Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) are required for maintenance of immune tolerance and immune homeostasis. Quantitative or functional Treg deficiency has been correlated with autoimmune disease, allergy, allograft rejection and graft versus host disease. Conversely, increased Treg can suppress tumor immunity resulting in tumor progression. Treg express a large number of cellular markers that reflect their level of maturation, functionality, activation and migratory capacity. Nevertheless, it has not previously been possible to integrate the expression of these various markers and correlate their expression with human Treg differentiation in vivo.Methods: We used single cell mass cytometry (CyTOF) to simultaneously study 36 phenotypic and functional markers of human Treg in samples obtained from umbilical cord blood (CB) (n=4) and healthy adult donors (n=10). Wanderlust trajectory detection algorithm was used to analyze temporal positioning of Treg across development. To quantify Treg heterogeneity we used ACCENSE; an analysis tool that combines a nonlinear dimensionality reduction algorithm (t-SNE) with spectral clustering algorithm and automated cell classification into subpopulations.Results: Using Wanderlust to characterize Treg maturation, the majority of CB Treg were naive (CD45RA+) and CB memory Treg (CD45RA-) were poorly differentiated with minimal expression of activation (HLA-DR) and pro-apoptotic (CD95) markers (Figure 1A). Adult Treg contained few naive cells and mature Treg effector cells expressed high levels of activation and pro-apoptotic markers. (Figure 1B). Using Wanderlust together with spearman correlation, 5 discrete stages of Treg maturation were identified; 1) recent thymic emigrants (RTE), 2) naive, 3) effector, 4) activated and 5) terminal effector. RTE Treg defined by expression of CD45RA, CD31 and CCR7, also expressed markers of proliferation (KI67) and functionality (Tbet, PDL-1, Helios) at low levels but lacked functional CTLA4 and TIM-3. Naive Treg lacked expression of CD31 but expressed other markers characteristic of RTE. Effector Treg expressed increased levels of CD95, CTLA-4, CCR7, GITR, Helios and FoxP3 but lacked HLA-DR. Activated effector Treg expressed the highest levels of FoxP3, Helios and Ki67, along with functional markers (CD28, CXCR3, ICOS, GITR, CD39, CTLA-4, TIM-3) and homing molecules (vascular endothelial CCR5, gut addressin ACT-1, skin addressins CCR4, CLA). Activated Treg express the highest levels and diversity of functional markers along with the ability to migrate to different tissues. Lastly, terminal effector Treg express a more restricted set of functional and homing markers (CD28, CTLA-4, ICOS and CCR5) with less diversity. Markers of exhaustion (PD-1 and TIM-3) are also expressed by effector, activated and terminal effector Treg. Pro-apoptotic markers (CD95high BCL2low) are primarily expressed by activated and terminal effector Treg.Using ACCENSE to evaluate Treg diversity allowed further identification of discrete Treg sub-populations within each maturation state. RTE and naive Treg appear very homogeneous and appear as a single cluster in both CB and adults. In contrast, effector, activated and terminal effector Treg are more heterogeneous and are visualized as 9 distinct clusters (Figure 1C, D). This clustering reflects distinct subsets of memory Treg that co-express various combinations of functional markers in our panel. All 9 Treg effector populations are present in CB, but at much lower levels. Treg effector cell diversity is maintained but does not increase as Treg mature and expand in adults and RTE/naive Treg become less prevalent.Conclusion: Our study is the first to quantify human Treg heterogeneity based on expression of a large set of activation, proliferation, tissue homing and functional markers in conjunction with stages of Treg maturation and differentiation. These studies define 5 stages of Treg maturation and 10 clusters of Treg diversity based on differential expression of phenotypic and functional markers. Similar approaches can be applied to describe maturation and diversity of other cell populations. Further application of this CyTOF panel can be used to study Treg maturation and diversity after hematopoietic stem cell transplantation and in immune and inflammatory diseases, to identify specific defects that may contribute to immune dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.