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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.

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 β-chain in mediating β-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 TCRβ rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR β-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRβ- 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 β-chain during human 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 αβ 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 αβ/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 αβ T cell or a γδ T cell, even before the lineage-dictating TCR signaling events. This article is categorized under: Immune System DiseasesStem Cells and Development Immune System DiseasesGenetics/Genomics/Epigenetics.

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.

This Pillars of Immunology article is a commentary on “MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus,” a pivotal article written by G. Anderson, E. J. Jenkinson, N. C. Moore, and J. J. Owen, and published in Nature, in 1993 https://www.nature.com/articles/362070a0.

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources., T cells derived from stem cells can be harnessed for regenerative medicine and cancer immunotherapy, but current technologies limit production and translation. Here, the authors present a serum-free, stromal-cell free DLL4-coated microbead method for the scalable production of T-lineage cells from multiple sources of stem cells.

T-cell development is predicated on the successful rearrangement of the T-cell receptor (TCR) gene loci, which encode for antigen-specific receptors. Recombination Activating Gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T-cell development. Here, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCRβ chain in mediating β-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 TCRβ rearrangements. Nonetheless, RAG2-KO DPs retrovirally-transduced to express a rearranged TCRβ chain showed increased survival and proliferation as compared to control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRβ- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide new insights as to the distinct requirement for the TCRβ chain during human 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.

The transcription factor HEB is involved in multiple stages of intrathymic T cell development, complexing with stage-specific partners to regulate different genes in a context-specific manner. Remarkably, little is known about how HEB influences the differentiation of peripheral T cells, and even less so of how the transcriptional circuitry in early T cell development may be re-invoked to generate long-lasting immunity. While T cells are detected in the periphery of HEB-deficient mice (HEB cKO), the contribution of HEB to the function of T cells upon pathogen encounter is obscure. Thus, HEB cKO mice were challenged with LCMV to determine the impact of HEB in the activation and differentiation of CD8+ T cells into effector and memory lineages. Throughout the course of infection, HEB cKO mice exhibited critical differences in their antigen-specific CD8+ response and had enhanced levels of stem cell-like memory T cells (Tscm). This implicated a role for HEB downstream of T cell activation, which we further confirmed using well-established in vitro cultures supplemented with or without Tscm-inducing cytokines. Consistently, HEB transcripts were detected in publicly available scRNA datasets from P14 transgenic mice infected with LCMV. To greater define the transcriptional pattern of HEB, and of others that orchestrate thymic T cell development in the context of peripheral T cell immunity, we will conduct a scRNA-seq comparative bioinformatic analysis of developing thymocytes to splenic LCMV-specific CD8+ T cells. This will advance our understanding of how early transcriptional cascades, like those initiated by HEB, are propagated in downstream immunological reactions, which may lead to therapies that can revitalize T cell immunity. Supported by a grant from NIH (1P01AI102853-06 ) and the The AAI Intersect Fellowship Program for Computational Scientists and Immunologists

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‐1β, 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.

SummaryInduced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSCs, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-lymphocytes and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole genome sequencing (WGS) based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modeling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.

SUMMARY 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., In brief Ontogeny and TCR signal strength are known to influence γδ T cell differentiation. Chen et al. show that temporal control of Notch signaling in RBPJ-inducible mice affects innate γδ T cell differentiation. TCR signal strength and Notch influence IL-4 versus IL-17 γδ T cell programming, which correlate with low versus high Ccr9 expression, respectively., Graphical Abstract

