Single-Cell Analysis of the Human T Regulatory Population Uncovers Functional Heterogeneity and Instability within FOXP3+ Cells

Natural FOXP3+CD4+CD25High regulatory T cells are critical in immunological self-tolerance. Their characterization in humans is hindered by the failure to discriminate these cells from activated effector T cells in inflammation. To explore the relationship between FOXP3 expression and regulatory function at the clonal level, we used a single-cell cloning strategy of CD25-expressing CD4+ T cell subsets from healthy human donors. Our approach unveils a functional heterogeneity nested within CD4+CD25HighFOXP3+ T cells, and typically not revealed by conventional bulk assays. Whereas most cells display the canonical regulatory T (Treg) cell characteristics, a significant proportion of FOXP3+ T cells is compromised in its suppressive function, despite the maintenance of other phenotypic and functional regulatory T hallmark features. In addition, these nonsuppressive FOXP3+ T cells preferentially emerge from the CD45RO+ memory pool, and arise as a consequence of a rapid downregulation of FOXP3 expression upon T cell reactivation. Surprisingly, these dysfunctional Treg cells with unstable FOXP3 expression do not manifest overt plasticity in terms of inflammatory cytokine secretion. These results open a path to an extensive study of the functional heterogeneity of CD4+CD25HighFOXP3+ Treg cells and warrant caution in the sole use of FOXP3 as a clinical marker for monitoring of immune regulation in humans.