Multi-scale Dynamical Modeling of T Cell Development from an Early Thymic Progenitor State to Lineage Commitment.

Intrathymic development of committed progenitor (pro)-T cells from multipotent hematopoietic precursors offers an opportunity to dissect the molecular circuitry establishing cell identity in response to environmental signals. This transition encompasses programmed shutoff of stem/progenitor genes, upregulation of T cell specification genes, proliferation, and ultimately commitment. To explain these features in light of reported cis-acting chromatin effects and experimental kinetic data, we develop a three-level dynamic model of commitment based upon regulation of the commitment-linked gene Bcl11b. The levels are (1) a core gene regulatory network (GRN) architecture from transcription factor (TF) perturbation data, (2) a stochastically controlled chromatin-state gate, and (3) a single-cell proliferation model validated by experimental clonal growth and commitment kinetic assays. Using RNA fluorescence in situ hybridization (FISH) measurements of genes encoding key TFs and measured bulk population dynamics, this single-cell model predicts state-switching kinetics validated by measured clonal proliferation and commitment times. The resulting multi-scale model provides a mechanistic framework for dissecting commitment dynamics.

Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Single-Cell Analysis Reveals Regulatory Gene Expression Dynamics Leading to Lineage Commitment in Early T Cell Development.

Intrathymic T cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T cell genes, but the underlying steps have remained obscure. Single-cell profiling was used to define the order of regulatory changes, employing single-cell RNA sequencing (scRNA-seq) for full-transcriptome analysis, plus sequential multiplexed single-molecule fluorescent in situ hybridization (seqFISH) to quantitate functionally important transcripts in intrathymic precursors. Single-cell cloning verified high T cell precursor frequency among the immunophenotypically defined "early T cell precursor" (ETP) population; a discrete committed granulocyte precursor subset was also distinguished. We established regulatory phenotypes of sequential ETP subsets, confirmed initial co-expression of progenitor with T cell specification genes, defined stage-specific relationships between cell cycle and differentiation, and generated a pseudotime model from ETP to T lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to T cell specification.

A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment.

Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.

Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16.

Multipotent progenitor cells confirm their T cell-lineage identity in the CD4-CD8- double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.

Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice.

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel-deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel-deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4(+) and CD8(+) T cells from c-Rel-deficient NOD mice showed significantly decreased T-cell receptor-induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel-competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel-deficient NOD mice. The results suggest that c-Rel-dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel-dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell-dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity.

Hematopoiesis and T-cell specification as a model developmental system.

The pathway to generate T cells from hematopoietic stem cells guides progenitors through a succession of fate choices while balancing differentiation progression against proliferation, stage to stage. Many elements of the regulatory system that controls this process are known, but the requirement for multiple, functionally distinct transcription factors needs clarification in terms of gene network architecture. Here, we compare the features of the T-cell specification system with the rule sets underlying two other influential types of gene network models: first, the combinatorial, hierarchical regulatory systems that generate the orderly, synchronized increases in complexity in most invertebrate embryos; second, the dueling 'master regulator' systems that are commonly used to explain bistability in microbial systems and in many fate choices in terminal differentiation. The T-cell specification process shares certain features with each of these prevalent models but differs from both of them in central respects. The T-cell system is highly combinatorial but also highly dose-sensitive in its use of crucial regulatory factors. The roles of these factors are not always T-lineage-specific, but they balance and modulate each other's activities long before any mutually exclusive silencing occurs. T-cell specification may provide a new hybrid model for gene networks in vertebrate developmental systems.

Developmental gene networks: a triathlon on the course to T cell identity.

Cells acquire their ultimate identities by activating combinations of transcription factors that initiate and sustain expression of the appropriate cell type-specific genes. T cell development depends on the progression of progenitor cells through three major phases, each of which is associated with distinct transcription factor ensembles that control the recruitment of these cells to the thymus, their proliferation, lineage commitment and responsiveness to T cell receptor signals, all before the allocation of cells to particular effector programmes. All three phases are essential for proper T cell development, as are the mechanisms that determine the boundaries between each phase. Cells that fail to shut off one set of regulators before the next gene network phase is activated are predisposed to leukaemic transformation.

Transcriptional establishment of cell-type identity: dynamics and causal mechanisms of T-cell lineage commitment.

Precursor cell entry into the T-cell developmental pathway can be divided into two phases by the closure of T-lineage commitment. As cells decide against the last alternative options to the T-cell fate, they turn on the transcription factor Bcl11b and silence expression of a group of multipotent progenitor regulatory factors that include hematopoietic transcription factor PU.1. Functional perturbation tests show that Bcl11b is needed for commitment while PU.1 actively participates in keeping open access to alternative fates, until it is silenced; however, PU.1 and Bcl11b both contribute positively to T-cell development. Our recent work reviewed here sheds light on the transcriptional regulatory network that determines the timing and irreversibility of Bcl11b activation, the ways that Notch signaling from the thymic microenvironment restricts the action of PU.1 to prevent it from diverting cells to non-T fates, and the target genes that PU.1 still regulates under the influence of Notch signaling to contribute to T-cell generation. We argue that T-cell development depends on the sequential operation of two interlaced, but mutually antagonistic, gene regulatory networks, one initially supporting expansion before commitment and the other imposing a "terminal" differentiation process on committed cells.

