ABSTRACT: Single cell RNA sequencing (scRNA seq) is a powerful approach for dissecting cellular heterogeneity in both human and veterinary medicine. Although prior canine studies have profiled peripheral leukocytes at the single-cell level, the accurate identification, functional characterization, and clinical relevance of specific immune subsets remain underexplored. Here, we aimed to generate a comprehensive single cell atlas of healthy canine peripheral blood leukocytes and to uncover the molecular mechanisms that underpin their homeostasis. We performed scRNA seq on peripheral leukocytes from six clinically healthy, small breed adult dogs using the 10x Genomics Chromium platform. Sequencing reads were aligned and annotated on canFam4, and data were processed in Seurat (v.5.1.0) with standard quality control, normalization, and integration workflows. Downstream bioinformatic and statistical analyses were conducted in RStudio (v.4.2.0). After filtering, we obtained 30,040 high quality single cell transcriptomes, which clustered into 51 distinct immune subsets. Leveraging canFam4 improved recovery of single-cell and key lineage and functional markers, such as CD14⁺CD33+ monocytes, XCR1⁺CD1D⁺ dendritic cells, CEACAM1+CD24⁺ neutrophils, and IL32⁺BATF⁺ regulatory T cells previously underrepresented in canFam3.1 based studies. We also identified interferon enriched CD14⁺ monocyte and CD4⁺ T subsets implicated in canine myxomatous mitral valve disease. Analysis of CD274⁺ myeloid and PDCD1⁺ T cell clusters provided evidence roles for IL 10 signaling, malate metabolism (MDH1/MDH2), and viral response pathways (LAG3, CD47, TBX21) in maintaining homeostasis. Finally, cohort specific differences in T cell exhaustion scores and proliferative (cycling) T cell fractions pointed to variable antigenic exposures. The single cell transcriptomic landscape of circulating canine leukocytes unveiled novel, clinically relevant immune subsets and the molecular pathways that maintain immune homeostasis. Our dataset and analytical framework represent valuable resources for translational research, particularly in cancer immunology.
