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

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.

Interaction between γδTCR signaling and the E protein-Id axis in γδ T cell development.

γδ T cells acquire their functional properties in the thymus, enabling them to exert rapid innate-like responses. To understand how distinct γδ T cell subsets are generated, we have developed a Two-Stage model for γδ T cell development. This model is predicated on the finding that γδTCR signal strength impacts E protein activity through graded upregulation of Id3. Our model proposes that cells enter Stage 1 in response to a γδTCR signaling event in the cortex that activates a γδ T cell-specific gene network. Part of this program includes the upregulation of chemokine receptors that guide them to the medulla. In the medulla, Stage 1 cells receive distinct combinations of γδTCR, cytokine, and/co-stimulatory signals that induce their transit into Stage 2, either toward the γδT1 or the γδT17 lineage. The intersection between γδTCR and cytokine signals can tune Id3 expression, leading to different outcomes even in the presence of strong γδTCR signals. The thymic signaling niches required for γδT17 development are segregated in time and space, providing transient windows of opportunity during ontogeny. Understanding the regulatory context in which E proteins operate at different stages will be key in defining how their activity levels impose functional outcomes.

E2A regulates neural ectoderm fate specification in human embryonic stem cells.

E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.

Cutting Edge: TCR-ß Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.

Integration of T-cell receptor, Notch and cytokine signals programs mouse γδ T-cell effector differentiation.

γδ T-cells perform a wide range of tissue- and disease-specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2-/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T-cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1ß, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets.

A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology.

A key role for IL-7R in the generation of microenvironments required for thymic dendritic cells.

Interleukin-7 receptor (IL-7R) signaling is critical for multiple stages of T-cell development, but a role in the establishment of the mature thymic architecture needed for T-cell development and thymocyte selection has not been established. Crosstalk signals between developing thymocytes and thymic epithelial cell (TEC) precursors are critical for their differentiation into cortical TECs (cTECs) and medullary TECs (mTECs). In addition, mTEC-derived factors have been implicated in the recruitment of thymic dendritic cells (DCs) and intrathymic DC development. We therefore examined corticomedullary structure and DC populations in the thymus of Il7r-/- mice. Analysis of TEC phenotype and spatial organization revealed a striking shift in the mTEC to cTEC ratio, accompanied by disorganized corticomedullary structure. Several of the thymic subsets known to have DC potential were nearly absent, accompanied by reductions in DC cell numbers. We also examined chemokine expression in the Il7r-/- thymus, and found a significant decrease in mTEC-derived CCR7 ligand expression, and high levels of cTEC-derived chemokines, including CCL25 and CXCL12. Although splenic DCs were similarly affected, bone marrow (BM) precursors capable of giving rise to DCs were unperturbed. Finally, BM chimeras showed that there was no intrinsic need for IL-7R signaling in the development or recruitment of thymic DCs, but that the provision of wild-type progenitors enhanced reconstitution of thymic DCs from Il7r-/- progenitors. Our results are therefore supportive of a model in which Il7r-dependent cells are required to set up the microenvironments that allow accumulation of thymic DCs.

Transcriptional priming of intrathymic precursors for dendritic cell development.

Specialized dendritic cells (DCs) within the thymus are crucial for the deletion of autoreactive T cells. The question of whether these cells arise from intrathymic precursors with T-cell potential has been hotly debated, and the regulatory pathways and signals that direct their development remain unclear. Here, we compared the gene expression profiles of thymic DC subsets with those of four early thymic precursor subsets: early T-cell precursors (ETPs), double-negative 1c (DN1c), double-negative 1d (DN1d) and double-negative 1e (DN1e) subsets. We found that the DN1d subset expressed Spi-B, HEBCan, Ccr7 and Ccr4, similar to thymic plasmacytoid DCs, whereas the DN1e subset expressed Id2, Ccr7 and Ccr4, similar to thymic conventional DCs. The expression of Ccr7 and Ccr4 in DN1d and DN1e cells suggested that they might be able to migrate towards the medulla (low in Dll proteins) and away from the cortex (high in Dll proteins) where early T-cell development occurs. We therefore assessed the sensitivity of developing DC precursors to Dll-Notch signaling, and found that high levels of Dll1 or Dll4 were inhibitory to DC development, whereas medium levels of Dll4 allowed DC development but not myeloid development. To evaluate directly the lineage potential of the ETP, DN1d and DN1e subsets, we injected them into nonirradiated congenic hosts intrathymically or intravenously, and found that they were all able to form medullary DCs in vivo. Therefore, DN1d and DN1e cells are transcriptionally primed to home to the thymus, migrate into DC-permissive microenvironments and develop into medullary DCs.

Gamma delta T-cell differentiation and effector function programming, TCR signal strength, when and how much?

