Cytokine-induced PD-L1 and PD-L2 expression is preserved but reverse signalling is altered in rheumatoid arthritis fibroblast-like synoviocytes.

Abstract

Background: Dysfunctional programmed cell death-1 (PD1) signalling may contribute to persistent immune activation in rheumatoid arthritis (RA). However, co-inhibitory molecule interactions between T cells and fibroblast-like synoviocytes (FLS) remain insufficiently understood. This study aimed to determine whether T cell activation and subset-associated cytokines differentially regulate the expression of PD-ligand 1 (PD-L1) and PD-L2 on FLS from RA patients compared with non-inflammatory (NI) controls, and whether PD1 engagement induces distinct transcriptional downstream responses in RA-FLS versus NI-FLS.

Methods: Primary FLS cell lines were cultured from synovial tissue of patients with established RA or NI controls undergoing arthroscopy due to previous injury. Cells were cultured with CD3+ T cells or stimulated with TNF, IFNγ, IL-4, or TGF-β, and PD-L1 and PD-L2 expression was analysed by flow cytometry. PD-L1 and PD-L2 knockout FLS were generated using CRISPR/Cas9. TNF-primed knockout or mock-transfected RA- and NI-FLS were treated with soluble PD1, and transcriptomic responses were analysed by total mRNA sequencing.

Results: Analysis of publicly available RNA-sequencing datasets showed that, in early RA, PD-L1 and PD-L2 expression were significantly higher in lymphoid compared to myeloid and fibroid synovial tissue pathotypes. In established RA, PD-L1 and PD-L2 expression in FLS showed a trend towards higher levels in lymphocyte-rich compared to lymphocyte-poor tissue. In vitro, activated but not resting T cells upregulated both PD-L1 and PD-L2 on FLS. Further, TNF and IFNγ induced PD-L1 expression up to fourfold compared with unstimulated FLS, whereas IL-4 and TGF-β had no effect. PD-L2 was induced by TNF, IFNγ, and IL-4 to similar levels. PD-L1 and PD-L2 expression did not differ between RA- and NI-FLS either in co-culture with T cells or under cytokine stimulation. Finally, soluble PD1-induced reverse signalling in FLS altered the expression of threefold more genes in NI-FLS compared to RA-FLS, indicating that RA-FLS might be less responsive to PD1 signalling. PD-L1 or PD-L2 knockout further showed that reverse signalling through each ligand modulates distinct gene sets in FLS.

Conclusions: Cytokine-induced PD-L1 and PD-L2 expression is preserved in FLS from established RA but PD1-ligand signalling is altered, potentially contributing to the loss of immune regulation in RA.