BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

Department of Zoology University of Oxford Oxford UK

The Pirbright Institute Ash Road Pirbright Woking Surrey United Kingdom

Veterinary Faculty Ludwig Maximillians University Planegg Germany

Veterinary Research Institute Brno Czech Republic

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$a Dixon, Robert $u Department of Zoology, University of Oxford, Oxford, UK

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$a Repertoire analysis of γδ T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires / $c R. Dixon, SG. Preston, S. Dascalu, PG. Flammer, SR. Fiddaman, K. McLoughlin, A. Boyd, J. Volf, I. Rychlik, MB. Bonsall, B. Kaspers, AL. Smith

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$a BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

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$a Preston, Stephen G $u Department of Zoology, University of Oxford, Oxford, UK

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$a Dascalu, Stefan $u Department of Zoology, University of Oxford, Oxford, UK $u The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United Kingdom

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$a Flammer, Patrik G $u Department of Zoology, University of Oxford, Oxford, UK

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$a Fiddaman, Steven R $u Department of Zoology, University of Oxford, Oxford, UK

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$a McLoughlin, Kirstie $u Department of Zoology, University of Oxford, Oxford, UK

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$a Boyd, Amy $u Department of Zoology, University of Oxford, Oxford, UK

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$a Volf, Jiri $u Veterinary Research Institute, Brno, Czech Republic

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$a Rychlik, Ivan $u Veterinary Research Institute, Brno, Czech Republic

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$a Bonsall, Michael B $u Department of Zoology, University of Oxford, Oxford, UK

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$a Kaspers, Bernd $u Veterinary Faculty, Ludwig Maximillians University, Planegg, Germany

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$a Smith, Adrian L $u Department of Zoology, University of Oxford, Oxford, UK. adrian.smith@zoo.ox.ac.uk

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