IL-17-producing γδ T-cells (γδT17) are critical components of the innate immune system, but the pathways that control their development are not well understood. We have previously shown that HEB transcription factors operate upstream of Sox4 and Sox13 to initiate the γδT17 program. However, the defects in fetal Vγ4+ γδT17 cells appear to be distinct from those in the Vγ6+ γδT17 cells. In the absence of HEB, fetal Vγ4+ γδT17 cells are unable to develop, whereas Vγ6+ γδT17 cells develop but are not able to produce IL-17. To further understand the molecular mechanisms that underlie these defects, we performed single cell RNA-seq on E18.5 γδTCR+ cells from WT and HEB-deficient mice, and did differential gene expression analysis on clusters expressing Vγ5, Vγ6, or Vγ4 mRNA. Our analysis revealed that in addition to Sox4 and Sox13, an entire suite of genes associated with the development and function of γδT17 cells was downregulated in HEB-deficient cells. Furthermore, Vδ4, which is normally restricted to cells expressing Vγ6 or Vγ5, was expressed at high levels in nearly all clusters, whereas the levels of Vγ4 and Vδ5 mRNA were severely decreased. These results suggest that direct control of Vγ and Vδ chain mRNA expression underlie the defect in Vγ4+ cell development, whereas Vγ6+ cells require a direct input from HEB into the gene program controlling γδT17 differentiation even in the presence of proper γδTCR pairing.

Gamma delta T cells (γδ T cells) are key players in tissue barrier homeostasis and immunity. Understanding the molecular mechanisms by which γδ T cells are programmed to develop into the γδT17 lineage is essential to develop strategies for controlling IL-17 activity during pathogenic conditions. Previously we showed that the E protein transcription factor HEB is an essential regulator of the γδT17 lineage, and that the generation of fetal γδT17 cells occurs through a HEB-dependent fetal-restricted pathway that does not involve upregulation of CD73 (Pathway 2). Id3, an antagonist of E proteins, inhibits key regulators of γδT17 development. Therefore, we hypothesized that the loss of Id3 would enhance γδT17 development. We characterized adult Id3-RFP knock-in mice, and found that the Id3−/− recapitulated the previously established expansion of Vγ1+ γδ T cells. However, the adult Id3+/− thymus showed a re-emergence of Pathway 2, suggesting that reduced Id3 levels promotes the fetal HEB-dependent γδT17 program. Moreover, there was an increase in Vγ1-Vγ4- γδ T cells, which suggests the appearance of Vγ6+ cells that are normally restricted to the fetal thymus. Interestingly, Vγ4+ cells and Vγ1-Vγ4-cells in Id3+/− mice displayed Pathway 2 profiles, while Vγ1+ cells exhibited the normal adult thymic developmental profile leading to mature CD73+ γδ T cells in both Id3+/− and Id3−/− mice. Future studies will examine whether reduced HEB in the Id3+/− mice inhibits Pathway 2 in the adult thymus, and obtain more definitive evidence as to whether the Id3+/− γδ T cells in the adult thymus develop toward the IL-17 fate. In conclusion, our studies indicate that the balance between E proteins and Id3 regulates the developmental programming pathway of γδ T 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.

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.

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 2 c-kit 1 (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/EBPa 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.

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.

γδ T cells form an integral part of the immune system of various lymphoid and mucosal tissues in mice and humans. Recently, it has become apparent that unlike αβ T cell receptor (TCR)-bearing T cells, these innate-like lymphocytes undergo functional programming during their development in the fetal and adult thymus. Distributed throughout the body, γδ T cells act as first responders to various environmental and pathogenic perturbations. Therefore, understanding the development and functional programming of these γδ T cells has broad implications on health and disease. Here, we review a complex network of molecular mechanisms that govern the cellular processes involved in γδ T cell development and concurrent installation of IFNγ and IL-17 effector programs, including TCR signaling strength, genetic and epigenetic programs, E protein activity, and various microenvironmental factors.

The basic helix-loop-helix (bHLH) transcription factors HEB and E2A are critical mediators of gene regulation during lymphocyte development. We have cloned a new transcription factor, called HEBAlt, from a pro-T cell cDNA library. HEBAlt is generated by alternative transcriptional initiation and splicing from the HEB gene locus, which also encodes the previously characterized E box protein HEBCan. HEBAlt contains a unique N-terminal coding exon (the Alt domain) that replaces the first transactivation domain of HEBCan. Downstream of the Alt domain, HEBAlt is identical to HEBCan, including the DNA binding domain. HEBAlt is induced in early thymocyte precursors and down-regulated permanently at the double negative to double positive (DP) transition, whereas HEBCan mRNA expression peaks at the DP stage of thymocyte development. HEBAlt mRNA is up-regulated synergistically by a combination of HEBCan activity and Delta-Notch signaling. Retroviral transduction of HEBAlt or HEBCan into hemopoietic stem cells followed by OP9-DL1 coculture revealed that HEBAlt-transduced precursors generated more early T lineage precursors and more DP pre-T cells than control transduced cells. By contrast, HEBCan-transduced cells that maintained high level expression of the HEBCan transgene were inhibited in expansion and progression through T cell development. HEB−/− fetal liver precursors transduced with HEBAlt were rescued from delayed T cell specification, but HEBCan-transduced HEB−/− precursors were not. Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors.

