Background: In rheumatic diseases, like systemic sclerosis and rheumatoid arthritis, myofibroblasts drive fibrosis of tissues (e.g skin, lungs and synovium). This fibrosis is an important cause of morbidity and mortality via tissue stiffening and hardening leading to loss of organ function. In such affected tissues, increased presence of CD8+ T cells has been observed, but these cells have a yet unknown role in myofibroblast formation and activation.

Objectives: Our purpose was to examine the role of CD8+ T cells in pro-fibrotic mediated myofibroblast activation and contraction in the developed humanized model of connective tissue disease pathology.

Methods: Primary skin fibroblasts and peripheral blood mononuclear cells (PBMCs) or sorted CD8+ T cells were co-cultured in a 3D collagen type 1 hydrogel for up to 96 hours (n=20). Allogeneic mismatching was used as a model to study auto-immune connective tissue biology. T cell activation and cytokine expression was evaluated using flow cytometry. Fibroblast activation was analyzed using immunohistochemistry (IHC) and qPCR and activation-induced contraction was measured macroscopically. Cell death was evaluated using flow cytometry (7AAD/AnnexinV) and IHC (activated caspace-3 and γH2AX). Activation of intracellular signaling pathways in fibroblasts was measured using luciferase reporter cell lines.

Results: Co-culture of fibroblasts with PBMCs strongly induced fibroblast contractility and this was further accompanied by an elevated myofibroblast phenotype as measured by increased expression of collagen type 1, fibroblast activation protein (FAP), αSMA and IL-6. Both CD4+ and CD8+ T cells were activated as a result of co-culture as identified by increased CD25 and CD69 expression and elevated IL-2 and IFN-γ production. Notably, enhanced fibroblast cell death was not observed, excluding cytolytic processes as a driving mechanism. Upon co-culture with either sorted CD4+ or CD8+ T cells, CD8+ T cells more strongly induced fibroblast contraction and activation than CD4+ T cells. This was associated with raised IL-6 production by CD8+ T cells and STAT3/TGFβ-induced signaling in fibroblasts. In addition, blocking cytokine release of CD8+ T cells with brefeldin A blocked the CD8+ T cell induced STAT3-signaling in fibroblasts but did not affect their cytolytic capacity. The use of JAK/STAT3-inhibitor tofacitinib or the TGFβ/SMAD2/3 inhibitor SB-505124 blocked the activated fibroblast phenotype. Notably, combined use of both inhibitors illustrated an additive effect and fully blocked myofibroblast activation and contraction.

Conclusion: CD8+ T cells drive myofibroblast contraction and activation not via cytotoxicity related programs but via cytokine release. This sheds light on novel mechanisms of immune cell mediated tissue fibrosis. Furthermore, our results suggest that combining JAK/STAT3 inhibition with an anti-fibrotic agent might be promising in mitigating immune cell mediated tissue fibrosis in connective tissue rheumatic disorders and has added value over blocking these pathways individually. This observation is believed to facilitate clinical practice in cases of patients with a fibrotic signature (endotype) that is associated with refractory disease [1].

REFERENCES: [1] Rivellese, F., Surace, A.E.A., Goldmann, K. et al. Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial. Nat Med 28 , 1256–1268 (2022). https://doi.org/10.1038/s41591-022-01789-0

Acknowledgements: NIL.

Disclosure of Interests: None declared.