Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes
IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal...
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Frontiers Media S.A.
2024-11-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1438004/full |
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| author | Raneesh Ramarapu Raneesh Ramarapu Judit M. Wulcan Haiyang Chang Peter F. Moore William Vernau Stefan M. Keller |
| author_facet | Raneesh Ramarapu Raneesh Ramarapu Judit M. Wulcan Haiyang Chang Peter F. Moore William Vernau Stefan M. Keller |
| author_sort | Raneesh Ramarapu |
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| description | IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species. |
| format | Article |
| id | doaj-art-f0b91dfbbe624e66a4b88a1a10bd40f1 |
| institution | Kabale University |
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| language | English |
| publishDate | 2024-11-01 |
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| series | Frontiers in Immunology |
| spelling | doaj-art-f0b91dfbbe624e66a4b88a1a10bd40f12024-11-15T04:36:40ZengFrontiers Media S.A.Frontiers in Immunology1664-32242024-11-011510.3389/fimmu.2024.14380041438004Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytesRaneesh Ramarapu0Raneesh Ramarapu1Judit M. Wulcan2Haiyang Chang3Peter F. Moore4William Vernau5Stefan M. Keller6Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesDepartment of Mathematics and Statistics, University of Guelph, Guelph, ON, CanadaDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United StatesIntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.https://www.frontiersin.org/articles/10.3389/fimmu.2024.1438004/fullfelineT cellssingle cell RNA-sequencing (scRNA-seq)T-cell receptor repertoirecross species analysismyeloid Cells |
| spellingShingle | Raneesh Ramarapu Raneesh Ramarapu Judit M. Wulcan Haiyang Chang Peter F. Moore William Vernau Stefan M. Keller Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes Frontiers in Immunology feline T cells single cell RNA-sequencing (scRNA-seq) T-cell receptor repertoire cross species analysis myeloid Cells |
| title | Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes |
| title_full | Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes |
| title_fullStr | Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes |
| title_full_unstemmed | Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes |
| title_short | Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes |
| title_sort | single cell rna sequencing of feline peripheral immune cells with v d j repertoire and cross species analysis of t lymphocytes |
| topic | feline T cells single cell RNA-sequencing (scRNA-seq) T-cell receptor repertoire cross species analysis myeloid Cells |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1438004/full |
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