., 1986; TRGV5 TRGV5 (P)Chrom. 13A2Chrom. 13A2 Pelkonen et al., [107]; TRGJ4 TRGC4 TRGJ4 Pelkonen et al., TRGV6 TRGV6 TRGC4 Vernooij et al., [68]; TRGV1 (7+1O) 1987; TRGV7 TRGV1TRGV7 (7+1O) One locus Mus musculus TRGV2 (O) TRGJ1 One locus Mus musculus VernooijMassari et et al., al., TRGV2 (O) TRGJ1 TRGC Massari et al., LAGOMORPHA Rabbit TRGV3 TRGJ2 TRGC 2012;1993; LAGOMORPHA Rabbit TRGV3 TRGJ2 Chrom. 10 2012; TRGV4 (O) Chrom. 10 TRGV4 (O) TRGV1 (7+1O) TRGV1 (7+1O) One locus One locus Oryctolagus cuniculus TRGV2 (O) TRGJ1 TRGV2 (O) TRGJ1 Oryctolagus cuniculus Massari et al., LAGOMORPHA Rabbit TRGC TRGC Massari et al., [109]; TRGV3 LAGOMORPHTRGJ2 A Rabbit TRGV3 TRGJ2 2012; Chrom. 10 Chrom. 10 TRGV4 (O) TRGV4 (O)

TRGV1 (7+1O) Oryctolagus cuniculus One locus Oryctolagus cuniculus TRGV2 (O) TRGJ1 Massari et al., TRGV1 (O) TRGC TRGV1 (O) LAGOMORPHA Rabbit TRGV1TRGV3 (O) TRGJ2 2012; TRGV2 Chrom. 10 TRGV2 TRGV2 TRGV3TRGV4 (O) TRGV3 TRGV3 TRGV6 (P) TRGV6 (P) TRGV6 (P) TRGJ1-1 Oryctolagus cuniculus TRGJ1-1 TRGV8TRGV1 (O) TRGJ1-1 TRGV8 TRGV8 TRGJ1-2 One locus TRGJ1-2 Rhesus TRBVATRGV2 (P) TRGJ1-2 TRGC1One locus One locus Lefranc M-P., TRBVA (P) Rhesus TRBVATRGC1 (P) TRGJ2-1 TRGC1 Lefranc M-P., PRIMATA Rhesus monkey PRIMATATRGJ2-1 TRGV9TRGV3 TRGJ2-1 TRGC2 2014;Lefranc M-P., [2]; TRGV9 PRIMATA monkey TRGV9TRGC2 TRGJ2-2 (P) TRGC2 Chrom. 3 2014; TRGJ2-2 (P) monkey TRGV10TRGV6 (P) (O) TRGJ2-2 (P) Chrom. 3 Chrom. 3 TRGV10 (O) TRGV10 (O) TRGJ2-3TRGJ1-1 TRGJ2-3 TRGVBTRGV8 (P) TRGJ2-3 TRGVB (P) TRGVB (P) TRGJ1-2 One locus Macaca mulatta Rhesus TRGV11TRBVATRGV1 (O)(P) (P) TRGC1 Macaca mulatta Lefranc M-P., TRGV11 (P) TRGV11 (P) TRGJ2-1 Macaca mulatta PRIMATA TRGVCTRGV9TRGV2 (P) TRGC2 2014; TRGVC (P) monkey TRGVC (P) TRGJ2-2 (P) Chrom. 3 TRGVDTRGV10TRGV3 (P) (O) TRGVD (P) TRGVDTRGV6 (P)(P) TRGJ2-3 TRGVB (P) TRGJ1-1 TRGV8 Macaca mulatta TRGV1 (O) TRGV1TRGV11 (O) (P) TRGJ1-2 One locus Rhesus TRGV1TRBVA (O)(P) TRGC1 Lefranc M-P., TRGV2 TRGV2TRGVC (P) TRGJ2-1 PRIMATA TRGV2TRGV9 TRGC2 2014; TRGV3 monkey TRGV3TRGVD (P) TRGJ2-2 (P) Chrom. 