Allogeneic hematopoietic stem cell transplantation (HSCT) is an increasingly used intensive therapy to treat patients with hematological malignancies and other life-threatening hematological and genetic diseases. Acute graft-versus-host disease (aGVHD) is the main complication of HSCT and an important cause of mortality. Cellular therapy with CD4+FOXP3+ regulatory T cells (Treg), a population with potent immunosuppressive properties, has proven highly effective in mouse models of GVHD; however, translation of this approach into humans has been difficult because of the identification of several distinct subsets of human Treg and the observation that an inflammatory environment may cause conversion of human Treg into effector T cells with pro-inflammatory functions. However, the notion of Treg cell plasticity remains highly controversial because of the heterogeneity of the Treg population and because clear evidence for lineage reprogramming is not available. The question whether Treg are a stable lineage is of paramount importance for the potential application of Treg therapy in the clinic.The main goal of this project is to promote the fundamental knowledge of human Treg subpopulations and to define their role in preventing aGVHD. We will characterize the heterogeneity of the Treg compartment using an efficient and robust single-cell analysis pipeline. Since the lymphopenic environment and inflammation created by conditioning treatment prior to HSCT may favor Treg conversion, we will determine a potential plasticity of Treg subpopulations in cell-based assay systems and by studying chromatin modifications at the loci of lineage-specific transcription factors and cytokine genes. Furthermore, we will analyze the role of Treg subsets in aGVHD, to define potential correlations with disease severity and response to treatment. This approach may identify new biomarkers for early diagnosis and steroid-resistant GVHD.