Selective Activation of Tumor Necrosis Factor Receptor II Induces Antiinflammatory Responses and Alleviates Experimental Arthritis Roman Fischer , 1 Marcel Proske, 2 Ma€ elle Duffey, 2 Hubert Stangl, 3 George F. Martinez, 4 Nathalie Peters, 2 Alexandra Kraske, 2 Rainer H. Straub, 3 John R. Bethea, 4 Roland E. Kontermann, 2 and Klaus Pfizenmaier 2 Objective. Treg cells modulate immune responses and can suppress the development of autoimmune dis- eases. Tumor necrosis factor receptor II (TNFRII) has been recognized as a key receptor on these cells that facil- itates expansion and stabilization of CD4+ Treg cells. The purpose of the present study was to investigate the thera- peutic activity of a novel TNFRII agonist in experimental arthritis as well as the role of different Treg cell subsets. Methods. A novel mouse TNFRII–selective fusion protein (EHD2-sc-mTNF R2 ) was generated by genetic en- gineering. Mouse T cells were incubated together with interleukin-2 and/or EHD2-sc-mTNF R2 , and the effects on Treg cells were analyzed by flow cytometry. Mice with colla- gen-induced arthritis (CIA) were treated with EHD2-sc- mTNF R2 or saline, and the therapeutic effects were moni- tored and characterized. Results. Selective activation of TNFRII was found to expand both CD4+ and CD8+ Treg cells. Moreover, TNFRII activation elevated the number of CD4+CD25+ and CD8+CD25+ Treg cells and increased the number of FoxP3-expressing cells in CD8+, but not CD4+, Treg cells, indicating different mechanisms of TNFRII-induced expan- sion of diverse T cell subsets with suppressive activity. In the CIA model, we demonstrated that administration of the TNFRII agonist EHD2-sc-mTNF R2 led to the expansion of both CD4+ and CD8+ Treg cells in vivo and induced anti- inflammatory responses that alleviated arthritis. Conclusion. Our findings support the use of TNFRII-selective therapeutics as an effective approach to the treatment of arthritic disease and possibly other inflammatory and autoimmune diseases. Tumor necrosis factor (TNF) is a multifunctional cytokine with pleiotropic functions. It is a master regulator of the immune system and a key player in the initiation and orchestration of inflammation and immunity. TNF is syn- thesized as a trimeric transmembrane protein (tmTNF) that can be proteolytically processed into soluble circulating TNF homotrimers (sTNF). Interestingly, sTNF and tmTNF differ in their capability to activate the 2 TNF receptors (TNFRs): TNFRI and TNFRII. Whereas TNFRI can be activated by both sTNF and tmTNF, TNFRII is dependent on tmTNF to be robustly activated (1). Deregulation of TNF expression and signaling can cause chronic inflammation, which may result in the development of autoimmune diseases and tissue damage (2–6). Indeed, elevated TNF levels have been associated with several inflammatory diseases, such as rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease; therapeutic agents that neutralize TNF are being success- fully used to treat these diseases (7,8). Surprisingly, how- ever, a clinical trial with an anti-TNF drug that blocks both sTNF and tmTNF in multiple sclerosis patients resulted in disease exacerbation and had to be stopped (9). Moreover, the approved TNF inhibitors can cause severe side effects, including opportunistic infections, reactivation of tuberculosis, development of autoimmune disease, increased susceptibility to the development of A video abstract of this article can be found at https://bcove.video/2JAAUnz and https://vimeo.com/262882079. Dr. Fischer’s work was supported by the Carl Zeiss Founda- tion (grant Az. 0563-2.8./508/2) and the DFG (research fellowship FI 2138/1-1). Drs. Stangl and Straub’s work was supported by DFG grants STR 511/26-1 and STR 511/34-1. 1 Roman Fischer, PhD: University of Stuttgart, Stuttgart, Germany, and Drexel University, Philadelphia, Pennsylvania; 2 Marcel Proske, MSc, Ma€ elle Duffey, MSc, Nathalie Peters, BTA, Alexandra Kraske, VT, Roland E. Kontermann, PhD, Klaus Pfizenmaier, PhD: University of Stuttgart, Stuttgart, Germany; 3 Hubert Stangl, PhD, Rainer H. Straub, MD: University Hospital Regensburg, Regensburg, Germany; 4 George F. Martinez, BSc, John R. Bethea, PhD: Drexel University, Philadelphia, Pennsylvania. Address correspondence to Roman Fischer, PhD, Depart- ment of Biology, Drexel University, 3245 Chestnut Street, Philadel- phia, PA 19104. E-mail: rf428@drexel.edu. Submitted for publication August 1, 2017; accepted in revised form January 9, 2018. 722 ARTHRITIS & RHEUMATOLOGY Vol. 70, No. 5, May 2018, pp 722–735 DOI 10.1002/art.40413 © 2018, American College of Rheumatology