γδ T-cells boast an impressive functional repertoire that can paint them as either champions or villains depending on the environmental and immunological cues. Understanding the function of the various effector γδ subsets necessitates tracing the developmental program of these subsets, including the point of lineage bifurcation from αß T-cells. Here, we review the importance of signals from the T-cell receptor (TCR) in determining αß versus γδ lineage fate, and further discuss how the molecular components of this pathway may influence the developmental programming of γδ T-cells functional subsets. Additionally, we discuss the role of temporal windows in restricting the development of IL-17 producing γδ T-cell subtypes, and explore whether fetal and adult hematopoietic progenitors maintain the same potential for giving rise to this important subset.

Context-dependent regulation of hematopoietic lineage choice by HEBAlt.

Hematopoietic development is controlled by combinatorial interactions between E-protein transcription factors and other lineage regulators that operate in the context of gene-regulatory networks. The E-proteins HEB and E2A are critical for T cell and B cell development, but the mechanisms by which their activities are directed to different genes in each lineage are unclear. We found that a short form of HEB, HEBAlt, acts downstream of Delta-like (DL)-Notch signaling to promote T cell development. In this paper, we show that forced expression of HEBAlt in mouse hematopoietic progenitors inhibited B cell development, but it allowed them to adopt a myeloid fate. HEBAlt interfered with the activity of E2A homodimers and with the expression of the transcription factor Pax5, both of which are critical for B cell development. However, when combined with DL-Notch signaling, HEBAlt enhanced the generation of T cell progenitors at the expense of myeloid cells. The longer form of HEB, HEBCan, also inhibited E47 activity and Pax5 expression, but it did not collaborate with DL-Notch signaling to suppress myeloid potential. Therefore, HEBAlt can suppress B cell or myeloid potential in a context-specific manner, which suggests a role for this factor in maintaining T lineage priming prior to commitment.

HEB in the spotlight: Transcriptional regulation of T-cell specification, commitment, and developmental plasticity.

The development of T cells from multipotent progenitors in the thymus occurs by cascades of interactions between signaling molecules and transcription factors, resulting in the loss of alternative lineage potential and the acquisition of the T-cell functional identity. These processes require Notch signaling and the activity of GATA3, TCF1, Bcl11b, and the E-proteins HEB and E2A. We have shown that HEB factors are required to inhibit the thymic NK cell fate and that HEBAlt allows the passage of T-cell precursors from the DN to DP stage but is insufficient for suppression of the NK cell lineage choice. HEB factors are also required to enforce the death of cells that have not rearranged their TCR genes. The synergistic interactions between Notch1, HEBAlt, HEBCan, GATA3, and TCF1 are presented in a gene network model, and the influence of thymic stromal architecture on lineage choice in the thymus is discussed.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

Direct regulation of TCR rearrangement and expression by E proteins during early T cell development.

γδ T cells are widely distributed throughout mucosal and epithelial cell-rich tissues and are an important early source of IL-17 in response to several pathogens. Like αß T cells, γδ T cells undergo a stepwise process of development in the thymus that requires recombination of genome-encoded segments to assemble mature T cell receptor (TCR) genes. This process is tightly controlled on multiple levels to enable TCR segment assembly while preventing the genomic instability inherent in the double-stranded DNA breaks that occur during this process. Each TCR locus has unique aspects in its structure and requirements, with different types of regulation before and after the αß/γδ T cell fate choice. It has been known that Runx and Myb are critical transcriptional regulators of TCRγ and TCRδ expression, but the roles of E proteins in TCRγ and TCRδ regulation have been less well explored. Multiple lines of evidence show that E proteins are involved in TCR expression at many different levels, including the regulation of Rag recombinase gene expression and protein stability, induction of germline V segment expression, chromatin remodeling, and restriction of the fetal and adult γδTCR repertoires. Importantly, E proteins interact directly with the cis-regulatory elements of the TCRγ and TCRδ loci, controlling the predisposition of a cell to become an αß T cell or a γδ T cell, even before the lineage-dictating TCR signaling events. This article is categorized under: Immune System Diseases > Stem Cells and Development Immune System Diseases > Genetics/Genomics/Epigenetics.

Fetal Thymic Organ Culture (FTOC) Optimized for Gamma-Delta T Cell Studies.

Fetal thymic organ culture (FTOC) provides a method for analyzing T cell development in a physiological context outside the animal. This technique enables studies of genetically altered mice that are embryonic or neonatal lethal, in addition to bypassing the complication of migration of successive waves of T cells out of the thymus. The hanging drop method involves depletion of thymocytes from host lobes using deoxyguanosine, followed by reconstitution with hematopoietic progenitors. This method has become standard for analysis of fetal liver precursors, bone marrow precursors, and early thymocytes. However, difficulties are encountered in the analysis of γδ T cell precursors using this method. We have developed a modification of FTOC in which partial depletion of hematopoietic precursors by shortened deoxyguanosine treatment, coupled with the use of TCRδ-deficient host lobes, enables engraftment and development of fetal γδTCR+ thymocytes. This method allows comparisons of development and functional differentiation of γδ T cell precursors between cells of different genotypes or treatments, in the context of a permissive thymic microenvironment.

Regulation of the Signal-Dependent E Protein HEBAlt Through a YYY Motif Is Required for Progression Through T Cell Development.