Transcriptional regulation of T-cell development involves successive interactions between complexes of transcriptional regulators and their binding sites within the regulatory regions of each gene. The regulatory modules that control expression of T-lineage genes frequently include binding sites for a core set of regulators that set the T-cell-specific background for signal-dependent control, including GATA-3, Notch/CSL, c-myb, TCF-1, Ikaros, HEB/E2A, Ets, and Runx factors. Additional regulators in early thymocytes include PU.1, Id-2, SCL, Spi-B, Erg, Gfi-1, and Gli. Many of these factors are involved in simultaneous regulation of non-T-lineage genes, T-lineage genes, and genes involved in cell cycle control, apoptosis, or survival. Potential and known interactions between early thymic transcription factors such as GATA-3, SCL, PU.1, Erg, and Spi-B are explored. Regulatory modules involved in the expression of several critical T-lineage genes are described, and models are presented for shifting occupancy of the DNA-binding sites in the regulatory modules of pre-Talpha, T-cell receptor beta (TCRbeta), recombinase activating genes 1 and 2 (Rag-1/2), and CD4 during T-cell development. Finally, evidence is presented that c-kit, Erg, Hes-1, and HEBAlt are expressed differently in Rag-2(-/-) thymocytes versus normal early thymocytes, which provide insight into potential regulatory interactions that occur during normal T-cell development.

Specification of mammalian T lymphocytes involves prolonged developmental plasticity even after lineage-specific gene expression begins. Expression of transcription factor PU.1 may maintain some myeloid-like developmental alternatives until commitment. Commitment could reflect PU.1 shutoff, resistance to PU.1 effects, and/or imposition of a suicide penalty for diversion. Here, we describe subclones from the SCID.adh murine thymic lymphoma, adh.2C2 and adh.6D4, that represent a new tool for probing these mechanisms. PU.1 can induce many adh.2C2 cells to undergo diversion to a myeloid-like phenotype, in an all-or-none fashion with multiple, coordinate gene expression changes; adh.6D4 cells resist diversion, and most die. Diversion depends on the PU.1 Ets domain but not on known interactions in the PEST or Q-rich domains, although the Q-rich domain enhances diversion frequency. Protein kinase C/MAP kinase stimulation can make adh.6D4 cells permissive for diversion without protecting from suicide. These results show distinct roles for regulated cell death and another stimulation-sensitive function that establishes a threshold for diversion competence. PU.1 also diverts normal T-cell precursors from wild type or Bcl2-transgenic mice to a myeloid-like phenotype, upon transduction in short-term culture. The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development.

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.

The runt family transcription factors Runx1 and Runx3 are expressed in developing murine thymocytes. We show that enforced expression of full-length Runx1 in CD4−CD8− thymocytes results in a profound suppression of immature CD4/CD8 double-positive thymocytes and mature CD4 single-positive thymocytes compared with controls. This effect arises from Runx1- or Runx3-mediated repression of CD4 expression, and is independent of positively selecting signals. Runx1 is able to repress CD4 in CD4/CD8 double-positive thymocytes, but not in mature splenic T cells. Runx-mediated CD4 repression is independent of association with the corepressors Groucho/TLE or Sin3. Two domains are required for complete Runx-mediated CD4 repression. These are contained within Runx1 aa 212–262 and 263–360. The latter region contains the nuclear matrix targeting sequence, which is highly conserved among runt family transcription factors across species. The presence of the nuclear matrix targeting sequence is required for Runx-mediated CD4 repression, suggesting that Runx transcription factors are stabilized on the CD4 silencer via association with the nuclear matrix.

