Atypical TCR delta found in sharks, amphibians, birds, and monotremes and TCR mu found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VH delta/V mu) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCR alpha/delta gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 J alpha, nine J delta, eight D delta, two C alpha, and five C delta gene segments in the TCR alpha/delta loci of the Chinese alligator, representing the most complicated TCR alpha/delta gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VH delta genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VH delta genes could be related to bird VH delta genes, VH delta/V mu genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCR delta/TCR mu genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.

AbstractAtypical TCRδ found in sharks, amphibians, birds, and monotremes and TCRμ found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VHδ/Vμ) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCRα/δ gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 Jα, nine Jδ, eight Dδ, two Cα, and five Cδ gene segments in the TCRα/δ loci of the Chinese alligator, representing the most complicated TCRα/δ gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VHδ genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VHδ genes could be related to bird VHδ genes, VHδ/Vμ genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCRδ/TCRμ genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.

All jawed vertebrates have four T cell receptor (TCR) chains that are expressed by thymus-derived lymphocytes and play a major role in animal immune defence. However, few studies have investigated the TCR chains of crocodilians compared with those of birds and mammals, despite their key evolutionary position linking amphibians, reptiles, birds and mammals. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization, evolution and expression of TRB and TRG loci in Alligator sinensis. According to the sequencing data, the Alligator sinensis TRB locus spans approximately 500 Kb of genomic DNA containing two D-J-C clusters and 43 V gene segments and is organized as V beta((39))-pJ beta 1-pC beta 1-pD beta 1-D beta 2- J beta 2((12))-C beta 2-V beta((4)), whereas the TRG locus spans 115 Kb of DNA genomic sequence consisting of 18 V gene segments, nine J gene segments and one C gene segment and is organized in a classical translocon pattern as V gamma((18)) -J gamma((9))-C gamma. Moreover, syntenic analysis of TRB and TRG chain loci suggested a high degree of conserved synteny in the genomic regions across mammals, birds and Alligator sinensis. By analysing the cloned TRB/TRG cDNA, we identified the usage pattern of V families in the expressed TRB and TRG. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed TRB and TRG sequences. Phylogenetic analysis revealed that TRB and TRG loci possess distinct evolutionary patterns. Most Alligator sinensis V subgroups have closely related orthologues in chicken and duck, and a small number of Alligator sinensis V subgroups have orthologues in mammals, which supports the hypothesis that crocodiles are the closest relatives of birds and mammals. Collectively, these data provide insights into TCR gene evolution in vertebrates and improve our understanding of the Alligator sinensis immune system. (C) 2018 Elsevier Ltd. All rights reserved.

Crocodilians are regarded as possessing a powerful immune system. However, the composition and action of the crocodilian immune system have remained unclear until now. Cathelicidins, the principal family of host defense peptides, play pivotal roles in vertebrate immune defense against microbial invasions. However, cathelicidins from crocodilians have not been extensively studied to date. In the present study, six novel cathelicidins (As-CATH1–6) were identified and characterized from the endangered Chinese alligator (Alligator sinensis). As-CATH1–6 exhibit no sequence similarity with any of the known cathelicidins. Structure analysis indicated that As-CATH1–3 adopt a random coil secondary conformation, whereas As-CATH4–6 were predicted to mainly adopt an amphipathic α-helix conformation. Among them, As-CATH4–6 exhibited potent, broad-spectrum and rapid antimicrobial activity by inducing the disruption of cell membrane integrity. They also exhibited strong ability to prevent the formation of bacterial biofilms and eradicate preformed biofilms. Furthermore, As-CATH4–6 exhibited potent anti-inflammatory activity by inhibiting the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and pro-inflammatory cytokines in mouse peritoneal macrophages. They directly neutralized LPS toxicity and therefore inhibited the binding of LPS to the TLR4 receptor and the subsequent activation of inflammatory response pathways. In a peritonitis mice model, As-CATH2–6 provided effective protection against bacterial infection through enhanced immune cell recruitment. In the host Chinese alligator, As-CATH1–6 are mainly expressed in immune organs and epithelial tissues. Bacterial infection significantly enhances their expression, which implies an important role in host anti-infective response. Taken together, the diversity and multiple functions of As-CATH1–6 partially reveal the powerful immune system of the Chinese alligator.