T-cell commitment inheritance-an agent-based multi-scale model.

T-cell development provides an excellent model system for studying lineage commitment from a multipotent progenitor. The intrathymic development process has been thoroughly studied. The molecular circuitry controlling it has been dissected and the necessary steps like programmed shut off of progenitor genes and T-cell genes upregulation have been revealed. However, the exact timing between decision-making and commitment stage remains unexplored. To this end, we implemented an agent-based multi-scale model to investigate inheritance in early T-cell development. Treating each cell as an agent provides a powerful tool as it tracks each individual cell of a simulated T-cell colony, enabling the construction of lineage trees. Based on the lineage trees, we introduce the concept of the last common ancestors (LCA) of committed cells and analyse their relations, both at single-cell level and population level. In addition to simulating wild-type development, we also conduct knockdown analysis. Our simulations predicted that the commitment is a three-step process that occurs on average over several cell generations once a cell is first prepared by a transcriptional switch. This is followed by the loss of the Bcl11b-opposing function approximately two to three generations later. This is when our LCA analysis indicates that the decision to commit is taken even though in general another one to two generations elapse before the cell actually becomes committed by transitioning to the DN2b state. Our results showed that there is decision inheritance in the commitment mechanism.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.

Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction.

Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with a high signal-to-background ratio in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of three different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with one-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

T-cell commitment inheritance - an agent-based multi-scale model.

T-cell development provides an excellent model system for studying lineage commitment from a multipotent progenitor. The intrathymic development process has been thoroughly studied. The molecular circuitry controlling it has been dissected and the necessary steps like programmed shut off of progenitor genes and T-cell genes upregulation have been revealed. However, the exact timing between decision-making and commitment stage remains unexplored. To this end, we implemented an agent-based multi-scale model to investigate inheritance in early T-cell development. Treating each cell as an agent provides a powerful tool as it tracks each individual cell of a simulated T-cell colony, enabling the construction of lineage trees. Based on the lineage trees, we introduce the concept of the last common ancestors (LCA) of committed cells and analyse their relations, both at single-cell level and population level. In addition to simulating wild-type development, we also conduct knockdown analysis. Our simulations showed that the commitment is a three-step process over several cell generations where a cell is first prepared by a transcriptional switch. This is followed by the loss of the Bcl11b-opposing function two to three generations later which is when the decision to commit is taken. Finally, after another one to two generations, the cell becomes committed by transitioning to the DN2b state. Our results showed that there is inheritance in the commitment mechanism.

Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms.

Runx factors are essential for lineage specification of various hematopoietic cells, including T lymphocytes. However, they regulate context-specific genes and occupy distinct genomic regions in different cell types. Here, we show that dynamic Runx binding shifts in mouse early T cell development are mostly not restricted by local chromatin state but regulated by Runx dosage and functional partners. Runx cofactors compete to recruit a limited pool of Runx factors in early T progenitor cells, and a modest increase in Runx protein availability at pre-commitment stages causes premature Runx occupancy at post-commitment binding sites. This increased Runx factor availability results in striking T cell lineage developmental acceleration by selectively activating T cell-identity and innate lymphoid cell programs. These programs are collectively regulated by Runx together with other, Runx-induced transcription factors that co-occupy Runx-target genes and propagate gene network changes.

Cold Spring Harbor Laboratory 2022: emerging insights and viewpoints in immunology.

Some of the current and former organizers of the Cold Spring Harbor Laboratory (CSHL) 'Gene Expression and Signaling in the Immune System' (GESIS) meeting offer opinions on emerging questions in immunology, discussing the strong value of this recurring scientific meeting in the field.

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.

Multi-modular structure of the gene regulatory network for specification and commitment of murine T cells.

T cells develop from multipotent progenitors by a gradual process dependent on intrathymic Notch signaling and coupled with extensive proliferation. The stages leading them to T-cell lineage commitment are well characterized by single-cell and bulk RNA analyses of sorted populations and by direct measurements of precursor-product relationships. This process depends not only on Notch signaling but also on multiple transcription factors, some associated with stemness and multipotency, some with alternative lineages, and others associated with T-cell fate. These factors interact in opposing or semi-independent T cell gene regulatory network (GRN) subcircuits that are increasingly well defined. A newly comprehensive picture of this network has emerged. Importantly, because key factors in the GRN can bind to markedly different genomic sites at one stage than they do at other stages, the genes they significantly regulate are also stage-specific. Global transcriptome analyses of perturbations have revealed an underlying modular structure to the T-cell commitment GRN, separating decisions to lose "stem-ness" from decisions to block alternative fates. Finally, the updated network sheds light on the intimate relationship between the T-cell program, which depends on the thymus, and the innate lymphoid cell (ILC) program, which does not.

Single-cell deletion analyses show control of pro-T cell developmental speed and pathways by Tcf7, Spi1, Gata3, Bcl11a, Erg, and Bcl11b.

As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.

Transcription factors specifically control change.

