CELLULAR, MOLECULAR AND FUNCTIONAL IDENTITY OF exTREGS

• Applicants: Professor Dr. Jochen Hühn; Professorin Dr. Julia Polansky-Biskup
• Subject Area: Immunology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 569008721

CD4+Foxp3+ regulatory T cells (Tregs) play an important role in the maintenance of immune tolerance, tissue homeostasis and the prevention of autoimmune and chronic inflammatory diseases. On the other hand, they have a detrimental role in anti-tumor immunity. Although still controversial, there is evidence that a fraction of Tregs lose expression of their lineage-specification transcription factor Foxp3, as well as their suppressive function, and may even acquire effector functions, a phenomenon that is more pronounced during inflammation. However, very little is known about the biology, abundance and function of these "exTreg" cells due to the lack of known specific markers. Using genetically engineered mice and a novel methodological approach, our preliminary data show that it is now possible to unambiguously identify exTregs by flow cytometry and single cell (sc)RNAseq. Based on this, we propose a research project to elucidate the biology of exTregs in mice. We will characterize their phenotype, transcriptome, epigenome, function and differentiation mechanism in both homeostasis and models of autoimmune disease (experimental autoimmune encephalomyelitis), cancer (MCA205 tumor) and infection (influenza). The first part of this project will consist of determining the proportion and phenotype of exTregs by multiparametric flow cytometry in different lymphoid and non-lymphoid tissues at homeostasis and in inflamed tissues in the three pathological conditions mentioned above. In addition, we will generate and analyze multi-omics data to assess the transcriptome (bulk RNAseq and scRNAseq), epigenetic profile (bulk ATACseq and DNA methylome) and T cell receptor repertoire (scTCRseq) of exTregs in homeostasis and in the three pathologies. In the second part, we will determine the functional properties of exTregs by studying their ability to produce cytokines, proliferate or exert a suppressive function in vitro. We will also test their potential pathogenic effector or protective suppressive function in vivo in the adoptive transfer colitis model. Finally, we will identify transcription factors and cis-regulatory elements specific for exTregs and analyze the role of these factors in the differentiation and maintenance of exTregs using state-of-the-art genetic- and epigenetic-editing techniques. This project will allow us to answer key questions about exTregs such as: What is the proportion of conventional CD4+Foxp3- T cells that are actually exTregs in different tissues and inflammatory contexts? What is the phenotype and function of exTregs? How do they contribute to inflammatory processes? What is the molecular mechanism involved in the generation of exTregs? Our project will unambiguously identify and functionally characterize the intriguing and much debated exTreg subset. Findings from this project will shed light on novel players and mechanisms in the regulation and function of the immune system and in the pathophysiology of inflammatory diseases.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Dr. Benoit Salomon, Ph.D.