The E protein transcription factors E2A and HEB are critical for many developmental processes, including T cell development. We have shown that the Tcf12 locus gives rise to two distinct HEB proteins, with alternative (HEBAlt) and canonical (HEBCan) N-terminal domains, which are co-expressed during early T cell development. While the functional domains of HEBCan have been well studied, the nature of the HEBAlt-specific (Alt) domain has been obscure. Here we provide compelling evidence that the Alt domain provides a site for the molecular integration of cytokine signaling and E protein activity. Our results indicate that phosphorylation of a unique YYY motif in the Alt domain increases HEBAlt activity by 10-fold, and that this increase is dependent on Janus kinase activity. To enable in vivo studies of HEBAlt in the T cell context, we generated ALT-Tg mice, which can be induced to express a HA-tagged HEBAlt coding cassette in the presence of Cre recombinases. Analysis of ALT-Tg mice on the Vav-iCre background revealed a minor change in the ratio of ISP cells to CD8+ SP cells, and a mild shift in the ratio of T cells to B cells in the spleen, but otherwise the thymus, spleen, and bone marrow lymphocyte subsets were comparable at steady state. However, kinetic analysis of T cell development in OP9-DL4 co-cultures revealed a delay in early T cell development and a partial block at the DN to DP transition when HEBAlt levels or activity were increased. We also observed that HEBCan and HEBAlt displayed significant differences in protein stability that were resolved in the thymocyte context. Finally, a proteomic screen identified STAT1 and Xpo1 as potential members of HEBAlt-containing complexes in thymocytes, consistent with JAK-induced activation of HEBAlt accompanied by translocation to the nucleus. Thus, our results show that the Alt domain confers access to multiple layers of post-translational control to HEBAlt that are not available to HEBCan, and thus may serve as a rheostat to tune E protein activity levels as cells move through different thymic signaling environments during T cell development.

Developmental progression of fetal HEB(-/-) precursors to the pre-T-cell stage is restored by HEBAlt.

Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. We have identified a unique form of HEB, called HEBAlt, which is expressed only during the early stages of T-cell development, whereas HEBCan is expressed throughout T-cell development. Here, we show that HEB(-/-) precursors are inhibited at the ß-selection checkpoint of T-cell development due to impaired expression of pTα and function of CD3ε, both of which are necessary for pre-TCR signaling. Transgenic expression of HEBAlt in HEB(-/-) precursors, however, upregulated pTα and allowed development to CD4(+) CD8(+) stage in fetal thymocytes. Moreover, HEBAlt did overcome the CD3ε signaling defect in HEB(-/-) Rag-1(-/-) thymocytes. The HEBAlt transgene did not permit Rag-1(-/-) precursors to bypass ß-selection, indicating that it was not acting as a dominant negative inhibitor of other E-proteins. Therefore, our results provide the first mechanistic evidence that HEBAlt plays a critical role in early T-cell development and show that it can collaborate with fetal thymic stromal elements to create a regulatory environment that supports T-cell development past the ß-selection checkpoint.

HEBAlt enhances the T-cell potential of fetal myeloid-biased precursors.

Hematopoiesis is controlled by the interplay between transcription factors and environmental signals. One of the primary determinants of the T-lineage choice is Delta-like (DL)-Notch signaling, which promotes T-cell development and inhibits B-cell development. We have found that the transcription factor HEBAlt is up-regulated in early hematopoietic precursors in response to DL-Notch signaling and that it can promote early T-cell development. Here, we identified a population of lineage-negative Sca-1⁻c-kit(+) (LK) cells in the mouse fetal liver that rapidly gave rise to myeloid cells and B cells but exhibited very little T-cell potential. However, forced expression of HEBAlt in these precursors restored their ability to develop into T cells. We also showed that Ikaros and Notch1 are up-regulated in response to HEBAlt over-expression and that activated Notch1 enhances the ability of LK cells to enter the T-cell lineage. Furthermore, the myeloid transcription factor C/EBPα is down-regulated in response to HEBAlt. We therefore propose that HEBAlt plays a role in the network that enforces the T-lineage fate and limits myeloid fate during hematopoiesis.

Ontogenic timing, T cell receptor signal strength, and Notch signaling direct γδ T cell functional differentiation in vivo.

γδ T cells form an integral arm of the immune system and are critical during protective and destructive immunity. However, how γδ T cells are functionally programmed in vivo remains unclear. Here, we employ RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T cell receptor (TCR) signal strength, and Notch signaling. We find skewing of Vγ1+ cells toward the PLZF+Lin28b+ lineage at the fetal stage. Generation of interleukin-17 (IL-17)-producing γδ T cells is favored during, although not exclusive to, the fetal stage. Surprisingly, Notch signaling is dispensable for peripheral γδ T cell IL-17 production. Strong TCR signals, together with Notch, promote IL-4 differentiation. Conversely, less strong TCR signals promote Notch-independent IL-17 differentiation. Single-cell transcriptomic analysis reveals differential programming instilled by TCR signal strength and Notch for specific subsets. Thus, our results precisely define the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T cell functional programming in vivo.