New Molecular Insights into Immune Cell Development.

During development innate lymphoid cells and specialized lymphocyte subsets colonize peripheral tissues, where they contribute to organogenesis and later constitute the first line of protection while maintaining tissue homeostasis. A few of these subsets are produced only during embryonic development and remain in the tissues throughout life. They are generated through a unique developmental program initiated in lympho-myeloid-primed progenitors, which lose myeloid and B cell potential. They either differentiate into innate lymphoid cells or migrate to the thymus to give rise to embryonic T cell receptor-invariant T cells. At later developmental stages, adaptive T lymphocytes are derived from lympho-myeloid progenitors that colonize the thymus, while lymphoid progenitors become specialized in the production of B cells. This sequence of events highlights the requirement for stratification in the establishment of immune functions that determine efficient seeding of peripheral tissues by a limited number of cells.

Transcriptional Control of Dendritic Cell Development.

The dendritic cells (DCs) of the immune system function in innate and adaptive responses by directing activity of various effector cells rather than serving as effectors themselves. DCs and closely related myeloid lineages share expression of many surface receptors, presenting a challenge in distinguishing their unique in vivo functions. Recent work has taken advantage of unique transcriptional programs to identify and manipulate murine DCs in vivo. This work has assigned several nonredundant in vivo functions to distinct DC lineages, consisting of plasmacytoid DCs and several subsets of classical DCs that promote different immune effector modules in response to pathogens. In parallel, a correspondence between human and murine DC subsets has emerged, underlying structural similarities for the DC lineages between these species. Recent work has begun to unravel the transcriptional circuitry that controls the development and diversification of DCs from common progenitors in the bone marrow.

Transcriptional regulation of innate and adaptive lymphocyte lineages.

The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function.

The Source and Dynamics of Adult Hematopoiesis: Insights from Lineage Tracing.

The generation of all blood cell lineages (hematopoiesis) is sustained throughout the entire life span of adult mammals. Studies using cell transplantation identified the self-renewing, multipotent hematopoietic stem cells (HSCs) as the source of hematopoiesis in adoptive hosts and delineated a hierarchy of HSC-derived progenitors that ultimately yield mature blood cells. However, much less is known about adult hematopoiesis as it occurs in native hosts, i.e., without transplantation. Here we review recent advances in our understanding of native hematopoiesis, focusing in particular on the application of genetic lineage tracing in mice. The emerging evidence has established HSCs as the major source of native hematopoiesis, helped to define the kinetics of HSC differentiation, and begun exploring native hematopoiesis in stress conditions such as aging and inflammation. Major outstanding questions about native hematopoiesis still remain, such as its clonal composition, the nature of lineage commitment, and the dynamics of the process in humans.

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.

Pan-cellular organelles and suborganelles-from common functions to cellular diversity?

Cell diversification is at the base of increasing multicellular organism complexity in phylogeny achieved during ontogeny. However, there are also functions common to all cells, such as cell division, cell migration, translation, endocytosis, exocytosis, etc. Here we revisit the organelles involved in such common functions, reviewing recent evidence of unexpected differences of proteins at these organelles. For instance, centrosomes or mitochondria differ significantly in their protein composition in different, sometimes closely related, cell types. This has relevance for development and disease. Particularly striking is the high amount and diversity of RNA-binding proteins at these and other organelles, which brings us to review the evidence for RNA at different organelles and suborganelles. We include a discussion about (sub)organelles involved in translation, such as the nucleolus and ribosomes, for which unexpected cell type-specific diversity has also been reported. We propose here that the heterogeneity of these organelles and compartments represents a novel mechanism for regulating cell diversity. One reason is that protein functions can be multiplied by their different contributions in distinct organelles, as also exemplified by proteins with moonlighting function. The specialized organelles still perform pan-cellular functions but in a cell type-specific mode, as discussed here for centrosomes, mitochondria, vesicles, and other organelles. These can serve as regulatory hubs for the storage and transport of specific and functionally important regulators. In this way, they can control cell differentiation, plasticity, and survival. We further include examples highlighting the relevance for disease and propose to examine organelles in many more cell types for their possible differences with functional relevance.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development.

The challenges in recapitulating in vivo human T cell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34+ progenitor and the more differentiated CD34- fractions in the human postnatal thymus. CD34+ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual T cell commitment. The earliest progenitors in the differentiation trajectory were CD7- and expressed a stem-cell-like transcriptional profile, but had also initiated T cell priming. Clustering analysis identified a CD34+ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined T cell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human T cell development.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

Characterization of Transcriptional Regulatory Networks that Promote and Restrict Identities and Functions of Intestinal Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. ILCs exhibit phenotypic and functional plasticity in response to environmental stimuli, yet the transcriptional regulatory networks (TRNs) that control ILC function are largely unknown. Here, we integrate gene expression and chromatin accessibility data to infer regulatory interactions between transcription factors (TFs) and genes within intestinal type 1, 2, and 3 ILC subsets. We predicted the "core" TFs driving ILC identities, organized TFs into cooperative modules controlling distinct gene programs, and validated roles for c-MAF and BCL6 as regulators affecting type 1 and type 3 ILC lineages. The ILC network revealed alternative-lineage-gene repression, a mechanism that may contribute to reported plasticity between ILC subsets. By connecting TFs to genes, the TRNs suggest means to selectively regulate ILC effector functions, while our network approach is broadly applicable to identifying regulators in other in vivo cell populations.

