Introduction: The domestic cat (Felis catus) is a valued companion animal and the second most popular pet (over 46.5 million US households). They are also an important model system for virally-induced cancers (feline leukemia virus) and virally-mediated immunodeficiency (feline immunodeficiency virus). However, species specific research limitations such as a lack of reagents and immune cell markers limit our ability to utilize these models to their full capacity. The goal of this study is to characterize the transcriptomic landscape of circulating feline T cells and other captured leukocytes utilizing CD5 (only available selective feline T cell marker) flow cytometry enriched single cell RNA-sequencing and V(D)J repertoire analysis in clinically healthy domestic shorthair cats between the ages of 6 months and 9 years as well as contextualize them with the leukocytes of other species. Methods: 5 mL of peripheral whole blood was collected from 4 healthy cats (6 months, 1 year, 4 years, 9 years) and were housed at the UC Davis Nutrition and Pet Care Center. Samples were enriched for T cells by flow cytometry using a mouse anti-feline CD5 monoclonal antibody. The GEX library was constructed using the Chromium Next GEM Single Cell 5’ Kit v2 (10x Genomics). The V(D)J library for all 4 T cell receptor loci was prepared using the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Sequencing was done via Illumina NovaSeq S4 and pre-processed using the Cell Ranger for alignment to the feline genome (felis_catus_9.0). Processing and downstream analyses were conducted via Seurat v5. Additional annotated datasets for cross species T cell integration were acquired from peer-reviewed literature. Results: Unsupervised clustering of GEX data revealed 7 major populations – T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved different populations of naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells for the first time. Cross species analysis revealed relatively high conservation of T cell subtypes along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated marked differences in CD8+ cytotoxic T cells from other alpha/beta T cell subsets including productive TRG transcript expression. Among the myeloid cells, we resolved 3 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. Discussion: Our study is the first to characterize the subtypes of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species integration. Additionally, we also characterize subtypes in the myeloid cells expanding our understanding of the feline immune system. We have also demonstrated species immune cell relatedness which can provide an important foundation for further translational immunology, pathogenesis, translational treatments, and species-tropism of pathogens in veterinary medicine and research.
Overall design: Flow sort of PBMCs with CD5+ enrichment from 4 healthy domestic shorthair cats followed by 5' scRNAseq with V(D)J analysis (ab TCR & gd TCR)
Less...