BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.
- MeSH
- adaptivní imunita genetika MeSH
- fylogeneze MeSH
- genetické lokusy genetika MeSH
- genomika metody MeSH
- kočky genetika imunologie MeSH
- lidé MeSH
- lymfoidní tkáň metabolismus MeSH
- receptory antigenů T-buněk klasifikace genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- kočky genetika imunologie MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Evolution of indolent to aggressive lymphoma has been described in dogs but is difficult to distinguish from the de novo development of a second, clonally distinct lymphoma. Differentiation of these scenarios can be aided by next generation sequencing (NGS)-based assessment of clonality of lymphocyte antigen receptor genes. CASE PRESENTATION: An 8-year-old male intact Mastiff presented with generalized lymphadenomegaly was diagnosed with nodal T zone lymphoma (TZL) based on cytology, histopathology, immunohistochemistry and flow cytometry. Thirteen months later, the dog re-presented with progressive lymphadenomegaly, and based on cytology and flow cytometry, a large B cell lymphoma (LBCL) was diagnosed. Sequencing-based clonality testing confirmed the de novo development of a LBCL and the persistence of a TZL. CONCLUSIONS: The occurrence of two distinct lymphoid neoplasms should be considered if patient features and tumor cytomorphology or immunophenotype differ among sequential samples. Sequencing-based clonality testing may provide conclusive evidence of two concurrent and distinct clonal lymphocyte populations, termed most appropriately "composite lymphoma".
- MeSH
- antitumorózní látky alkylující aplikace a dávkování terapeutické užití MeSH
- antitumorózní látky hormonální aplikace a dávkování terapeutické užití MeSH
- chlorambucil aplikace a dávkování terapeutické užití MeSH
- difúzní velkobuněčný B-lymfom komplikace patologie veterinární MeSH
- lymfom T-buněčný komplikace patologie veterinární MeSH
- nemoci psů patologie MeSH
- prednison aplikace a dávkování terapeutické užití MeSH
- psi MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80-90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.
- MeSH
- B-lymfocyty cytologie MeSH
- buněčné klony * MeSH
- DNA primery * MeSH
- psi genetika imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- zvířata MeSH
- Check Tag
- psi genetika imunologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The ability to mount adaptive immune responses to a diverse array of pathogens is essential to maintaining the health of an individual. The outcome of adaptive immune responses is influenced by the pool of available lymphocyte antigen receptors. Understanding the composition and dynamics of immune repertoires is hence of relevance to characterizing physiologic immunological processes as well as understanding disease pathogenesis. The dog is increasingly recognized as a model for human disease. The objective of this study was to utilize NGS for comprehensive and unbiased analysis of the IGH repertoire in healthy dogs. First, the IGH locus was searched in silico for previously unidentified genes. Second, IGH transcripts from major lymphoid organs were amplified using a 5'RACE approach without V/J primer bias. Third, amplicons were sequenced on an Illumina MiSeq platform, and data were analyzed using the ARResT/Interrogate platform. Data analysis included V/J usage, V-J pairing biases, isotype frequency, CDR3 diversity, convergent recombination, and public repertoires. The results of this study provide a comprehensive IGH repertoire analysis for healthy dogs. These data will allow further improvement of V/J gene-specific primer sets and will serve as baseline for future studies investigating immune repertoires in health and disease.
- MeSH
- imunoglobulinové izotypy genetika MeSH
- psi MeSH
- receptory antigenů genetika MeSH
- sekvenční analýza DNA MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
