Pacheco-Olvera DL et al. (2025), Genomic characterization of the T-cell receptor...

XB-ART-61555

Front Immunol 2025 Sep 30;16:1656386. doi: 10.3389/fimmu.2025.1656386.

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Genomic characterization of the T-cell receptor loci in Ambystoma mexicanum.

Pacheco-Olvera DL , Saint Remy-Hernández S , Godoy-Lozano EE , Téllez-Sosa J , Valdovinos-Torres H , Curiel-Quesada E , López-Macías C , Martínez-Barnetche J .

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BACKGROUND: Amphibians are valuable models for comparative immunology. In the caudate Ambystoma mexicanum, the architecture of immunoglobulin loci resembles that of the anuran Xenopus tropicalis, although some antibody gene features are absent. Evidence supports the presence of T lymphocytes in axolotl, the expression of T cell receptor alpha, beta, and delta chains, and a restricted diversity in the delta chain. Here, we describe the T cell receptor loci in the A. mexicanum genome and compare them with X. tropicalis and other tetrapods. METHODS: T cell receptor loci were mapped and annotated in the A. mexicanum genome (UKY_AMEXF1_1) using reference sequences from axolotl, X. tropicalis, human, and mouse. Gene models were refined with RNA sequencing data from spleen, lung, and liver. RESULTS: The T cell receptor alpha and delta locus in axolotl shows an overall conserved structure compared with other tetrapods. The alpha locus contained a higher number of variable genes than the beta and delta loci, with a predominance of functional genes (ratio 3.06). No gene encoding the pre-T cell receptor chain alpha was identified. The delta locus harbored two conventional variable genes, but no expression was detected in RNA sequencing data, suggesting pseudogenization. Neither delta chain diversity genes nor gamma chain elements were found in the genome or spleen transcriptome. The beta locus displayed structural similarity to that of other tetrapods and included five translocons with diversity, joining, and constant segments. One constant gene consisted of two exons encoding two constant domains. Functional variable genes predominated in the beta locus (ratio 3.6). CONCLUSION: Our study reveals conserved but distinctive features of axolotl T cell receptor loci, including restricted delta-chain diversity, absence of gamma chain and pre-T cell receptor alpha, and structural novelty in the beta locus. These findings provide new insights into the evolution of T cell receptors in amphibians and offer a genomic framework to explore the links between adaptive immunity and tissue regeneration in A. mexicanum.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: amph aoah atl2 bbs9 bmper clcn1 cnpy3 eif4a3 elmo1 epdr1 fbxo42 gnmt gpr141 klc4 klhdc3 lactbl1 mea1 nobox npsr1 nt5c3a polr1b pou6f2 rp9 rpl7l1 sfrp4 slc2a1 stard3nl trac trav ttl velo1