3 TRGV3TRGV10 (O) Lefranc, and TRGV4 TRGV4 TRGJ2-3 Lefranc, and TRGV4TRGVB (P) Rabbitts, 1985 TRGV5 TRGV5TRGV1 (O) Macaca mulatta Rabbitts, 1985 TRGJP1 TRGV5TRGV11 (P) TRGJP1 Lefranc, 1986a TRGV5P (P) TRGV5PTRGV2 (P) TRGJP1 Lefranc, 1986a TRGJP TRGV5PTRGVC (P) (P) TRGJP One locus One locus Lefranc,1986b,cLefranc, and Rabbitts, [42]; TRGV6 (P) TRGV6TRGV3TRGC1 (P) TRGJP TRGC1 One locus Lefranc,1986b,c Human TRGJ1 TRGV6TRGVD (P) (P) TRGJ1 TRGC1 Lefranc,Lefranc andandLefranc, [43–45]; TRGV7 (P) Human TRGV7TRGV4TRGC2 (P) TRGJ1 TRGC2 Lefranc and TRGJP2 Human TRGV7 (P) TRGJP2 TRGC2Chrom. 7p14Chrom. 7p14 Rabbitts,Lefranc 19891985 and Rabbitts, [49]; TRGV8 TRGV8TRGV5 TRGJP2 Chrom. 7p14 Rabbitts, 1989 TRGJ2 TRGV8 TRGJ2TRGJP1 Lefranc,Lefranc 1986aand TRGVA (P) TRGVATRGV5PTRGV1 (O)(P) (P) TRGJ2 Lefranc and TRGVA (P) TRGJP One locus Lefranc,1986b,cLefranc et al., TRGV9 TRGV9TRGV6TRGV2 (P) TRGC1 Homo sapiens Lefranc et al., TRGV9 TRGJ1 HomoHomo sapiens Lefranc1989 and TRGV10 (O) Human TRGV10TRGV7TRGV3 (P) (O) TRGC2 1989 TRGV10 (O) TRGJP2 Chrom. 7p14 Rabbitts,Lefranc, 1989and TRGVBTRGV8TRGV4 (P) TRGVB (P) TRGVB (P) Rabbitts, 1985 TRGV11TRGV5 (O) TRGJ2 Lefranc and TRGV11 (O) TRGVA (P) TRGJP1 Lefranc, 1986a TRGV11TRGV5P (O)(P) Lefranc et al., TRGV9 Legend:TRGJP P = pseudogene, O = OneORF. locus Homo sapiens Lefranc,1986b,c Legend: PTRGV6= pseudogene, (P) Legend: O = ORF.P = pseudogene,TRGC1 O = ORF. 1989 TRGV10 (O) TRGJ1 Lefranc and Human TRGV7 (P) TRGC2 TRGVB (P) TRGJP2 Chrom. 7p14 Rabbitts, 1989 If the dolphinTRGV8 TRG represents the smallest and simplest locus within the mammalian superorder If the dolphinTRGV11 TRG represents (O) theTRGJ2 smallest and simplest locus within the mammalian superorderLefranc and Laurasiatheria (CetartiTRGVAodactyla/Perissodactyla/Carnivora), (P) the rabbit (Oryctolagus cuniculusis) TRG Laurasiatheria (Cetartiodactyla/Perissodactyla/Carnivora),Legend: P = pseudogene, O =the ORF. rabbit (Oryctolagus cuniculusis) TRGLefranc et al., TRGV9 Homo sapiens 1989 TRGV10 (O) If the dolphinTRGVB TRG represents (P) the smallest and simplest locus within the mammalian superorder Laurasiatheria (CetartiTRGV11odactyla/Perissodactyla/Carnivora), (O) the rabbit (Oryctolagus cuniculusis) TRG Legend: P = pseudogene, O = ORF.

