Healthy human enthesis stromal cells mediate immunoregulation via the CD39/CD73 adenosine ectonucleotidase pathway

OBJECTIVES: Entheseal inflammation (ligament and tendon insertions) and subclinical intestinal inflammation are both hallmarks of the seronegative spondyloarthropathy (SpA) diseases. While regulatory T cells (Tregs) are key to intestinal homeostasis, entheseal homeostatic mechanisms are poorly understood in humans. METHODS: Single-cell RNA sequencing of entheseal tissue, comparative transcriptomic analysis with intestinal tissue datasets, and functional analysis of entheseal stromal populations were undertaken. Functional immunomodulation by entheseal mesenchymal stromal cells (MSCs) was evaluated via coculture with activated T cells. CD39/CD73 pathway involvement was confirmed using pharmacological inhibition and transcriptional profiling. RESULTS: Single-cell RNA sequencing of 27,348 entheseal cells revealed a lower frequency of Tregs in the T cells of the enthesis (2.60% ± 0.36%) compared to those of the ileum (7.37% ± 4.47%) and colon (18% ± 8.59%). We found that entheseal fibroblasts expressed key immunomodulatory markers, including CD39 (ENTPD1) and CD73 (NT5E), which were further upregulated upon coculture with activated T cells. Entheseal MSCs significantly suppressed T cell proliferation (up to 89%, P < .0001) in an adenosine-dependent manner. Transcriptional profiling revealed that entheseal MSC cocultured with activated T cells upregulated genes of known functional importance in Treg, including IL10, IDO1, PTGS2, HLA-G, and CD274. In this assay, dual CD39/CD73 inhibition restored T cell proliferation by ∼48% (P = .0004), confirming that entheseal MSC-mediated immunomodulation acts in part through an adenosine-mediated mechanism. CONCLUSIONS: The normal spinal enthesis harbours an immunoregulatory cellular environment related to entheseal MSCs that utilises the CD39/CD73 adenosine ectonucleotidase axis that may help maintain local immune homeostasis, while the same adenosine ectonucleotidase immunoregulatory pathway is likely dependent on Treg function in the intestine. This has broad implications for understanding the cellular bases of immune dysregulation in the gut-joint axis and could help guide tissue-specific therapy in SpA.