Background/Purpose: In autoimmune diseases such as rheumatoid arthritis (RA), the inflammatory environment induces T regulatory cell (Treg) dysfunction and reduces the frequency of tolerogenic dendritic cells (tolDC), allowing inflammation to persist. In RA, clinical response to anti-TNF therapy is associated with improved Treg function and reemergence of tolDC, however many patients experience progressive loss of immune control and secondary loss of response (~50% within 3-5 years). Therefore, therapies that both suppress inflammation and drive persistent immune tolerance will likely elicit more durable disease suppression. Recent studies have demonstrated that TNFR2 promotes Treg suppressive activity and tolDC function. Herein, we demonstrate that small-molecule TNF inhibition prevents pro-inflammatory TNFR1-dependent signaling, while preserving beneficial membrane TNF (mTNF)/TNFR2 activity leading to enhanced Treg and tolDC activity.

Methods: HEK293, K562 cells and HUVEC cells were used to assess the activity of small-molecule inhibitors on TNFR1/TNFR2-specific signaling. HeLa cells expressing a mutant non-cleavable TNF were generated to express mTNF to activate TNFR2 signaling. Treg and tolDC were co-cultured with mTNF expressing HeLa cells and function was evaluated using cytokine and phenotypic marker levels by flow cytometry and cytokine bead array. Collagen antibody and collagen II-induced arthritis mouse models were used to demonstrate the activity of TNF signaling blockade.

Results: Small-molecule TNF inhibitors displayed a concentration-dependent reduction in soluble TNF/TNFR1-stimulated signaling assays including NF-κB activation and IL-8 secretion, equivalent to blockade using anti-TNF biologics. In addition, in murine models of arthritis, TNF small-molecule inhibition showed equivalent disease suppression to an anti-TNF biologic. In contrast, mTNF-stimulated TNFR2 activity was retained with small-molecule inhibitors but abolished using anti-TNF biologics. TNFR2 activation increased the abundance of highly suppressive FoxP3^highHelios⁺ cells in Treg co-cultures with mTNF-expressing HeLa cells and elevated expression of tolerogenic cytokines IL-10 and TGF-β. These phenotypes were blocked using an anti-TNF biologic but were retained using a small-molecule TNF inhibitor. These cytokines have been shown to promote generation of tolDC and activation of TNFR2 promoted IL-10 production from tolDC. Treg and tolDC suppressive function has been shown to restrict T cell expression of pathogenic cytokines such as IL-17. Interestingly, anti-TNF treatment led to enhanced IL-17 levels in the biologic-treated group but not with small-molecule inhibition. These data demonstrate that TNF small-molecule inhibition is effective at inhibiting pro-inflammatory signaling and providing equivalent disease control to an anti-TNF biologic while promoting suppressive Treg and tolDC function.

Conclusion: TNFR2 promotes immune tolerance by supporting anti-inflammatory Treg and tolDC activity. This may allow TNF small-molecule inhibition to provide more durable disease suppression than anti-TNF biologics by providing superior control of pathogenic cytokines such as IL-17.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/small-molecule-inhibitors-of-tnf-reduce-pro-inflammatory-disease-activity-while-retaining-tnfr2-signaling-which-preserves-the-suppressive-capacity-of-regulatory-t-cells-and-promotes-immune-tolerance/