The Histone Methyltransferase SETDB1 Controls T Helper Cell Lineage Integrity by Repressing Endogenous Retroviruses.

Upon activation, naive CD4+ T cells differentiate into distinct T cell subsets via processes reliant on epigenetically regulated, lineage-specific developmental programs. Here, we examined the function of the histone methyltransferase SETDB1 in T helper (Th) cell differentiation. Setdb1-/- naive CD4+ T cells exhibited exacerbated Th1 priming, and when exposed to a Th1-instructive signal, Setdb1-/- Th2 cells crossed lineage boundaries and acquired a Th1 phenotype. SETDB1 did not directly control Th1 gene promoter activity but relied instead on deposition of the repressive H3K9me3 mark at a restricted and cell-type-specific set of endogenous retroviruses (ERVs) located in the vicinity of genes involved in immune processes. Refined bioinformatic analyses suggest that these retrotransposons regulate Th1 gene cis-regulatory elements or act as Th1 gene enhancers. Thus, H3K9me3 deposition by SETDB1 ensures Th cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network.

The nature of embryonic stem cells.

Mouse embryonic stem (ES) cells perpetuate in vitro the broad developmental potential of naïve founder cells in the preimplantation embryo. ES cells self-renew relentlessly in culture but can reenter embryonic development seamlessly, differentiating on schedule to form all elements of the fetus. Here we review the properties of these remarkable cells. Arising from the stability, homogeneity, and equipotency of ES cells, we consider the concept of a pluripotent ground state. We evaluate the authenticity of ES cells in relation to cells in the embryo and examine their utility for dissecting mechanisms that confer pluripotency and that execute fate choice. We summarize current knowledge of the transcription factor circuitry that governs the ES cell state and discuss the opportunity to expose molecular logic further through iterative computational modeling and experimentation. Finally, we present a perspective on unresolved questions, including the challenge of deriving ground state pluripotent stem cells from non-rodent species.

Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells.

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4+CD8+ T cells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of T cell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Speed and navigation control of thymocyte development by the fetal T-cell gene regulatory network.

T-cell differentiation is a tightly regulated developmental program governed by interactions between transcription factors (TFs) and chromatin landscapes and affected by signals received from the thymic stroma. This process is marked by a series of checkpoints: T-lineage commitment, T-cell receptor (TCR)ß selection, and positive and negative selection. Dynamically changing combinations of TFs drive differentiation along the T-lineage trajectory, through mechanisms that have been most extensively dissected in adult mouse T-lineage cells. However, fetal T-cell development differs from adult in ways that suggest that these TF mechanisms are not fully deterministic. The first wave of fetal T-cell differentiation occurs during a unique developmental window during thymic morphogenesis, shows more rapid kinetics of differentiation with fewer rounds of cell division, and gives rise to unique populations of innate lymphoid cells (ILCs) and invariant γδT cells that are not generated in the adult thymus. As the characteristic kinetics and progeny biases are cell-intrinsic properties of thymic progenitors, the differences could be based on distinct TF network circuitry within the progenitors themselves. Here, we review recent single-cell transcriptome data that illuminate the TF networks involved in T-cell differentiation in the fetal and adult mouse thymus.

Development of γδ T cells in the thymus - A human perspective.

γδ T cells are increasingly emerging as crucial immune regulators that can take on innate and adaptive roles in the defence against pathogens. Although they arise within the thymus from the same hematopoietic precursors as conventional αß T cells, the development of γδ T cells is less well understood. In this review, we focus on summarising the current state of knowledge about the cellular and molecular processes involved in the generation of γδ T cells in human.

The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance.

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative "slender" forms differentiate into cell cycle arrested "stumpy" forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

Tet2 deletion in CD4+ T cells disrupts Th1 lineage commitment in memory cells and enhances T follicular helper cell recall responses to viral rechallenge.

Following viral clearance, antigen-specific CD4+ T cells contract and form a pool of distinct Th1 and Tfh memory cells that possess unique epigenetic programs, allowing them to rapidly recall their specific effector functions upon rechallenge. DNA methylation programing mediated by the methylcytosine dioxygenase Tet2 contributes to balancing Th1 and Tfh cell differentiation during acute viral infection; however, the role of Tet2 in CD4+ T cell memory formation and recall is unclear. Using adoptive transfer models of antigen-specific wild type and Tet2 knockout CD4+ T cells, we find that Tet2 is required for full commitment of CD4+ T cells to the Th1 lineage and that in the absence of Tet2, memory cells preferentially recall a Tfh like phenotype with enhanced expansion upon secondary challenge. These findings demonstrate an important role for Tet2 in enforcing lineage commitment and programing proliferation potential, and highlight the potential of targeting epigenetic programing to enhance adaptive immune responses.

Enhancer, transcriptional, and cell fate plasticity precedes intestinal determination during endoderm development.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

Three-step transcriptional priming that drives the commitment of multipotent progenitors toward B cells.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.