Transcription factors are defined by their sequence-specific binding to DNA and by their selective impacts on gene expression, depending on specific binding sites. The factor binding motifs in the DNA should thus represent a blueprint of regulatory logic, suggesting that transcription factor binding patterns on the genome (e.g., measured by ChIP-seq) should indicate which target genes the factors are directly controlling. However, although genetic data confirm high impacts of transcription factor perturbation in embryology, transcription factors bind to far more sites than the number of genes they dynamically regulate, when measured by direct perturbation in a given cell type. Also, deletion of carefully chosen transcription factor binding sites often gives disappointingly weak results. In a new study in the previous issue of Genes & Development, Lo and colleagues (pp. 1079-1095) reconcile these contradictions by using an elegant experimental system to directly compare the roles of transcription factor-binding site interaction in gene regulation maintenance with roles of the same factor-site interactions in gene regulation through developmental change. They examine Oct4:Sox2 shared target genes under maintained versus reinduced pluripotency conditions within the same cell clone. The results show that the same factor-site interaction impacts can appear modest in assays in developmental steady-state but are far more important as regulatory catalysts of developmental change.

Stage-specific action of Runx1 and GATA3 controls silencing of PU.1 expression in mouse pro-T cells.

PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be down-regulated during T lineage commitment. The transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown. We show that increasing Runx1 and/or GATA3 down-regulates Spi1 expression in pro-T cells, while deletion of these factors after Spi1 down-regulation reactivates its expression. Leveraging the stage specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro-T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro-T cells during commitment. Thus, Runx1 and GATA3 work stage specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T lineage commitment.

Epigenetic Dynamics in the Function of T-Lineage Regulatory Factor Bcl11b.

The transcription factor Bcl11b is critically required to support the development of diverse cell types, including T lymphocytes, type 2 innate lymphoid cells, neurons, craniofacial mesenchyme and keratinocytes. Although in T cell development its onset of expression is tightly linked to T-lymphoid lineage commitment, the Bcl11b protein in fact regulates substantially different sets of genes in different lymphocyte populations, playing strongly context-dependent roles. Somewhat unusually for lineage-defining transcription factors with site-specific DNA binding activity, much of the reported chromatin binding of Bcl11b appears to be indirect, or guided in large part by interactions with other transcription factors. We describe evidence suggesting that a further way in which Bcl11b exerts such distinct stage-dependent functions is by nucleating changes in regional suites of epigenetic modifications through recruitment of multiple families of chromatin-modifying enzyme complexes. Herein we explore what is - and what remains to be - understood of the roles of Bcl11b, its cofactors, and how it modifies the epigenetic state of the cell to enforce its diverse set of context-specific transcriptional and developmental programs.

Logic and lineage impacts on functional transcription factor deployment for T-cell fate commitment.

Transcription factors are the major agents that read the regulatory sequence information in the genome to initiate changes in expression of specific genes, both in development and in physiological activation responses. Their actions depend on site-specific DNA binding and are largely guided by their individual DNA target sequence specificities. However, their action is far more conditional in a real developmental context than would be expected for simple reading of local genomic DNA sequence, which is common to all cells in the organism. They are constrained by slow-changing chromatin states and by interactions with other transcription factors, which affect their occupancy patterns of potential sites across the genome. These mechanisms lead to emergent discontinuities in function even for transcription factors with minimally changing expression. This is well revealed by diverse lineages of blood cells developing throughout life from hematopoietic stem cells, which use overlapping combinations of transcription factors to drive strongly divergent gene regulation programs. Here, using development of T lymphocytes from hematopoietic multipotent progenitor cells as a focus, recent evidence is reviewed on how binding specificity and dynamics, transcription factor cooperativity, and chromatin state changes impact the effective regulatory functions of key transcription factors including PU.1, Runx1, Notch-RBPJ, and Bcl11b.

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.

Runx1 and Runx3 drive progenitor to T-lineage transcriptome conversion in mouse T cell commitment via dynamic genomic site switching.

Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental "ratchet" mechanism making commitment irreversible.

Single-cell insights into the hematopoietic generation of T-lymphocyte precursors in mouse and human.

T-Cell development is a major branch of lymphoid development and a key output of hematopoiesis, especially in early life, but the molecular requirements for T-cell potential have remained obscure. Considerable advances have now been made toward solving this problem through single-cell transcriptome studies, interfaced with in vitro differentiation assays that monitor potential efficiently at the single-cell level. This review focuses on a series of recent reports studying mouse and human early T-cell precursors, both in the developing fetus and in stringently purified postnatal samples of intrathymic and prethymic T-lineage precursors. Cross-comparison of results reveals a robustly conserved core program in mouse and human, but with some informative and provocative variations between species and between ontogenic states. Repeated findings are the multipotent progenitor regulatory signature of thymus-seeding cells and the proximity of the T-cell program to dendritic cell programs, especially to plasmacytoid dendritic cells in humans.