Background: The enthesis is a critical target tissue in spondyloarthritis (SpA), characterised by inflammation and post-inflammatory osteogenesis [1]. Multipotential stromal mesenchymal stem cells (MSCs) with multilineage differentiation capacity, including stromal immune regulation, may play a crucial role in immune regulation and tissue repair at the enthesis. Although our prior work demonstrated that entheseal MSCs regulate T cell activation and proliferation via the CD39/CD73 ectonucleotidase pathway [2]. The effect of MSCs on T-cell IL-17 and TNF cytokine regulation, central to SpA pathogenesis, remains unclear.

Objectives: To investigate the immunomodulatory effects of MSCs on CD4 ⁺ CD25 ⁻ T cells, focusing on their influence on IL-17A, IL-17F, TNF and other cytokines production under co-culture conditions.

Methods: MSCs were isolated from healthy soft tissue enthesis (spinous processes and interspinous ligament regions) and bone marrow aspirates (BM-MSCs). Cells were expanded to passages 3–5 and co-cultured with CD4 ⁺ CD25 ⁻ T cells stimulated with CD3/CD28 beads at a 1:1 ratio for 5 days (n=5). T cell proliferation was assessed via CFSE staining, while IL-17A and IL-17F levels were quantified using intracellular staining and ELISA (n=7). TNF-α, IFN-γ, and IL-22 cytokines were measured using LegendPlex. RNA-seq was performed on stromal MSCs post-co-culture to identify differentially expressed genes and pathways (n=3). Additional RNA-seq experiments analysed MSCs exposed to IL-17A + TNF versus TNF alone for 48 hours (n=3).

Results: MSC co-culture suppressed T cell proliferation by 80% (P < 0.0001), demonstrating their strong immunosuppressive capacity. This suppression was accompanied by significant reductions in TNF-α, IFN-γ, and IL-22 levels (P < 0.001). However, a paradoxical effect was observed, with IL-17A production significantly upregulated in co-cultured T cells (P < 0.0001), while IL-17F levels were markedly reduced (P = 0.0014). Flow cytometry revealed an increase in IL-17A-positive T cells in the co-culture condition compared to non-stimulated controls, whereas IL-17F-positive T cells were reduced compared to stimulated controls. This cytokine profile was also evident in iliac crest bone marrow-MSCs were co-cultured with T cells, suggesting the phenomenon is not specific to entheseal MSCs. RNA-seq analysis of MSCs post-co-culture with T-cell supernatants identified 1,234 differentially expressed genes (FDR < 0.05). Key upregulated genes included IL1B (log2 FC = 9.00, Padj = 1.69 × 10 ⁻¹⁵ ) and IL6 (log2 FC = 6.48, Padj = 1.38 × 10 ⁻⁷ ), both critical for Th17 differentiation and IL-17A production. Elevated PD-L1 (CD274) and IDO1 expression suggested an immunosuppressive microenvironment favouring selective cytokine expression. Additionally, NFKBIZ (log2 FC = 3.20, Padj = 8.60 × 10 ⁻⁶ ), a regulator of NF-κB signalling, was significantly upregulated, highlighting its role in IL-17A transcription. RNA-seq of MSCs exposed to IL-17A + TNF versus TNF alone reinforced these findings, with consistent NFKBIZ upregulation and significant CXCL3 expression, indicating that IL-17A amplifies a pro-inflammatory microenvironment. Pathway analysis of MSC revealed enrichment in T cell receptor signalling (NES = 2.48, P < 0.0001) and cytokine-mediated signalling, with upregulation of CCL20, which facilitates Th17 cell recruitment.

Conclusion: Despite potent suppression of T cell proliferation, MSCs promote selective chemokine and cytokine signalling that enhances IL-17A production via NF-κB-driven pathways, while reducing IL-17F. These findings highlight the dual immunosuppressive and potential pro-inflammatory or regulatory aspect of IL-17A for entheseal MSCs- T-cell cross talk in tissue homeostasis in SpA.

REFERENCES: [1] Benjamin, M. et al., Scand J Med Sci Sports, 2009. 19(4): p. 520-7.

[2] Altaie A, et al. Annals of the Rheumatic Diseases, 83, 110 (2024).

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.