If the dolphin TRG represents the smallest and simplest locus within the mammalian superorder Laurasiatheria (Cetartiodactyla/Perissodactyla/Carnivora), the rabbit (Oryctolagus cuniculusis) TRG

Genes 2020, 11, 624 17 of 28

In contrast to the evolutionary conservation of the TRB and TRA/TRD loci, dynamic evolution of the TRG locus is demonstrated within the primate lineage. More recently, the TRG locus has been determined in four nonhuman primate species: in great apes, Pan troglodytes and Pongo pygmaeus; in Macaca mulatta (an Old World monkey) (Table4, Supplementary Figure S3C); and in Callithrix jacchus (a New World monkey) [110]. Although the structure of the locus is still maintained with respect to the human locus, substantial diversification can be observed. In particular, the TRGV1 subgroup region is highly dynamic; it maintains a flanking position in all species, but the genes have undergone substantial duplications/deletions in which the orthology between genes has become less clear. For example, in the M. mulatta locus (Table4, Supplementary Figure S3C), the number of TRGV1 genes is more contained compared to human, with five genes of which only three are functional. While the first three genes show an evident orthology with respect to the first three human genes, the last ones seem to have been generated by a recent species-specific duplication [110]. In contrast, the TRGV9-TRGV10-TRGV11 region, which retains a central position in the locus, appears to be conserved with a high level of homology between the primate species, although TRGV11 was not found in C. jacchus, and TRGV10 is functional in Pan troglodytes. Two TRGC genes are present in all species, while the number and distribution of TRGJ varies, as in the case of M. mulatta where, unlike the human locus, two TRGJ genes are before the TRGC1 gene and three TRGJ (one is not functional), are upstream of the TRGC2 gene. In M. mulatta the TRGC1 and TRGC2 genes have three and five exons, respectively, and the presence of polymorphisms has not been determined.

7. Phylogenetic Analysis Highlights a Diverse Mode of Evolution of the V, J, and C Subfamily Genes The rearranged V-D-J and V-J genes encode the V-β and V-α, or the V-δ and V-γ domains, respectively, which form the antigen-binding site of the αβ or γδ TR receptor, while the C genes encode the C-region which comprises the C-domain (C-β and C-α, or C-δ and C-γ), the connecting region, the transmembrane region which anchors each receptor chain in the membrane, and a very short (absent for the delta chain) cytoplasmic region [1]. CD3 proteins are associated to the αβ or γδ TR for the signal transduction. Thus, the TcR of the T cells, constituting the TR + CD3 coreceptors, is the equivalent of the BcR of the B cells, constituting the IG and CD79 coreceptors. The different roles of the types of genes that make up the receptor seems to influence each one’s way of evolving. Generally, the genes encoding the variable domain seems to follow evolutionary dynamics, shared among species, that preserve the sequence of the orthologous genes; whereas, the evolution of the C genes is reflective of the phylogenetic history of each species. This observation (or consideration) comes from analysis of the TRB and TRA/TRD loci, which retain a conserved general gene arrangement and, although these regions appear to have been evolving dynamically in each eutherian mammalian species, a gross order of genes is still maintained. As a matter of fact, phylogenetic analysis conducted on the TRBV and TRAV genes showed that the V genes from different mammals intermingle rather than forming separate clades, proving that duplications of ancestral genes followed by diversification is the major mode of evolution [26,27,35,37,40]. This rule also fits also the TRG genes, despite the structure of the TRG locus differing considerably across species. Indeed, the first phylogenetic investigations involving mammalian TRG genes showed that the sheep TRGV genes also form groupings with human and bovine genes [85]. Subsequent studies led over time to the identification of the genomic organization of the TRG locus in many other mammals such as rabbits, dogs, camels, dolphins, and cats, and confirmed the first evolutionary indication, despite the discovery of a great diversity of the gene arrangement within the TRG region of the various species [27,38,96,109]. The tree shown in Figure3 recapitulates the evolutionary relationship between the mammalian TRGV genes by combining pre-existing phylogenetic data. Genes 2020, 11, x FOR PEER REVIEW 18 of 28 ancestral genes followed by diversification is the major mode of evolution [26,27,35,37,40]. This rule also fits also the TRG genes, despite the structure of the TRG locus differing considerably across species. Indeed, the first phylogenetic investigations involving mammalian TRG genes showed that the sheep TRGV genes also form groupings with human and bovine genes [85]. Subsequent studies led over time to the identification of the genomic organization of the TRG locus in many other mammals such as rabbits, dogs, camels, dolphins, and cats, and confirmed the first evolutionary indication, despite the discovery of a great diversity of the gene arrangement within the TRG region of the various species [27,38,96,109]. The tree shown in Figure 3 recapitulates the evolutionary Genes 2020, 11, 624 18 of 28 relationship between the mammalian TRGV genes by combining pre-existing phylogenetic data.

