Background/Purpose:  Primary Sjögren’s Syndrome (pSS) is a systemic auto-immune disease, leading to an exocrinopathy of mainly salivary and lachrymal glands. T- and B-cell-driven immunity is critically involved in its immunopathology. Recently we demonstrated synergistic T- and B-cell activation upon T-cell triggering and TLR7/9-driven B-cell activation in pSS patients, accompanied by synergistic induction of immunoglobulins and IFNɣ- and IL-17- producing T-cells. In addition, TLR7/9-expressing activated pDCs associated with increased type I IFNs and IFN-inducible genes are increased pSS patients. Several studies have shown that the DMARDs leflunomide (LEF) and hydroxychloroquine (HCQ) inhibit immune activation in pSS, but given separately only show moderate efficacy^(1-4). However, LEF and HCQ target different pathways with overlapping, but also potentially additive mechanisms, where LEF primarily targets T- and B-cells and HCQ TLR7/9-driven B-cell and pDC activation.  

Objective: To assess the additive effects of LEF and HCQ on CD4 T- and B-cell activation and Th cytokine and IFN-α secretion in vitro, employing T cell receptor/TLR9-triggered PBMC.

Methods:  PBMCs from healthy persons and pSS patients were cultured with antigen (SEB), TLR9-agonist (CPG-C) and their combination (n=11), in presence or absence of different dosages of LEF, HCQ and their combination (at least n=4). Proliferation of T- and B-cells (using Cell Trace Violet) and release of IFN-α, IFN-γ and IL-17 was measured (Luminex).

Results: In line with robust T and B cell activation, IFN-ɣ and IL-17 production and synergistic IFN-α production, indicative of pDC activity, was achieved by a combination of SEB and TLR9 ( (all p<0.001). Both HCQ and LEF potently and dose-dependently inhibited B- and T-cell proliferation and IFN-α production. Mean inhibition of IFN-a was 99% and 85% at 10 μM HCQ and 100 μM LEF, resp and 100% using LEF/HCQ combination (both p<0.05). At the latter concentrations HCQ and LEF additively inhibited IFN-ɣ production (mean: HCQ 41%, LEF 35%,  and combination 75%, all p<0.05; both HCQ and LEF vs combination p<0.05) and IL-17 production (mean: HCQ 68%, LEF 77%, and combination 85%, all p<0.05, HCQ vs combi p=0.05 and LEF vs combi p=0.19). Both T- and B-cell proliferation were additively and significantly inhibited by a combination of HCQ and LEF (all p<0.05).

Conclusion: HCQ and LEF robustly inhibited lymphocyte proliferation and cytokine production with additive inhibition of Th1 and Th17 cytokine secretion (IFN-γ and IL-17) and T- and B-cell proliferation. These findings could indicate the potential surplus value of combination therapy with HCQ and LEF for patients with pSS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/leflunomidehydroxychoroquine-combination-treatment-additively-inhibits-t-cell-receptortoll-like-receptor-9-triggered-th1-and-th17-cytokine-secretion/