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	Figure 1. The TRA-TRD locus in Ambystoma mexicanum is located in the centromeric portion of chr13p (20.7Mbp). (A) Gene density plot of chromosome 13p, where the TRA-TRD cluster is highligted (box); dark blue colors indicate low gene density. (B) Overview of the whole TRA-TRD locus (264.6-285.3 Mbp), showing non-TR genes (black) in proximal flank, TRAC and TRDC genes (blue), TRAJ and TRDJ genes (yellow), and TRAV and TRDV genes (red). (C) Close-up of the TRDC-J gene cluster (267.8-280.7 Mbp). (D) Detailed view of theTRAJ cluster (281-281.2 Mbp). (E) Spleen RNA-seq coverage histogram of the TRDC-J region. (F) Spleen RNA-seq coverage histogram of the TRAC-J region. Note that in the E and F panels the color intensity shifts from blue to red whith read counts increasing in the spleen transcriptome.
	Figure 2. Synteny of the TRA-TRD locus in Ambystoma mexicanum compared to other tetrapods. Schematic representation of TRA-TRD locus in human (Homo sapiens, GRCh38.p14), mouse (Mus musculus, GRCm39), opossum (Monodelphis domestica, ASM229v1), frog (Xenopus tropicalis, UCB_Xtro_10.0), and axolotl (A. mexicanum, UKYF1_1) are shown. Solid-filled symbols represent the TRA locus, while open symbols correspond to the TRD locus. Constant regions are indicated in blue, J gene cluster is depicted in dark yellow, and V clusters are highlighted in red. D genes are displayed as green rectangles, and non-TR genes are depicted in black. Interestingly, in the axolotl, the IGHC locus (purple) is not linked to the TRAD cluster as observed in X. tropicalis and spans across the centromere (depicted as a gray circle). The figures are not to scale, and the same scheme is applied to all species for consistency. Gene orientation in mammals and axolotl is 5’-3’and in X. tropicalis is 3’-5’.
	Figure 3. ynteny of the TRB locus in Ambystoma mexicanum compared to other tetrapods. Schematic representation of the TRB locus in human (Homo sapiens, GRCh38.p14), mouse (Mus musculus, GRCm39), opossum (Monodelphis domestica, ASM229v1), frog (Xenopus tropicalis, UCB_Xtro_10.0), and axolotl (A. mexicanum, UKYF1_1). The TRBC genes (constant regions) are shown in blue, the TRBJ cluster in yellow, and the TRBV cluster in red. TRBD genes are depicted in green, while non-TR genes are represented in black. The figure is not to scale, and the color scheme is consistent across all species for clarity. Gene orientation in mammals is 5’-3’and in amphibians is 3’-5’.
	Figure 4. The TRB locus of Ambystoma mexicanum is located in the telomeric portion of chromosome 3p (4.82 Mbp). (A) Gene density plot of chromosome 3, where the TRB locus is encoded (highlighted with a box). Dark blue regions indicate areas of low gene density. (B) Overview of the TRB locus (30.03-34.85 Mbp), showing non-TR genes in black. Proximal and distal flanking genes include TRPV and PRSS, respectively.Gaps are represented in gray. TRBC genes are shown in blue, TRBJ genes in yellow, TRBD genes in orange, and TRBV genes in red. (C) Zoomed view (32.03–32.9 Mbp) of the TRB cluster A, showing detailed gene organization. (D) Close-up of the TRBC-J-D functional genes. (E) Close-up view of the TRBC_003 gene, which includes two Cβ-domain exons. (F) Spleen RNA-seq coverage histogram of the TRBC-J-D region, showing transcriptional activity. (G) Spleen RNA-seq coverage histogram of the TRBC-C2-J-D region, indicating transcription levels in this area. Note that in the E and F panels the color intensity shifts from blue to red whith read counts increasing in the spleen transcriptome.
	Figure 5. Absence of the TRG locus in the Ambystoma mexicanum genome (UKYF1_1). Schematic representation illustrating the absence of the TRG locus. In humans, the TRG locus is located on chromosome 7 (36.4–50.1 Mb), while in Xenopus tropicalis, it is located on chromosome 6 (61.99–63.99 Mb). The V cluster is shown in red, the J cluster in yellow, and the constant (C) region in blue, regardless of functionality. Non-TR genes are depicted in black. In A. mexicanum, analysis of chromosome 5p (834.5–863.2 Mb) reveals a complete absence of the TRG locus. However, strong synteny is observed with the genomic neighborhood found in both humans and X. tropicalis. The proximal flanking genes include AMPH, POU6F2, NPSR1, BMPER, BBS9, NT5C3A, RP9, and VELO1, while the distal flanking genes are STARD3NL, EPDR1, SFP4, GPR141, ELMO1, AOAH, ANLN, and MATCAP2. Notably, the orientation of these flanking genes is inverted in X. tropicalis compared to A. mexicanum and humans, which share not only the same gene content but also a conserved gene orientation. This suggests that, despite the loss of the TRG locus in A. mexicanum, the surrounding genomic architecture remains highly conserved.
	Figure 6. Absence of the PTCRA locus in the Ambystoma mexicanum genome (UKYF1_1). Schematic representation of the PTCRA locus across representative genomes. Black arrows indicate syntenic genes, white arrows represent non-syntenic genes, and red arrows depict the PTCRA gene ortholog. In mammals (Homo sapiens and Monodelphis domestica), there is perfect synteny in the locus. In sauropsids, such as Gallus gallus, Taeniopygia castanotis (chicken and zebra finch) loci are syntenic; in Anolis carolinensis (lizard), only POLR1B, TLL upstream and downstream CNPY3, GNMT, PEX6, and CUL7 are conserved. In X. tropicalis synteny is observed but there is no evidence of PTCRA. In the case of A. mexicanum, we identified all neighboring genes of the PTCRA locus; however, a rearrangement of the entire chromosome is observed, leading to the absence of PTCRA.
