SAT0001 FOSL-2 IS A REPRESSOR OF FOXP3 EXPRESSION DURING TREG DEVELOPMENT AND CONTROLS AUTOIMMUNITY

Background Fos like 2 (Fosl-2) is a transcription factor belonging to the AP-1 transcription complex. We have recently described a Fosl-2 transgenic (tg) mouse model which develops a multi-organ inflammatory and autoimmune phenotype. In these mice, we have characterized a decrease in regulatory T cells (Tregs), which preceded the activation of T cells and the development of inflammation. Objectives To analyze how Fosl-2 reduces the Treg population and triggers autoimmunity in Fosl-2 tg mice. Methods We used previously generated Fosl-2 tg overexpressing and Fosl-2 T cell specific knock out (Fosl-2 ko) mice. For mixed bone marrow transfer experiments, lethally irradiated recipients received a one to one mix of Fosl-2 tg (CD45.2) and wt (CD45.1) bone marrow to create Fosl-2 tg-wt chimera. We addressed the contribution of Tregs to the inflammatory phenotype by co-transferring Fosl-2 tg CD4 T cells and wt Tregs to Rag2-/- recipients. Treg and T cell populations were analyzed by flow cytometry and inflammation was addressed by CD45 staining on paraffin-embedded sections. RNA-sequencing was used to compare wt, Fosl-2 tg and Fosl-2 ko CD4 T cells after 24 hours of stimulation with anti-CD3 (2μg/ml) and anti-CD28 (2μg/ml). Results We first addressed whether Fosl-2 affected Tregs in a cell intrinsic way using mixed bone marrow experiments. In these animals, the CD45.2 Fosl-2 tg CD4 T cells showed a much lower proportion of Tregs compared to the CD45.1 wt population, both in the spleen (0.73%±0.15 vs 31.6%±3.6, P=0.062) and thymus (1.3%±1.04 vs 3.23%±0.78, P=0.001). This demonstrates that Fosl-2 overexpression represses Treg development in a cell intrinsic way. In T cell transfer experiments, Rag2-/- mice receiving 106 Fosl-2 tg CD4 cells developed lung inflammation 5 weeks after transfer, confirming that T cells are inducers of inflammation in Fosl-2 tg mice. Moreover, co-transfer of either 3*105 or 106 wt Treg cells resulted in a dose dependent reduction of inflammation. These data indicated that the decrease in the Treg population in Fosl-2 tg mice is responsible for the induction of inflammation. We then analysed Fosl-2 transcriptional targets in T cells by RNA-seq. Using a fold change > 1.5 and False Discovery Rate (FDR) of 0.05, we identified 191 differentially expressed genes in both Fosl-2 tg and Fosl-2 ko cells compared to wt. Interestingly, one of the top target genes of Fosl-2 was FoxP3. This unbiased approach thus revealed that FoxP3 expression is repressed by Fosl-2, with a 6.5 fold reduction in Fosl-2 tg cells and a 2.5 fold increase in Fosl-2 ko cells. This effect was confirmed on the protein level with a reduction in FoxP3 induction in Fosl-2 tg cells treated with TGFβ. The repression of Treg development observed in Fosl-2 tg mice could thus be explained by a direct transcriptional control of FoxP3 expression. Additionally, we found that Fosl-2 repressed a set of genes important for Tregs and other T helper cells. This included Nr4a2, a transcription factor involved in Treg development, IRF8 and Eomes, two genes involved in the polarization of Th1 and Th17 cells, or Ccl1 a chemokine important for Treg homeostasis. Conclusion Fosl-2 is involved in the control of FoxP3 expression in T cells. Through this, overexpression of Fosl-2 represses Treg development and induces a Treg dependent autoimmune phenotype in mice. This mechanism could thus be involved in the pathogenesis of autoimmune diseases and might represent a therapeutic target to modulate the Treg population. Disclosure of Interests Florian Renoux: None declared, Mara Stellato: None declared, Alexander Vogetseder: None declared, Riyung Huang: None declared, Arun Subramaniam: None declared, Przemyslaw Blyszczuk: None declared, Jörg Distler: None declared, Gabriela Kania: None declared, Onur Boyman: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders