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

[INTRODUCTION] 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.

[METHODS] 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.

[RESULTS AND DISCUSSION] Analysis of 30,040 high-quality transcriptomes revealed 51 distinct immune subsets, including monocytes, dendritic cells, neutrophils, and regulatory T cells, which were underrepresented in canFam3.1-based studies. Interferon-enriched monocytes and T subsets associated with myxomatous mitral valve disease were also identified. Functional enrichment analyses suggested that is associated with attenuated TCR signaling, whereas was associated with malate metabolism pathways in T cells and reduced expression in T cells linked to antiviral responses. , which encodes PD-L1 was linked to IL-10 production in neutrophils, and 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.

[CONCLUSION] To our knowledge, this study represents the first comprehensive, gene-resolved single-cell analysis to reveal 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.