Figure 3. Evolutionary relationships of the eutherian mammalian TRGV genes. The TRGV as well Figure 3. Evolutionary relationships of the eutherian mammalian TRGV genes. The TRGV as well as as the TRGJ (Figure4) and TRGC (Figure5) gene sequences used for the phylogenetic analysis the TRGJ (Figure 4) and TRGC (Figure 5) gene sequences used for the phylogenetic analysis were were retrieved from the IMGT® database (IMGT Repertoire, http://www.imgt.org) if annotated, retrieved from the IMGT® database (IMGT Repertoire, http://www.imgt.org) if annotated, or from the or from the GenBank database using the accession numbers reported in the reference articles listed GenBank database using the accession numbers reported in the reference articles listed in Table 2 and in Tables2 and4. Chicken (Galgal) TRG genes were used as the outgroup [66]. For simplicity, Table 4. Chicken (Galgal) TRG genes were used as the outgroup [66]. For simplicity, we included in we included in the analysis the TRGV genes belonging to a single species for each mammalian the analysis the TRGV genes belonging to a single species for each mammalian suborder in which the suborder in which the genomic organization of the TRG locus has been inferred. One member gene genomic organization of the TRG locus has been inferred. One member gene for each of the chicken for each of the chicken (Galgal) TRGV subgroups was used as an outgroup. Multiple alignments of (Galgal) TRGV subgroups was used as an outgroup. Multiple alignments of the V-region nucleotide the V-region nucleotide sequences of functional genes and in-frame pseudogenes were carried out with sequences of functional genes and in-frame pseudogenes were carried out with the MUSCLE program the MUSCLE program [111]. The evolutionary analyses were conducted in MEGA7 [112]. We used [111]. The evolutionary analyses were conducted in MEGA7 [112]. We used the neighbor-joining (NJ) the neighbor-joining (NJ) method to reconstruct the phylogenetic tree [113]. The percentage of replicate method to reconstruct the phylogenetic tree [113]. The percentage of replicate trees in which the trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches to the branches [114]. The trees are drawn to scale, with branch lengths in the same units as those [114]. The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary of the evolutionary distances used to infer the phylogenetic trees. The evolutionary distances were distances used to infer the phylogenetic trees. The evolutionary distances were computed using the computed using the p-distance method [115], and the units are the number of base differences per p-distance method [115], and the units are the number of base differences per site. The analysis site. The analysis involved 66 nucleotide sequences. All positions containing gaps and missing data wereinvolved eliminated. 66 nucleotide There sequences. were a total All of positions 176 positions containing in the gaps final dataset.and missing Monophyletic data were eliminated. groupings describedThere were in a the total text of are 176 indicated positions by in capitalthe final letters. dataset. The Monophyletic blue and red groupings branches ofdescribed the tree highlightin the text twoare indicated major groupings by capital of letters. the mammalian The blue and genes. red branches The IMGT of the six-letter tree high standardizedlight two major abbreviations groupings for species (Homsap (human), Musmus (mouse), Felcat (cat), Oviari (sheep), Camdro (dromedary), Turtru (dolphin), Orycun (rabbit)) and nine-letter abbreviations for subspecies (Canlupfam, dog) taxa are used. Genes 2020, 11, 624 19 of 28 Genes 2020, 11, x FOR PEER REVIEW 20 of 28

