ThPOK is a critical multifaceted regulator of myeloid lineage development.

The transcription factor ThPOK (encoded by Zbtb7b) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention.

Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4+ T cell identity and function.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.

The role of ThPOK in control of CD4/CD8 lineage commitment.

During alphabeta T cell development, cells diverge into alternate CD4 helper and CD8(+) cytotoxic T cell lineages. The precise correlation between a T cell's CD8 and CD4 choice and its TCR specificity to class I or class II MHC was noted more than 20 years ago, and establishing the underlying mechanism has remained a focus of intense study since then. This review deals with three formerly discrete topics that are gradually becoming interconnected: the role of TCR signaling in lineage commitment, the regulation of expression of the CD4 and CD8 genes, and transcriptional regulation of lineage commitment. It is widely accepted that TCR signaling exerts a decisive influence on lineage choice, although the underlying mechanism remains intensely debated. Current evidence suggests that both duration and intensity of TCR signaling may control lineage choice, as proposed by the kinetic signaling and quantitative instructive models, respectively. Alternate expression of the CD4 and CD8 genes is the most visible manifestation of lineage choice, and much progress has been made in defining the responsible cis elements and transcription factors. Finally, important clues to the molecular basis of lineage commitment have been provided by the recent identification of the transcription factor ThPOK as a key regulator of lineage choice. ThPOK is selectively expressed in class II-restricted cells at the CD4(+)8(lo) stage and is necessary and sufficient for development to the CD4 lineage. Given the central role of ThPOK in lineage commitment, understanding its upstream regulation and downstream gene targets is expected to reveal further important aspects of the molecular machinery underlying lineage commitment.

Multifaceted control of T cell differentiation by STIM1.

In T cells, stromal interaction molecule (STIM) and Orai are dispensable for conventional T cell development, but critical for activation and differentiation. This review focuses on novel STIM-dependent mechanisms for control of Ca2+ signals during T cell activation and its impact on mitochondrial function and transcriptional activation for control of T cell differentiation and function. We highlight areas that require further work including the roles of plasma membrane Ca2+ ATPase (PMCA) and partner of STIM1 (POST) in controlling Orai function. A major knowledge gap also exists regarding the independence of T cell development from STIM and Orai, despite compelling evidence that it requires Ca2+ signals. Resolving these and other outstanding questions ensures that the field will remain active for many years to come.

Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Noncanonical mode of ERK action controls alternative αß and γδ T cell lineage fates.

Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.

Suppression of Ca2+ signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

Role of a selecting ligand in shaping the murine γδ-TCR repertoire.

Unlike αß-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.

Functional Conservation of a Developmental Switch in Mammals since the Jurassic Age.

ThPOK is a "master regulator" of T lymphocyte lineage choice, whose presence or absence is sufficient to dictate development to the CD4 or CD8 lineages, respectively. Induction of ThPOK is transcriptionally regulated, via a lineage-specific silencer element, SilThPOK. Here, we take advantage of the available genome sequence data as well as site-specific gene targeting technology, to evaluate the functional conservation of ThPOK regulation across mammalian evolution, and assess the importance of motif grammar (order and orientation of TF binding sites) on SilThPOK function in vivo. We make three important points: First, the SilThPOK is present in marsupial and placental mammals, but is not found in available genome assemblies of nonmammalian vertebrates, indicating that it arose after divergence of mammals from other vertebrates. Secondly, by replacing the murine SilThPOK in situ with its marsupial equivalent using a knockin approach, we demonstrate that the marsupial SilThPOK supports correct CD4 T lymphocyte lineage-specification in mice. To our knowledge, this is the first in vivo demonstration of functional equivalency for a silencer element between marsupial and placental mammals using a definitive knockin approach. Finally, we show that alteration of the position/orientation of a highly conserved region within the murine SilThPOK is sufficient to destroy silencer activity in vivo, demonstrating that motif grammar of this "solid" synteny block is critical for silencer function. Dependence of SilThPOK function on motif grammar conserved since the mid-Jurassic age, 165 Ma, suggests that the SilThPOK operates as a silenceosome, by analogy with the previously proposed enhanceosome model.

Marked induction of the helix-loop-helix protein Id3 promotes the gammadelta T cell fate and renders their functional maturation Notch independent.

alphabeta and gammadelta T cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify fate and promote the development of these lineages remain poorly understood. Here, we show that differential activation of the extracellular signal-related kinase (ERK)-early growth response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression served to regulate adoption of the gammadelta fate. Moreover, Id3 was both necessary and sufficient to enable gammadelta-lineage cells to differentiate independently of Notch signaling and become competent IFNgamma-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the gammadelta fate and its maturation in the thymus.

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas.