Loss of T cell progenitor checkpoint control underlies leukemia initiation in Rag1-deficient nonobese diabetic mice.

NOD mice exhibit major defects in the earliest stages of T cell development in the thymus. Genome-wide genetic and transcriptome analyses were used to investigate the origins and consequences of an early T cell developmental checkpoint breakthrough in Rag1-deficient NOD mice. Quantitative trait locus analysis mapped the presence of checkpoint breakthrough cells to several known NOD diabetes susceptibility regions, particularly insulin-dependent diabetes susceptibility genes (Idd)9/11 on chromosome 4, suggesting common genetic origins for T cell defects affecting this trait and autoimmunity. Genome-wide RNA deep-sequencing of NOD and B6 Rag1-deficient thymocytes revealed the effects of genetic background prior to breakthrough, as well as the cellular consequences of the breakthrough. Transcriptome comparison between the two strains showed enrichment in differentially expressed signal transduction genes, prominently tyrosine kinase and actin-binding genes, in accord with their divergent sensitivities to activating signals. Emerging NOD breakthrough cells aberrantly expressed both stem cell-associated proto-oncogenes, such as Lmo2, Hhex, Lyl1, and Kit, which are normally repressed at the commitment checkpoint, and post-ß-selection checkpoint genes, including Cd2 and Cd5. Coexpression of genes characteristic of multipotent progenitors and more mature T cells persists in the expanding population of thymocytes and in the thymic leukemias that emerge with age in these mice. These results show that Rag1-deficient NOD thymocytes have T cell defects that can collapse regulatory boundaries at two early T cell checkpoints, which may predispose them to both leukemia and autoimmunity.

Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αß T cell development in nonobese diabetic mice.

The first TCR-dependent checkpoint in the thymus determines αß versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo. Cultured CD4 and CD8 double-negative cells from NOD mice exhibited major defects in the generation of CD4 and CD8 double-positive αß T cells, whereas γδ T cell development from bipotent precursors was enhanced. Limiting dilution and single-cell experiments show that the divergent effects on αß and γδ T cell development did not spring from biased lineage choice but from increased proliferation of γδ T cells and impaired accumulation of αß T lineage double-positive cells. In vivo, NOD early T cell subsets in the thymus also show characteristics indicative of defective ß-selection, and peripheral αß T cells are poorly established in mixed bone marrow chimeras, contrasting with strong γδ T as well as B cell repopulation. Thus, NOD T cell precursors reveal divergent, lineage-specific differentiation abnormalities in vitro and in vivo from the first TCR-dependent developmental choice point, which may have consequences for subsequent lineage decisions and effector functions.

Fine-scale staging of T cell lineage commitment in adult mouse thymus.

T cell development is marked by the loss of alternative lineage choices accompanying specification and commitment to the T cell lineage. Commitment occurs between the CD4 and CD8 double-negative (DN) 2 and DN3 stages in mouse early T cells. To determine the gene regulatory changes that accompany commitment, we sought to distinguish and characterize the earliest committed wild-type DN adult thymocytes. A transitional cell population, defined by the first downregulation of surface c-Kit expression, was found to have lost the ability to differentiate into dendritic cells and NK cells when cultured without Notch-Delta signals. In the presence of Notch signaling, this subset generates T lineage descendants in an ordered precursor-product relationship between DN2, with the highest levels of surface c-Kit, and c-Kit-low DN3 cells. These earliest committed cells show only a few differences in regulatory gene expression, compared with uncommitted DN2 cells. They have not yet established the full expression of Notch-related and T cell differentiation genes characteristic of DN3 cells before beta selection. Instead, the downregulation of select stem cell and non-T lineage genes appears to be key to the extinction of alternative lineage choices.

Transcription factor expression dynamics of early T-lymphocyte specification and commitment.