FigureFigure 4. 4.Evolutionary Evolutionary relationships relationships of of the the eutherian eutherian mammalian mammalianTRGJ TRGJgenes. genes. The The TRGJ TRGJ coding coding sequencessequences of of all all mammalianmammalian species species were were included included in inthe the tree. tree. A chicken A chicken (Galgal) (Galgal) TRGJ geneTRGJ wasgene used wasas usedoutgroup as outgroup [66]. Multiple [66]. alignments Multiple alignments of the gene of sequences the gene were sequences carried were out carriedusing the out MUSCLE using theprogram MUSCLE [111]. program The evolutionary [111]. The evolutionary analyses were analyses conducted were in conducted MEGA7 [112]. in MEGA7 We used [112 the]. We neighbor- used the neighbor-joining (NJ) method to reconstruct the phylogenetic tree [113]. The percentage of replicate joining (NJ) method to reconstruct the phylogenetic tree [113]. The percentage of replicate trees in trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the to the branches [114]. The trees are drawn to scale, with branch lengths in the same units as those branches [114]. The trees are drawn to scale, with branch lengths in the same units as those of the of the evolutionary distances used to infer the phylogenetic trees. The evolutionary distances were evolutionary distances used to infer the phylogenetic trees. The evolutionary distances were computed using the p-distance method [115] and the units are the number of base differences per computed using the p-distance method [115] and the units are the number of base differences per site. site. The analysis involved 67 nucleotide sequences. All positions containing gaps and missing data The analysis involved 67 nucleotide sequences. All positions containing gaps and missing data were were eliminated. There were a total of 39 positions in the final dataset. The C-proximal TRGJ genes eliminated. There were a total of 39 positions in the final dataset. The C-proximal TRGJ genes are are shown in red; the C-distal TRGJ genes are shown in blue. The TRGJ genes occupying the middle shown in red; the C-distal TRGJ genes are shown in blue. The TRGJ genes occupying the middle position of the J cluster formed by three genes within each own TRG locus are marked with a black position of the J cluster formed by three genes within each own TRG locus are marked with a black circle. The IMGT six-letter standardized abbreviations for species (Homsap (human), Musmus (mouse), circle. The IMGT six-letter standardized abbreviations for species (Homsap (human), Musmus Felcat (cat), Bostau (bovine), Oviari (sheep), Camdro (dromedary), Turtru (dolphin), Orycun (rabbit)) (mouse), Felcat (cat), Bostau (bovine), Oviari (sheep), Camdro (dromedary), Turtru (dolphin), Orycun and nine-letter standardized abbreviation for subspecies (Canlupfam, dog) taxa are used. (rabbit)) and nine-letter standardized abbreviation for subspecies (Canlupfam, dog) taxa are used. Despite the dynamic evolution of each TRG locus, the TRGV genes of the different species The red branch consists of all the J genes (paralogous genes within and between each species intermingle with each other to form monophyletic groups (A–G) rather than separate species-specific and orthologous genes between species) occupying the position closest to the constant gene (C- clades. Indeed, each branch groups corresponding genes (or gene subgroups/subfamilies) of all species proximal) within the V-J-(J)-J-C clusters, as well as all the mouse TRGJ genes, which are single in each with a clear orthology, irrespective of their genomic organization, indicating their occurrence from a TRGC cassette (Figure 1A, Figure 2, and Supplementary Figures S2 and S3). In the blue branch, all common ancestor and a strong selective pressure to maintain their function. For instance, Branch A the J (paralogous and orthologous) genes occupying the farthest position from the constant gene (C- groups corresponding genes which have been preserved in all species of mammals as a result of a distal) are present together with the TRGJ genes that are in the middle of the J cluster formed by three strong functional constraint. Other monophyletic clades (B–G) group corresponding genes present genes within the human, sheep, camel, and dolphin TRG loci (black circles in Figure 4). These genes in some but not in all species in accordance with the birth-and-death model of multigene family form a paraphyletic group that differentiates the TRGJ genes of carnivores from those of other evolution, which explains that some duplicated genes are retained in the genome for a long time, while mammalian species. These data highlight an evident functional constraint of the J genes, which play others are deleted or become pseudogenes. This evolutionary model also explains the emergence of a role in the recombination process and structurally contribute to the V domain of the receptor [2].

