SAT0043 ANALYSIS OF THE ROLE OF RORΓT+FOXP3+ (TR17) IN THE REGULATION OF AUTOIMMUNE ARTHRITIS
Authors: Kotona Furuyama, Yuya Kondo, Masahiro Yokosawa, Masaru Shimizu, Hiroto Tsuboi, Isao Matsumoto, Takayuki Sumida
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> RORγt<sup>+</sup>Foxp3<sup>+</sup> regulatory T (Treg) cells, designated as Tr17 is one of the new subset of Treg cells, having the potential to regulate the development of experimental autoimmune encephalomyelitis (EAE) thorough a specific repression of Th17 mediated inflammation (1). The function of Tr17 remain unclear in the development of other autoimmune diseases such as collagen induced arthritis (CIA). <h3>Objectives:</h3> To clarify the role of RORγt<sup>+</sup>Foxp3<sup>+</sup> (Tr17) cells in the development CIA. <h3>Methods:</h3> 1) Lymphocytes in draining lymph node (LN) were harvested from C57BL/6 mice on 10 days after immunization of type II collagen (CII) emulsified with complete Freund's adjuvant. The expression of RORγt in Foxp3<sup>+</sup>Treg cells was analyzed by flow cytometry and compared them with lymphocytes of non-immunized mice. 2) At 10 days after CII immunization, C-C chemokine receptor type 6 (CCR6), CD25, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and glucocorticoid-induced TNF-receptor (GITR) expression on Tr17, RORγt<sup>-</sup>Treg cells, and RORγt<sup>+</sup>non-Treg cells in LN were analyzed by flow cytometry. 3) Lymphocytes were harvested from Foxp3<sup>IRES-gfp</sup> reporter mice on 10 days after first CII immunization. CD4<sup>+</sup>GFP<sup>+</sup> Treg and CD4<sup>+</sup>GFP<sup>-</sup> T cells were isolated and stimulated with anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb <i>in vitro</i>. The expression of IL-10 and IL-17 in RORγt<sup>+</sup>GFP<sup>+</sup> Tr17 cells was analyzed by flow cytometry and compared with that in RORγt<sup>-</sup>GFP<sup>+</sup> cells or RORγt<sup>+</sup>GFP<sup>-</sup> cells. 4) After the induction of CIA, lymphocytes in inflamed ankle joints and LN were harvested from C57BL/6 mice. The expression of RORγt in both Foxp3<sup>+</sup>Treg cells and Foxp3<sup>-</sup> non-Treg cells were analyzed by flow cytometry. 5) Foxp3<sup>cre</sup>RORγt<sup>fl/fl</sup> (conditional knock out; cKO) mice which deficit Tr17 cells were immunized with CII on days 0 and 21. Incidence and severity of CIA were analyzed and compered with Foxp3<sup>wt</sup>RORγt<sup>fl/fl</sup> (control) mice. 6) At 10 days post first CII immunization. Lymphocytes from control mice and cKO mice were cultured with or without 100 μg/mL of denatured CII for 72 h. IL-17 and IFNγ levels in supernatants measured by ELISA. <h3>Results:</h3> 1) Tr17 cells in draining lymph nodes were significantly increased in CII-immunized mice compared with non-immunized mice. 2) CCR6, CD25, CTLA-4, and GITR expression was elevated on Tr17 cells compared with RORγt<sup>-</sup>Treg cells and Th17 cells. 3) IL-10 producing cells were significantly increased in Tr17 cells compared with RORγt<sup>-</sup>Treg cells (12.57 +/- 1.135, p &lt; 0.001). On the other hand, IL-17 producing cells was tended to be decreased in Tr17 cells in spite of the high expression of RORγt compared with RORγt<sup>+</sup>Th17 cells (2.376 +/- 0.231, p = 0.176). 4) Tr17 cells and RORγt<sup>+</sup>non-Treg cells were increased and Foxp3<sup>+</sup>Treg cells were decreased in inflamed ankle joints compared with LN after the induction of CIA. 5) CIA tended to be exacerbated in cKO mice compared with control mice. 6) Production of IL-17 tended to be higher in cKO mice than in control mice. <h3>Conclusion:</h3> Tr17 cells were increased in the course of CIA and infiltrated into inflamed joints. Moreover, Tr17 cells had the potential to regulate the development of CIA thorough the high expression of suppressive molecules such as IL-10 and CTLA-4. <h3>Reference</h3> [1] Byong-Seok K. et al. Generation of RORγt+ Antigen-Specific T Regulatory 17 Cells from Foxp3+ Precursors in Autoimmunity. Cell Reports. 2017. 21, 195-207 <h3>Disclosure of Interests:</h3> Kotona Furuyama: None declared, Yuya Kondo: None declared, Masahiro Yokosawa: None declared, Masaru Shimizu: None declared, Hiroto Tsuboi: None declared, Isao Matsumoto: None declared, Takayuki Sumida Grant/research support from: Bristol-Myers Squibb, Speakers bureau: Bristol-Myers Squibb