Mammalian T lymphocytes are a prototype for development from adult pluripotent stem cells. While T-cell specification is driven by Notch signaling, T-lineage commitment is only finalized after prolonged Notch activation. However, no T-lineage specific regulatory factor has been reported that mediates commitment. We used a gene-discovery approach to identify additional candidate T-lineage transcription factors and characterized expression of >100 regulatory genes in early T-cell precursors using realtime RT-PCR. These regulatory genes were also monitored in multilineage precursors as they entered T-cell or non-T-cell pathways in vitro; in non-T cells ex vivo; and in later T-cell developmental stages after lineage commitment. At least three major expression patterns were observed. Transcription factors in the largest group are expressed at relatively stable levels throughout T-lineage specification as a legacy from prethymic precursors, with some continuing while others are downregulated after commitment. Another group is highly expressed in the earliest stages only, and is downregulated before or during commitment. Genes in a third group undergo upregulation at one of three distinct transitions, suggesting a positive regulatory cascade. However, the transcription factors induced during commitment are not T-lineage specific. Different members of the same transcription factor family can follow opposite trajectories during specification and commitment, while factors co-expressed early can be expressed in divergent patterns in later T-cell development. Some factors reveal new regulatory distinctions between alphabeta and gammadelta T-lineage differentiation. These results show that T-cell identity has an essentially complex regulatory basis and provide a detailed framework for regulatory network modeling of T-cell specification.

A gene regulatory network armature for T lymphocyte specification.

Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

Launching the T-cell-lineage developmental programme.

Multipotent blood progenitor cells enter the thymus and begin a protracted differentiation process in which they gradually acquire T-cell characteristics while shedding their legacy of developmental plasticity. Notch signalling and basic helix-loop-helix E-protein transcription factors collaborate repeatedly to trigger and sustain this process throughout the period leading up to T-cell lineage commitment. Nevertheless, the process is discontinuous with separately regulated steps that demand roles for additional collaborating factors. This Review discusses new evidence on the coordination of specification and commitment in the early T-cell pathway; effects of microenvironmental signals; the inheritance of stem-cell regulatory factors; and the ensemble of transcription factors that modulate the effects of Notch and E proteins, to distinguish individual stages and to polarize T-cell-lineage fate determination.

Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3.

GATA-3 is essential for T cell development from the earliest stages. However, abundant GATA-3 can drive T lineage precursors to a non-T cell fate, depending on Notch signaling and developmental stage. Here, overexpression of GATA-3 blocked the survival of pro-T cells when Notch-Delta signals were present but enhanced viability in their absence. In fetal thymocytes at the double-negative 1 (DN1) stage and DN2 stage but not those at the DN3 stage, overexpression of GATA-3 rapidly induced respecification to the mast cell lineage with high frequency by direct transcriptional 'reprogramming'. Normal DN2 thymocytes also showed mast cell potential when interleukin 3 and stem cell factor were added in the absence of Notch signaling. Our results suggest a close relationship between the pro-T cell and mast cell programs and a previously unknown function for Notch in T lineage fidelity.

Notch/Delta signaling constrains reengineering of pro-T cells by PU.1.

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through beta-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo beta-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment.

Progression of regulatory gene expression states in fetal and adult pro-T-cell development.

Precursors entering the T-cell developmental pathway traverse a progression of states characterized by distinctive patterns of gene expression. Of particular interest are regulatory genes, which ultimately control the dwell time of cells in each state and establish the mechanisms that propel them forward to subsequent states. Under particular genetic and developmental circumstances, the transitions between these states occur with different timing, and environmental feedbacks may shift the steady-state accumulations of cells in each state. The fetal transit through pro-T-cell stages is faster than in the adult and subject to somewhat different genetic requirements. To explore causes of such variation, this review presents previously unpublished data on differentiation gene activation in pro-T cells of pre-T-cell receptor-deficient mutant mice and a quantitative comparison of the profiles of transcription factor gene expression in pro-T-cell subsets of fetal and adult wildtype mice. Against a background of consistent gene expression, several regulatory genes show marked differences between fetal and adult expression profiles, including those encoding two basic helix-loop-helix antagonist Id factors, the Ets family factor SpiB and the Notch target gene Deltex1. The results also reveal global differences in regulatory alterations triggered by the first T-cell receptor-dependent selection events in fetal and adult thymopoiesis.

Developmental and molecular characterization of emerging beta- and gammadelta-selected pre-T cells in the adult mouse thymus.

The first checkpoint in T cell development, beta selection, has remained incompletely characterized for lack of specific surface markers. We show that CD27 is upregulated in DN3 thymocytes initiating beta selection, concomitant with intracellular TCR-beta expression. Clonal analysis determined that CD27high DN3 cells generate CD4+CD8+ progeny with more than 90% efficiency, faster and more efficiently than the CD27low majority. CD27 upregulation also occurs in gammadelta-selected DN3 thymocytes in TCR-beta-/- mice and in IL2-GFP transgenic reporter mice where GFP marks the earliest emerging TCR-gammadelta cells from DN3 thymocytes. With CD27 to distinguish pre- and postselection DN3 cells, a detailed gene expression analysis defined regulatory changes associated with checkpoint arrest, with beta selection, and with gammadelta selection. gammadelta selection induces higher CD5, Egr, and Runx3 expression as compared to beta selection, but it triggers less proliferation. Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing alphabeta and gammadelta T-lineage cells.