Genes 2020, 11, x FOR PEER REVIEW 21 of 28

In contrast to the intermingling of the TRGV and TRGJ genes from different species, the Genes 2020, 11, 624 20 of 28 mammalian TRGC genes evolved in a species-specific manner, and the sequences form distinct clades consistent with the current phylogeny [88,90,96]. The tree shown in Figure 5 recapitulates the newevolutionary genes that have relationships undergone of substantialthe TRGC genes diversification in the different through eutherian species-specific mammalian duplication species events, and as indicatedhighlights in the the species-specific tree by species-specific grouping. clustering of the genes (especially in the G and F clades).

Figure 5. Phylogenetic relationships of the eutherian mammalian TRGC genes. The TRGC coding Figure 5. Phylogenetic relationships of the eutherian mammalian TRGC genes. The TRGC coding nucleotide sequences from the different eutherian mammals were combined in the same alignment. nucleotide sequences from the different eutherian mammals were combined in the same alignment. The chicken (Galgal) TRGC gene was used as outgroup [66]. Multiple alignments of the gene sequences The chicken (Galgal) TRGC gene was used as outgroup [66]. Multiple alignments of the gene were carried out using the MUSCLE program [111]. The evolutionary analyses were conducted sequences were carried out using the MUSCLE program [111]. The evolutionary analyses were in MEGA7 [112]. We used the neighbor-joining (NJ) method to reconstruct the phylogenetic tree [113]. conducted in MEGA7 [112]. We used the neighbor-joining (NJ) method to reconstruct the The percentage of replicate trees in which the associated taxa clustered together in the bootstrap phylogenetic tree [113]. The percentage of replicate trees in which the associated taxa clustered test (100 replicates) is shown next to the branches [114]. The trees are drawn to scale, with branch together in the bootstrap test (100 replicates) is shown next to the branches [114]. The trees are drawn lengths in the same units as those of the evolutionary distances used to infer the phylogenetic trees. to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the The evolutionary distances were computed using the p-distance method [115] and the units are phylogenetic trees. The evolutionary distances were computed using the p-distance method [115] and thethe number units ofare base the number differences of base per differences site. The analysis per site. involved The analysis 38 nucleotide involved 38 sequences. nucleotide All sequences. positions containingAll positions gaps containing and missing gaps data and were missing eliminated. data were There eliminated. were aThere total were of 324 a total positions of 324 inpositions the final dataset.in the Thefinal IMGTdataset. six-letter The IMGT standardized six-letter standard abbreviationsized abbreviations for species for (Homsap species (Homsap (human), (human), Musmus (mouse),Musmus Felcat (mouse), (cat), Bostau Felcat (bovine),(cat), Bostau Oviari (bovine) (sheep),, CamdroOviari (sheep), (dromedary), Camdro Turtru (dromedary), (dolphin), Turtru Orycun (rabbit))(dolphin), and nine-letterOrycun (rabbit)) standardized and nine-letter abbreviations standa forrdized subspecies abbreviations (Canlupfam, for subspecies dog) taxa (Canlupfam, are used. dog) taxa are used. Moreover, looking at the tree as a whole, two major groupings of the mammalian genes are clearly distinguishableGene conversion (theseems blue to be and the red mechanism branches that of thehas treehomogenized in Figure 3the). TheTRGC blue sequences branch within groups geneseach exhibiting lineage (concerted a conserved evolution), nature maintaining across species a high with level a of clear similarity correspondence between the betweenTRGC protein genes, whereasisotypes the in red each part species. of the This tree containsconservationTRGV is constrainedgenes that haveby structural undergone and substantialfunctional requirements. diversification throughIn fact, duplications the constant portion within eachof the species. TR does Fornot bind example, antigen, the while blue it branch must interact includes with all monomorphic the artiodactyl TRGVstructuresgenes belonginglike the extracellular