CD4+ regulatory T cells (CD4Tregs) play a critical role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. IL-2 supports the proliferation and survival of CD4Tregs and previous studies have demonstrated that IL-2 induces selective expansion of CD4Tregs and improves clinical manifestations of chronic GVHD. However, mechanisms for selective activation of CD4Tregs and the effects of low-dose IL-2 on other immune cells are not well understood. Using mass cytometry, we demonstrate that low concentrations of IL-2 selectively induce STAT5 phosphorylation in Helios+ CD4Tregs and CD56 bright CD16–NK cells in vitro. Preferential activation and expansion of Helios+ CD4Tregs and CD56 bright CD16–NK cells was also demonstrated in patients with chronic GVHD receiving low-dose IL-2. With prolonged IL-2 treatment for 48 weeks, phenotypic changes were also observed in Helios–CD4Tregs. The effects of low-dose IL-2 therapy on conventional CD4+ T cells and CD8+ T cells were limited to increased expression of PD-1 on effector memory T cells. These studies reveal the selective effects of low-dose IL-2 therapy on Helios+ CD4Tregs and CD56 bright NK cells that constitutively express high-affinity IL-2 receptors as well as the indirect effects of prolonged exposure to low concentrations of IL-2 in vivo.