Investigating t cell recruitment in atherosclerosis using a novel human 3D tissue-culture model reveals the role of CXCL12 in intraplaque neovessels

Background Development of effective treatments for atherosclerosis requires new models that better predict the human immune response. Although T cells are abundant in human atherosclerotic lesions and play a key role in the pathogenesis, the mechanism involved in plaque infiltration remains ill defined. Methods We developed a three-dimensional tissue-culture model to study leukocyte recruitment to human atherosclerotic plaques. In this study, human atherosclerotic plaques obtained during carotid endarterectomy surgery were co-cultured with patient-matching T cells. Exogenous T cells were stained using a multi-factor staining strategy, which involved intracellular fluorescent cell tracker dyes combined with nuclear labels. Flow cytometry was used to assess the presence of the labeled cells within the plaques, and microscopic analysis was performed to examine their localization. Results Flow cytometry and microscopy cell-tracking analysis demonstrated that exogenous T cells successfully migrated into atherosclerotic plaques. Furthermore, infiltrated CD8+ T cells displayed a significant increase of CD69 expression, indicating their activation within the tissue. Blocking chemokine receptors, particularly CXCR4, significantly impaired T cell infiltration, demonstrating that exogenous CD8+ T cells invade plaques through chemotactic migration. Surprisingly, 3D microscopy combined with optical tissue clearing strategy revealed that CXCL12, the sole ligand of CXCR4, mainly accumulated in intraplaque neovessels. Single-cell RNA sequencing (scRNAseq) analysis further confirmed that endothelial cells from intraplaque neovessels were the primary source for CXCL12. Additionally, exogenous T cells were found within and in proximity to these neovessels, suggesting that the CXCL12/CXCR4 axis regulates T cell recruitment through intraplaque neovessels. Conclusions Overall, these findings shed new light on the mechanism of action of CXCL12 in atherosclerosis and demonstrated the potential of the model to advance our understanding of leukocyte accumulation in human atherosclerosis and assist in testing novel pharmacological therapies. ### Competing Interest Statement The authors have declared no competing interest.