to the TRGC5 part of cassette, the TRD which chain hasand beenthe CD3 shown coreceptors; to be the therefore, most evolutionarily it plays an ancientimportant [89]. Thisrole cassettein signal wouldtransduction. have been duplicated to generate a second one from which the other artiodactyl TRGC cassette developed. The red part of the tree includes the TRGV genes of the second cassette and all its derivatives. As another example, in the blue part of the tree can be found the human TRGV9, TRGV10, and TRGV11 genes, which have been proven to be conserved across the primate lineage and to occupy similar positions within the different TRG loci. Conversely, the human genes Genes 2020, 11, 624 21 of 28 grouped in the red part of the tree have undergone a highly dynamic evolution, making their orthology with the nonhuman primate genes unclear (Figure3). Overall, one can speculate that the TRGV genes present today in the different mammalian species derived from two different ancestors. The blue branch of the tree groups the genes derived from the oldest one, and they have mostly have maintained evolutionary stability, while, the red part consists of new repertoires of genes that evolved dynamically by duplication (and presumably deletion) even within closely related species. The peculiar genomic organization of TRG loci in cassettes, clusters, or semi-clusters favors the physical proximity of TRGV and TRGJ genes and prompted investigation of the phylogenetic behavior of the TRGJ with respect to TRGV and TRGC genes. Evolutionary analyses conducted with the human, sheep, cattle, dolphin, and dromedary TRGJ sequences showed the clustering of the TRGJ genes into two main groups in relation to the physical position of each J gene within its own cluster with respect to the TRGC gene [38,88,96]. An updated version of the same evolutionary analyses performed with the addition of the TRGJ sequences of mammalian species representative of Carnivora, Rodentia, and Lagomorpha orders (Figure4), confirmed the division of the TRGJ genes into two major monophyletic groupings. The red branch consists of all the J genes (paralogous genes within and between each species and orthologous genes between species) occupying the position closest to the constant gene (C-proximal) within the V-J-(J)-J-C clusters, as well as all the mouse TRGJ genes, which are single in each TRGC cassette (Figure1A, Figure2, and Supplementary Figures S2 and S3). In the blue branch, all the J (paralogous and orthologous) genes occupying the farthest position from the constant gene (C-distal) are present together with the TRGJ genes that are in the middle of the J cluster formed by three genes within the human, sheep, camel, and dolphin TRG loci (black circles in Figure4). These genes form a paraphyletic group that differentiates the TRGJ genes of carnivores from those of other mammalian species. These data highlight an evident functional constraint of the J genes, which play a role in the recombination process and structurally contribute to the V domain of the receptor [2]. In contrast to the intermingling of the TRGV and TRGJ genes from different species, the mammalian TRGC genes evolved in a species-specific manner, and the sequences form distinct clades consistent with the current phylogeny [88,90,96]. The tree shown in Figure5 recapitulates the evolutionary relationships of the TRGC genes in the different eutherian mammalian species and highlights the species-specific grouping. Gene conversion seems to be the mechanism that has homogenized the TRGC sequences within each lineage (concerted evolution), maintaining a high level of similarity between the TRGC protein isotypes in each species. This conservation is constrained by structural and functional requirements. In fact, the constant portion of the TR does not bind antigen, while it must interact with monomorphic structures like the extracellular part of the TRD chain and the CD3 coreceptors; therefore, it plays an important role in signal transduction. However, orthology between multiple TRGC genes can be maintained in more closely related species, as in cattle and sheep. In contrast, dromedary TRGC genes group apart from the other Cetartiodactyla suborders, for which a common ancestor gene can be hypothesized.

