Characterization of the chicken T cell receptor γ repertoire by high-throughput sequencing

Abstract

Background As one of “γδ-high” species, chicken is an excellent model for the study of γδ T cells in non-mammalian animals. However, a comprehensive characterization of the TCRγδ repertoire is still missing in chicken. The objective of this study was to characterize the expressed TCRγ repertoire in chicken thymus using high-throughput sequencing. Methods In this study, we first obtained the detailed genomic organization of the TCRγ locus of chicken based on the latest assembly of the red jungle fowl genome sequences (GRCg6a) and then characterized the TCRγ repertoire in the thymus of four chickens by using 5′ Rapid Amplification of cDNA Ends (5′ RACE) along with high-throughput sequencing (HTS). Results The chicken TCRγ locus contains a single Cγ gene, three functional Jγ segments and 44 Vγ segments that could be classified into six subgroups, each containing six, nineteen, nine, four, three and three members. Dot-plot analysis of the chicken TCRγ locus against itself showed that almost all the entire zone containing Vγ segments had arisen through tandem duplication events, and the main homology unit, containing 9 or 10 Vγ gene segments, has tandemly duplicated for four times. For the analysis of chicken TCRγ repertoire, more than 100,000 unique Vγ-region nucleotide sequences were obtained from the thymus of each chicken. After alignment to the germline Vγ and Jγ segments identified above, we found that the four chickens had similar repertoire profile of TCRγ. In brief, four Vγ segments (including Vγ3.7, Vγ2.13, Vγ1.6 and Vγ1.3) and six Vγ-Jγ pairs (including Vγ3.7-Jγ3, Vγ2.13-Jγ1, Vγ2.13-Jγ3, Vγ1.6-Jγ3, Vγ3.7-Jγ1 and Vγ1.6-Jγ1) were preferentially utilized by all four individuals, and vast majority of the unique CDR3γ sequences encoded 4 to 22 amino acids with mean 12.90 amino acids, which exhibits a wider length distribution and/or a longer mean length than CDR3γ of human, mice and other animal species. Conclusions In this study, we present the first in-depth characterization of the TCRγ repertoire in chicken thymus. We believe that these data will facilitate the studies of adaptive immunology in birds.

Unraveling the chicken T cell repertoire with enhanced genome annotation

“…Previous work has identified V(D)J and C genes in the chicken genome that are arranged in highly structured clusters: The TCR β locus (TRB) spans a region of approximately 210 kilobases (kb) on chromosome 1 ( 16 – 20 ), the TCR γ locus (TRG) spans a region of approximately 82 kb on chromosome 2 ( 19 , 21 – 23 ), and TCR α (TRA) and TCR δ (TRD) genes are arranged in a hybrid locus of approximately 800 kb on chromosome 27, with the TCR δ sequences nested between the Vα and Jα genes ( 13 , 14 , 19 , 24 ). In addition, a separate small TCR δ-like locus was identified on chromosome 10 comprised of a single cassette with one copy of immunoglobulin heavy chain (IgH) V-like VHδ, Dδ, Jδ and Cδ genes ( 25 ).…”

“…Furthermore, the challenge of comparability has been exacerbated by instances where the annotated sequences were not universally accessible in the public domain ( 14 , 19 ). Recent studies have provided robust annotations of the TCR β and TCR γ loci ( 20 , 22 , 23 ). However, comprehensive details regarding the TCR α/δ sequences are still unavailable.…”

“…TCR profiling enables the characterization of T cell diversity at baseline and facilitates quantification of clonal expansion and memory recall responses to microbial challenges ( 27 , 28 ). Current research endeavors have expanded this approach to chicken samples, offering a comprehensive analysis of private and public β and γ T cell populations across various tissues and microbial conditions ( 22 , 23 , 29 ). However, current knowledge about chicken α and δ chain sequences remains incomplete.…”

Correction to: Characterization of the chicken T cell receptor γ repertoire by high-throughput sequencing

“…Following publication of the original article [ 1 ], an error was identified in the Methods section.…”

“…All the changes requested are implemented in this correction and the original article [ 1 ] has been corrected.…”