A modular system for programming multistep activation of endogenous genes in stem cells.

Unlabelled: Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events.

Realization of the T Lineage Program Involves GATA-3 Induction of Bcl11b and Repression of Cdkn2b Expression.

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate.

Polymorphic Immune Mechanisms Regulate Commensal Repertoire.

Environmental influences (infections and diet) strongly affect a host's microbiota. However, host genetics may influence commensal communities, as suggested by the greater similarity between the microbiomes of identical twins compared to non-identical twins. Variability of human genomes and microbiomes complicates the understanding of polymorphic mechanisms regulating the commensal communities.

Cutting Edge: Lymphomyeloid-Primed Progenitor Cell Fates Are Controlled by the Transcription Factor Tal1.

Lymphoid specification is the process by which hematopoietic stem cells (HSCs) and their progeny become restricted to differentiation through the lymphoid lineages. The basic helix-loop-helix transcription factors E2A and Lyl1 form a complex that promotes lymphoid specification. In this study, we demonstrate that Tal1, a Lyl1-related basic helix-loop-helix transcription factor that promotes T acute lymphoblastic leukemia and is required for HSC specification, erythropoiesis, and megakaryopoiesis, is a negative regulator of murine lymphoid specification.

Transcription factor ID2 prevents E proteins from enforcing a naïve T lymphocyte gene program during NK cell development.

All innate lymphoid cells (ILCs) require the small helix-loop-helix transcription factor ID2, but the functions of ID2 are not well understood in these cells. We show that mature natural killer (NK) cells, the prototypic ILCs, developed in mice lacking ID2 but remained as precursor CD27CD11b cells that failed to differentiate into CD27CD11b cytotoxic effectors. We show that ID2 limited chromatin accessibility at E protein binding sites near naïve T lymphocyte-associated genes including multiple chemokine receptors, cytokine receptors, and signaling molecules and altered the NK cell response to inflammatory cytokines.

Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage.

Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway.

The chemokine receptor CCR9 controls the immigration of multipotent hematopoietic progenitor cells into the thymus to sustain T cell development. Postimmigration, thymocytes downregulate CCR9 and migrate toward the subcapsular zone where they recombine their TCR β-chain and γ-chain gene loci. CCR9 is subsequently upregulated and participates in the localization of thymocytes during their selection for self-tolerant receptor specificities.

E proteins and the regulation of early lymphocyte development.

Lymphopoiesis generates mature B, T, and NK lymphocytes from hematopoietic stem cells via a series of increasingly restricted developmental intermediates. The transcriptional networks that regulate these fate choices are composed of both common and lineage-specific components, which combine to create a cellular context that informs the developmental response to external signals. E proteins are an important factor during lymphopoiesis, and E2A in particular is required for normal T- and B-cell development.

Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors.

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFalpha stimulation. In cells from TRADD-deficient mice, TNFalpha-mediated apoptosis and TNFalpha-stimulated NF-kappaB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFalpha-mediated inflammatory responses in vivo.

Generation of immunocompetent T cells from embryonic stem cells.

Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors derive from the mesoderm, which gives rise to hemangioblasts that can differentiate into endothelial or endocardial precursors, or hematopoietic stem cells (HSCs). These HSCs, in turn, may either self-renew or differentiate into lineage-restricted progenitors, and ultimately mature effector cells.

T-cell potential and development in vitro: the OP9-DL1 approach.

In vivo, T cells develop in the thymus from bone marrow-derived hematopoietic progenitors. Similarly, T cells can develop in vitro in model systems that mimic thymic function. The recent development of the OP9-DL1 cell culture system, a two-dimensional T-inductive environment, has provided greater access to the processes of commitment and development in T lymphocytes.

In vitro generation of T lymphocytes from embryonic stem cells.

Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors have their origin in the mesoderm, which gives rise to hemangioblasts that can differentiate into endothelial or endocardial precursors or hematopoietic stem cells. These hematopoietic stem cells in turn may either self-renew or differentiate into lineage-restricted progenitors and ultimately mature effector cells.

Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.

The bone marrow and thymus, although both hemopoietic environments, induce very distinct differentiation outcomes. The former supports hemopoietic stem cell self-renewal and multiple hemopoietic lineages, while the latter supports T lymphopoiesis almost exclusively. This distinction suggests that the thymic environment acts to restrict the hemopoietic fates available to thymic immigrants.

In vitro generation of lymphocytes from embryonic stem cells.

Lymphocytes arise during ontogeny via a series of increasingly restricted intermediates. Initially, the mesoderm gives rise to hemangioblasts, which can differentiate into endothelial precursors, or hematopoietic stem cells (HSCs). HSCs can either self-renew or differentiate into lineage-restricted progenitors and, ultimately, to mature effector cells.

Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system.

In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors.

Embryonic stem (ES) cells can differentiate into most blood cells in vitro, providing a powerful model system to study hematopoiesis. However, ES cell-derived T lymphocytes have not been generated in vitro, and it was unresolved whether such potential is absent or merely difficult to isolate. Because the latter case might result from rapid commitment to non-T-cell fates, we isolated ES cell-derived prehematopoietic precursors for reconstitution of fetal thymic organ cultures.

Regulatory cytokine production stimulated by DNA vaccination against an altered form of glutamic acid decarboxylase 65 in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop a T-cell dependent autoimmune form of diabetes, in which glutamic acid decarboxylase 65 (GAD65) is an important islet target antigen. Intramuscular DNA vaccination with a plasmid encoding native GAD65 (a cytosolic antigen) did not significantly alter the incidence of diabetes, but vaccination against an altered form of GAD65 with a signal peptide (spGAD), which is secreted in vitro, was protective. The preventive effect was further enhanced by repeated injections of the spGAD plasmid.