IntroductionThe development and differentiation of immune cells is carefully orchestrated by an array of cytokines. Signal transducers and activators of transcription (Stats) represent a small but critical family of transcription factors that play important roles in transmitting cytokine signals. Consequently, Stats are critical for immunoregulation and the development of immune cells. 1,2 Stat5a and Stat5b are two closely related proteins that have overlapping functions with respect to lymphoid development and differentiation. 3,4 Gene targeting of Stat5a and Stat5b (collectively referred to as Stat5), results in impairment in the development of T, B, and natural killer (NK) cells. [5][6][7] In mice in which the amino termini of Stat5a and Stat5b are deleted (denoted as Stat5 ⌬N mice), major disruption of various immune cell parameters was noted. 8,9 However, residual Stat5 function permits T cell development, albeit suboptimally. 10 This contrasts with the complete absence of Stat5a/b, which results in dramatic reduction in thymocyte numbers, in part due to effects on lymphoid stem cell function. 5 T regulatory (Treg) cells comprise a population of cells enriched in CD4 ϩ CD25 ϩ T cells that suppresses T-cell proliferation and function and attenuates immune responses against self-or nonself-antigens. [11][12][13] Naturally arising Treg cells are produced in the thymus as a functionally distinct T-cell subpopulation, whereas adaptive Treg cells are induced from naive T cells after antigen exposure in the periphery. [14][15][16][17] In classic studies, mice develop organ-specific autoimmune disease following neonatal thymectomy, which is corrected by reconstitution with CD4 ϩ CD25 ϩ T cells. 13 The essential role of Treg cells in maintaining tolerance has been confirmed by findings that defective function of this subset is a feature of many models of autoimmunity. 18 More recently, it was discovered independently by several groups that a subset of CD4 ϩ CD25 ϩ T cells expresses the transcription factor Foxp3, which is necessary and sufficient for Treg cell development and function. [19][20][21][22] Foxp3 is highly conserved in mice and humans. Mutation of Foxp3 in mice (scurfy) results in early autoimmune disease, 23 whereas mutations of human Foxp3 are associated with a disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). 24 In mice, Foxp3 is a reliable marker for the Treg lineage.Multiple lines of evidence have indicated that IL-2 is an important growth factor for Treg development and maintenance. Mice lacking IL-2 or its receptor subunits, IL-2R␣ (CD25) and IL-2R␤ (CD122), have deficits in CD4 ϩ CD25 ϩ Treg cells and develop autoimmune disease similar to Foxp3 Ϫ/Ϫ mice. [25][26][27] However, IL-2 is dispensable for Treg cell development, as some Foxp3-expressing cells are present in Il2 Ϫ/Ϫ and Il2ra Ϫ/Ϫ mice, suggesting the involvement of other cytokines. 28 In vitro culture of CD4 ϩ T cells with transforming growth factor-␤1 (TGF-␤1) can promote the generation o...

SUMMARY Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insights. The comprehensive single cell dataset permits mapping of the mouse hematopoietic stem cell (HSC) differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.