	Figure 1. The TRA-TRD locus in Ambystoma mexicanum is located in the centromeric portion of chr13p (20.7Mbp). (A) Gene density plot of chromosome 13p, where the TRA-TRD cluster is highligted (box); dark blue colors indicate low gene density. (B) Overview of the whole TRA-TRD locus (264.6-285.3 Mbp), showing non-TR genes (black) in proximal flank, TRAC and TRDC genes (blue), TRAJ and TRDJ genes (yellow), and TRAV and TRDV genes (red). (C) Close-up of the TRDC-J gene cluster (267.8-280.7 Mbp). (D) Detailed view of theTRAJ cluster (281-281.2 Mbp). (E) Spleen RNA-seq coverage histogram of the TRDC-J region. (F) Spleen RNA-seq coverage histogram of the TRAC-J region. Note that in the E and F panels the color intensity shifts from blue to red whith read counts increasing in the spleen transcriptome.
	Figure 2. Synteny of the TRA-TRD locus in Ambystoma mexicanum compared to other tetrapods. Schematic representation of TRA-TRD locus in human (Homo sapiens, GRCh38.p14), mouse (Mus musculus, GRCm39), opossum (Monodelphis domestica, ASM229v1), frog (Xenopus tropicalis, UCB_Xtro_10.0), and axolotl (A. mexicanum, UKYF1_1) are shown. Solid-filled symbols represent the TRA locus, while open symbols correspond to the TRD locus. Constant regions are indicated in blue, J gene cluster is depicted in dark yellow, and V clusters are highlighted in red. D genes are displayed as green rectangles, and non-TR genes are depicted in black. Interestingly, in the axolotl, the IGHC locus (purple) is not linked to the TRAD cluster as observed in X. tropicalis and spans across the centromere (depicted as a gray circle). The figures are not to scale, and the same scheme is applied to all species for consistency. Gene orientation in mammals and axolotl is 5’-3’and in X. tropicalis is 3’-5’.
	Figure 3. Synteny of the TRB locus in Ambystoma mexicanum compared to other tetrapods. Schematic representation of the TRB locus in human (Homo sapiens, GRCh38.p14), mouse (Mus musculus, GRCm39), opossum (Monodelphis domestica, ASM229v1), frog (Xenopus tropicalis, UCB_Xtro_10.0), and axolotl (A. mexicanum, UKYF1_1). The TRBC genes (constant regions) are shown in blue, the TRBJ cluster in yellow, and the TRBV cluster in red. TRBD genes are depicted in green, while non-TR genes are represented in black. The figure is not to scale, and the color scheme is consistent across all species for clarity. Gene orientation in mammals is 5’-3’and in amphibians is 3’-5’.
	Figure 4. The TRB locus of Ambystoma mexicanum is located in the telomeric portion of chromosome 3p (4.82 Mbp). (A) Gene density plot of chromosome 3, where the TRB locus is encoded (highlighted with a box). Dark blue regions indicate areas of low gene density. (B) Overview of the TRB locus (30.03-34.85 Mbp), showing non-TR genes in black. Proximal and distal flanking genes include TRPV and PRSS, respectively.Gaps are represented in gray. TRBC genes are shown in blue, TRBJ genes in yellow, TRBD genes in orange, and TRBV genes in red. (C) Zoomed view (32.03–32.9 Mbp) of the TRB cluster A, showing detailed gene organization. (D) Close-up of the TRBC-J-D functional genes. (E) Close-up view of the TRBC_003 gene, which includes two Cβ-domain exons. (F) Spleen RNA-seq coverage histogram of the TRBC-J-D region, showing transcriptional activity. (G) Spleen RNA-seq coverage histogram of the TRBC-C2-J-D region, indicating transcription levels in this area. Note that in the E and F panels the color intensity shifts from blue to red whith read counts increasing in the spleen transcriptome.
	Figure 5. Absence of the TRG locus in the Ambystoma mexicanum genome (UKYF1_1). Schematic representation illustrating the absence of the TRG locus. In humans, the TRG locus is located on chromosome 7 (36.4–50.1 Mb), while in Xenopus tropicalis, it is located on chromosome 6 (61.99–63.99 Mb). The V cluster is shown in red, the J cluster in yellow, and the constant (C) region in blue, regardless of functionality. Non-TR genes are depicted in black. In A. mexicanum, analysis of chromosome 5p (834.5–863.2 Mb) reveals a complete absence of the TRG locus. However, strong synteny is observed with the genomic neighborhood found in both humans and X. tropicalis. The proximal flanking genes include AMPH, POU6F2, NPSR1, BMPER, BBS9, NT5C3A, RP9, and VELO1, while the distal flanking genes are STARD3NL, EPDR1, SFP4, GPR141, ELMO1, AOAH, ANLN, and MATCAP2. Notably, the orientation of these flanking genes is inverted in X. tropicalis compared to A. mexicanum and humans, which share not only the same gene content but also a conserved gene orientation. This suggests that, despite the loss of the TRG locus in A. mexicanum, the surrounding genomic architecture remains highly conserved.
	Figure 6. Absence of the PTCRA locus in the Ambystoma mexicanum genome (UKYF1_1). Schematic representation of the PTCRA locus across representative genomes. Black arrows indicate syntenic genes, white arrows represent non-syntenic genes, and red arrows depict the PTCRA gene ortholog. In mammals (Homo sapiens and Monodelphis domestica), there is perfect synteny in the locus. In sauropsids, such as Gallus gallus, Taeniopygia castanotis (chicken and zebra finch) loci are syntenic; in Anolis carolinensis (lizard), only POLR1B, TLL upstream and downstream CNPY3, GNMT, PEX6, and CUL7 are conserved. In X. tropicalis synteny is observed but there is no evidence of PTCRA. In the case of A. mexicanum, we identified all neighboring genes of the PTCRA locus; however, a rearrangement of the entire chromosome is observed, leading to the absence of PTCRA.