Differential Immune Cell Distribution Between Arterial and Venous Blood in Healthy Rats

We previously identified differentially expressed miRNAs between arterial and venous blood in rats; however, whether immune cell composition and transcriptional states differ along the arterial-venous axis remains unclear. Here, single-cell RNA sequencing of 119 481 PBMCs, complemented by flow cytometry and protein-level validation, was performed to characterize immune heterogeneity between arterial and venous blood. Cell composition analysis showed enrichment of T cells in arterial blood, whereas B cells, NK cells, and monocytes were more abundant in venous blood. At the subset level, naïve T cells were enriched in arterial blood, while CD8⁺ effector memory T cells were increased in venous blood. NK cell analysis demonstrated enrichment of cytotoxic NK subsets in venous blood and resting NK subsets in arterial blood, whereas monocyte subset proportions were comparable between compartments. Transcriptomic analysis revealed reduced JAK-STAT signaling in venous PBMCs, consistent with decreased STAT3 and NF-κB phosphorylation. Subset-specific analysis further showed reduced TNF/NF-κB signaling and enrichment of ribosome-associated pathways in venous T cells, enhanced cytotoxic, chemokine, and NF-κB-related programs in venous NK cells, and attenuated TNF/NF-κB signaling with increased ribosome-associated activity in monocytes, indicating functional reprogramming without compositional changes. Collectively, these findings demonstrate coordinated but cell type-specific immune adaptations along the arterial-venous axis, identifying blood sampling site as a critical determinant of immune readouts and a previously underappreciated source of variability in immunological studies.