High-resolution single-cell RNA sequencing using canFam4 reveals novel immune subsets and checkpoint programs in healthy dogs

Single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of immune heterogeneity. Although previous studies have mapped the single-cell transcriptomic atlases of peripheral leukocytes in healthy dogs, the identification and functional characterization of distinct immune subsets remain incomplete. We constructed a single-cell atlas of peripheral leukocytes from six healthy small-breed dogs using the 10x Genomics platform and the updated canFam4 genome. Analysis of 30,040 high-quality transcriptomes revealed 51 distinct immune subsets, including CD14{square}CD33{square} monocytes, XCR1{square}CD1D{square} dendritic cells, CEACAM1{square}CD24{square} neutrophils, and IL32{square}BATF{square} regulatory T cells, which were underrepresented in canFam3.1-based studies. Interferon-enriched CD14{square} monocytes and CD4{square} T subsets associated with myxomatous mitral valve disease were also identified. Functional analysis revealed that PDCD1 attenuates TCR signaling, LAG3 modulates malate metabolism in CD4{square} T cells, and suppresses TBX21 in CD8{square} T cells associated with viral response. CD274 encoding PD-L1 was linked to IL-10 production in neutrophils, and CTLA4 represented an initial activation of double-negative T subsets. T cell exhaustion scores and proliferative fractions varied across cohorts, reflecting differences in environmental antigenic exposures. Our study represents the first comprehensive, gene-resolved single-cell analysis that reveals immunoregulatory checkpoint mechanisms underlying immune homeostasis in healthy dogs. Our dataset will serve as a valuable resource for future comparative and translational immunology research in dogs.