8. Conclusions The great plasticity of the adaptive T-cell receptor repertoire in vertebrates is mainly due to gene duplication and to somatic rearrangement during T-cell differentiation. Ohno postulated that gene duplication plays a major role in evolution in his book “Evolution by Gene Duplication” [116]. Here, we refer to duplication-driven evolution of V, J, and C genes of the TRG locus in mammals and discuss the correlation between its genomic organization and the possible modalities of duplication in organisms belonging to the superorders of Cetartiodactyla (Ruminantia/Tylopoda/ Cetacea) and Carnivora in comparison with Rodentia, Lagomorpha, and Primata and with outgroups. Genes 2020, 11, 624 22 of 28

In this regard, a careful look at the genomic organization of TRG inside the outgroup (birds, fishes, reptiles, and marsupials) locus reveals that the duplications have affected (involved) the V and J genes individually by creating clusters. Hence, duplications have occurred in the region of the TRGJ genes with the main objective of obtaining a high number (seven in the marsupials and nine in the alligator) of functional genes (Table1). Duplications of TRGV genes have taken place in the upstream region of the J region, although remaining circumscribed in the physical area of the V region. In all examined cases (chicken, duck, shark, alligator, and opossum) except one (salmon), the TRGC remained unique (Table1). Unique among the analyzed vertebrate species is the organization of the TRG locus in humans and in Primata (Figure1, Table4) where J-gene duplications also involved the C gene, creating a duplicated J-C cluster located downstream of the V region. Moving from the “V-J-C-cluster” model to the structural “V-J-J-C” cassette typical of Cetartiodactyla (Ruminantia, Tylopoda, and Cetacea), the duplications involved V, J, and C genes physically tied together in the same chromosomal area, giving rise to “recombinational units” (seven cassettes in Bos taurus and six cassettes in Ovis aries) (Figure2 and Tables2 and3). The number of cassettes is reduced to three in Camelus dromedarius (Tylopoda). Moreover, a minimal structure V-J-C (2V, 3J, and 1C) with a high level of homology to the ancient Artiodactyla cassette defines the Tursiops truncatus (Cetacea) TRG locus. The cassette organization is also found in Carnivora, where the number of duplicate cassettes remains high (Table2). This representative number of eight in Canis lupus familiaris and six in Felis catus is comparable to that of ruminants. However, in contrast to the bovine and ovine TRG loci, the value of the functional/total genes ratio (Supplementary Table S1) found in Carnivora suggests that there is no correlation between the extensive duplications of the cassettes and a need for new functional genes in the adaptive immune response. A suggestive and intriguing peculiarity of the TRG locus compared to the other TR loci is the number of TRGC genes and the diversity in the corresponding protein structure. In particular, the TRGC genes can show a different exon–intron organization that is evident not only if we compare TRGC genes within the TRG loci of the diverse species, but even between genes within a single TRG locus. Their diversity is mainly linked to the different number of Exon 2 copies, which can encode the connecting region. The biological significance of these repetitive exons remains unclear [83,117]. One hypothesis is the connecting-region length variation may affect processes such as signal transduction or interactions with other molecules at the cell surface [118]. In fact, the TR is capable of delivering a variety of different signals related to the different functions attributed to γδ T cells [119]. However, despite the differences in the TRG locus structure and in the arrangement of the genes, phylogenetic analyses show a tightly evolutionary conserved relationship of the genes encoding the variable domain in diverse species, with genes that have been preserved in all mammalian species as a result of a strong functional constraint and the emergence of new genes that have undergone substantial diversification through species-specific duplication. All these features highlight the important role of the TRG chain in the adaptive immune response.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/6/624/s1, Figure S1: TRG locus representation in outgroups, Figure S2: TRG locus representation in Cetartiodactyla and Carnivora, Figure S3: TRG locus representation in Rodentia, Lagomorpha and Primata, Table S1: Ratio functional/total TRGV, TRGJ and TRGC genes. Author Contributions: Conceptualization, R.A., S.M. and S.C.; Writing—Original Draft Preparation, R.A., G.L. and S.C.; Writing—Review & Editing, S.M. and S.C.; Supervision, M.-P.L.; Resources and Funding Acquisition, R.A., F.G., A.C.J. and S.C. All authors have read and agreed to the published version of the manuscript. Funding: The financial support of the University of Bari and of University of Salento is gratefully acknowledged. This research was funded by “Salvaguardia di piccoli pelagici: una pesca sostenibile ed innovativa nel Basso Adriatico–SALV.ADRI”, finanziato nell’ambito dell’Avviso Pubblico del Fondo Europeo per gli Affari Marittimi e per la Pesca (FEAMP) 2014/2020 alla Misura alla 1.26 “Innovazione” (art. 26 del Reg. UE 508/2014) “Regione Puglia”. Genes 2020, 11, 624 23 of 28

Acknowledgments: We thank Angela Pala and Massimo Lacitignola for technical assistance in the manuscript preparation. Conflicts of Interest: The authors declare no conflict of interest.

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