Background/Purpose: Rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) are chronic autoimmune diseases that tend to flare repeatedly in the same joints, displaying joint-specific memory. We have identified CD8+ tissue resident memory T cells (TRM) in human arthritic joints and demonstrated in murine models that these long-lived TRM remain in synovium during remission and act as key mediators of arthritis flares. However, the factors that drive TRM formation in the synovium have not been characterized. IL-15 supports CD8 T cell activation and TRM development in other tissues. It is also elevated in synovial fluid and is expressed in the synovium in RA. Here, we explored the inflammatory drivers of IL-15 production by synovial stromal cells and investigated the role of IL-15 in synovial TRM development.

Methods: Fibroblast-like synoviocytes (FLS) isolated from human RA synovium and human umbilical vein endothelial cells (HUVEC) were grown in tissue culture and stimulated with TNF, IL-1beta, IL-6 or IFNgamma for 48 hours. Cells were collected, and IL-15 mRNA expression was assessed by quantitative PCR. To study human synovial TRM development, we developed a 3D synovial organoid that recapitulates the synovial stromal structure utilizing FLS and HUVEC encapsulated in Matrigel. CD8 memory T cells were isolated from peripheral blood of healthy donors and co-cultured with the synovial organoid for 2-3 weeks to generate TRM. TRM development was validated by cell surface markers, gene expression profile, free fatty acid uptake and migratory response to tissue egress signals. TNF was added to the culture media for 3-5 days to simulate inflammation, then removed for the duration in culture. IL-15 receptor alpha (IL-15Ra) or CD122 blocking antibodies were added to T cells 30 minutes prior to the formation of organoids, and they were replenished twice a week to inhibit IL-15 activity. After 3 weeks, cells were dissociated from the organoids and assayed for TRM surface phenotype by flow cytometry.

Results: TNF stimulation dramatically increased IL-15 gene expression in FLS by 30-fold, while it had a smaller effect on HUVEC (5-fold). IL-1beta also induced a 6-fold change in IL-15 expression in FLS, but IL-6 and IFNgamma did not. Culturing CD8 memory T cells in synovial organoids, particularly with TNF, supported the development of cells with TRM surface phenotype (CD45RO+CD62L-CCR7-HLADR-CD25-CD103+CD49a+) and TRM gene expression patterns. These TRM also had enhanced free fatty acid uptake, and they did not migrate across a transwell membrane in response to CCL21, further confirming TRM functionality. Inhibition of IL-15 activity with IL-15Ra or CD122 blocking antibodies reduced TRM development.

Conclusion: We developed a novel model for studying human synovial TRM by differentiating TRM within 3D synovial organoids composed of stromal cells from human RA synovium. We show that TNF preferentially induces FLS to produce IL-15 and demonstrate that IL-15 activity mediates TRM development in this synovial organoid system. As synovial TRM facilitate arthritis flares, effective methods to impede synovial TRM development may be valuable for durable disease control.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tnf-stimulated-production-of-il-15-by-fibroblast-like-synoviocytes-mediates-human-resident-memory-t-cells-development-in-synovial-organoid-model/