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αßTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

Antigen presentation to the T-cell receptor leads to sustained cytosolic Ca2+ elevation, which is critical for T-cell activation. We previously showed that in activated T cells, Ca2+ clearance is inhibited by the endoplasmic reticulum Ca2+ sensor stromal interacting molecule 1 (STIM1) via association with the plasma membrane Ca2+/ATPase 4 (PMCA4) Ca2+ pump. Having further observed that expression of both proteins is increased in activated T cells, the current study focused on mechanisms regulating both up-regulation of STIM1 and PMCA4 and assessing how this up-regulation contributes to control of Ca2+ clearance. Using a STIM1 promoter luciferase vector, we found that the zinc finger transcription factors early growth response (EGR) 1 and EGR4, but not EGR2 or EGR3, drive luciferase activity. We further found that neither STIM1 nor PMCA4 is up-regulated when both EGR1 and EGR4 are knocked down using RNA interference. Further, under these conditions, activation-induced Ca2+ clearance inhibition was eliminated with little effect on Ca2+ entry. Finally, we found that nuclear factor of activated T-cell (NFAT) activity is profoundly attenuated if Ca2+ clearance is not inhibited by STIM1. These findings reveal a critical role for STIM1-mediated control of Ca2+ clearance in NFAT induction during T-cell activation.-Samakai, E., Hooper, R., Martin, K. A., Shmurak, M., Zhang, Y., Kappes, D. J., Tempera, I., Soboloff, J. Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

CD4-CD8 lineage commitment is regulated by a silencer element at the ThPOK transcription-factor locus.

The transcription factor ThPOK is necessary and sufficient to trigger adoption of the CD4 lymphocyte fate. Here we investigate the regulation of ThPOK expression and its subsequent control of CD4+ T cell commitment. Treatment of immature thymocytes with anti-TCR (T cell receptor) showed that TCR signals were important in ThPOK induction and that the CD4+8lo stage was the likely target of the inductive TCR signal. We identified at the ThPOK locus a key distal regulatory element (DRE) that mediated its differential expression in class I- versus II-restricted CD4+8lo thymocytes. The DRE was both necessary for suppression of ThPOK expression in class I-restricted thymocytes and sufficient for its induction in class II-restricted thymocytes. Mutagenesis analysis defined an essential 80bp core DRE sequence and its potential regulatory motifs. We propose a silencer-dependent model of lineage choice, whereby inactivation of the DRE silencer by a strong TCR signal leads to CD4 commitment, whereas continued silencer activity leads to CD8 commitment.

Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells.

MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

TCR-mediated ThPOK induction promotes development of mature (CD24-) gammadelta thymocytes.

T lymphocytes develop into two major lineages characterized by expression of the alphabeta and gammadelta T cell receptor (TCR) heterodimers. Within each major lineage, further specialization occurs, resulting in distinct subsets that differ in TCR specificity, phenotype and functional attributes. Thus, in the murine thymus, two distinct subsets of mature (CD24-) gammadelta cells have been identified, that is NK1.1+ cells, which are enriched for Vgamma1.1 usage and selectively produce IFNgamma on stimulation, and CCR6+ cells, which are enriched for Vgamma2 usage produce IL17. The upstream signals and transcriptional pathways that promote development of these distinct gammadelta subsets remain relatively poorly understood. Here, we show that the Zn-finger transcription factor ThPOK has a critical function in the development of gammadelta thymocytes. Thus, lack of functional ThPOK causes a marked reduction in the percentage and absolute number of mature gammadelta thymocytes, and a particularly severe reduction of NK1.1+ cells. Conversely, constitutive ThPOK expression leads to a striking increase in mature NK1.1+ gammadelta thymocytes. Further, we show that ThPOK induction in gammadelta thymocytes is induced by strong TCR signals mediated by engagement with antibody or high-affinity endogenous ligands, and that an important ThPOK cis-acting element, the distal regulatory element (DRE), is sufficient for this TCR-dependent induction. These results show that ThPOK expression in gammadelta thymocytes is regulated in part by the strength of TCR signalling, identify ThPOK as an important mediator of gammadelta T cell development/maturation, and lend strong support to the view that development of a significant fraction of gammadelta T cells depends on TCR engagement/signalling.

Towards a molecular understanding of the differential signals regulating alphabeta/gammadelta T lineage choice.

While insights into the molecular processes that specify adoption of the alphabeta and gammadelta fates are beginning to emerge, the basis for control of specification remains highly controversial. This review highlights the current models attempting to explain T lineage commitment. Recent observations support the hypothesis that the T cell receptor (TCR) provides instructive cues through differences in TCR signaling intensity and/or longevity. Accordingly, we review evidence addressing the importance of differences in signal strength/longevity, how signals differing in intensity/longevity may be generated, and finally how such signals modulate the activity of downstream effectors to promote the opposing developmental fates.

Expanding roles for ThPOK in thymic development.

The role of the zinc finger transcription factor ThPOK (T-helper-inducing POZ-Kruppel-like factor) in promoting commitment of αß T cells to the CD4 lineage is now well established. New results indicate that ThPOK is also important for the development and/or acquisition of effector functions by other T cell subsets, including several not marked by CD4 expression, i.e. double-negative invariant natural killer T (iNKT) cells, γδ cells, and even memory CD8(+) T cells. There is compelling evidence that ThPOK expression in most or all of these cases is dependent on T-cell receptor signaling and that differences in relative TCR signal strength/length may induce different levels of ThPOK expression. The developmental consequences of ThPOK expression vary according to cell type, which may partly reflect differences in ThPOK levels and/or in transcriptional networks between cell types.