The fate of T cells differentiating into the CD4 or CD8 lineage is typically fixed when cells leave the thymus. However, CD4+ helper T cells can be reprogrammed to develop into CD4+CD8α+ cytotoxic T lymphocytes in the gut.

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 αβ T-cells. Here, we review the importance of signals from the T-cell receptor (TCR) in determining αβ 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.

The development of T cells and B cells from pluripotent hematopoietic precursors occurs through a stepwise narrowing of developmental potential that ends in lineage commitment. During this process, lineage-specific genes are activated asynchronously, and lineage-inappropriate genes, although initially expressed, are asynchronously turned off. These complex gene expression events are the outcome of the changes in expression of multiple transcription factors with partially overlapping roles in early lymphocyte and myeloid cell development. Key transcription factors promoting B-cell development and candidates for this role in T-cell development are discussed in terms of their possible modes of action in fate determination. We discuss how a robust, stable, cell-type–specific gene expression pattern may be established in part by the interplay between endogenous transcription factors and signals transduced by cytokine receptors, and in part by the network of effects of particular transcription factors on each other.

Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5′C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic (alpha, beta, gamma, and delta T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both alpha/beta and gamma/delta TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

In all vertebrate species examined to date, rearrangement and somatic modification of gene segmental elements that encode portions of the antigen-combining sites of immunoglobulins are integral components of the generation of antibody diversity. In the phylogenetically primitive cartilaginous fishes, gene segments encoding immunoglobulin heavy and light chain loci are arranged in multiple clusters, in which segmental elements are separated by only 300-400 bp. In some cases, segmental elements are joined in the germline of nonlymphoid cells (joined genes). Both genomic library screening and direct amplification of genomic DNA have been used to characterize at least 89 different type I light chain gene clusters in the skate, Raja. Analyses of predicted nucleotide sequences and predicted peptide structures are consistent with the distribution of genes into different sequence groups. Predicted amino acid sequence differences are preferentially distributed in complementarity-determining versus framework regions, and replacement-type substitutions exceed neutral substitutions. When specific germline sequences are related to the sequences of individual cDNAs, it is apparent that the joined genes are expressed and are potentially somatically mutated. No evidence was found for the presence of any type I light chain gene in Raja that is not germline joined. The type I light chain gene clusters in Raja appear to represent a novel gene system in which combinatorial and junctional diversity are absent.

IL-17 producing Vγ2+ γδ T cells (Vγ2 T cells) form an integral part of the immune system of various lymphoid and mucosal tissues in mice. The γδ T cells acquire the ability to produce IL-17 during their thymic development but the molecular basis of this programming is not well understood. Here, we aim to characterized the role of HEB in the development of IL-17 producing Vγ2 T cells by utilizing HEB conditional knockout mice on a vav-Cre background (HEB conKO). First, we observed that there was a block in the development of Vγ2 T cells in the fetal thymic organ culture (FTOC) from HEB conKO embryos at E14. However, in contrast to the near absence of fetal thymic Vγ2 T cells in FTOC, neonatal thymus of HEB conKO mice supported the development of Vγ2 T cells, albeit at a significantly lower frequency than the wildtype thymus. Further, the frequency of Vγ2 T cells in the thymus of adult mice was the equivalent to that in the wildtype thymus, showing a gradual increase in the development of Vγ2 T cells in the HEB conKO thymus with age. As IL-17 producing γδ T cells have been shown to be generated preferentially during fetal development, we characterized the functional subsets of Vγ2 T cells found in adult HEB conKO mice. In fact, there was a significant reduction in the frequency of RORγt+ Vγ2+ T cells in the thymus of adult HEB conKO mice, as well as in the lungs, spleen and lymph nodes. Furthermore, the HEB-deficient Vγ2 T cells in the lungs, spleen and lymph nodes exhibited a profound deficiency in their ability to produce IL-17 in response to stimulation with IL-1β, IL-23 and IL-21 or PMA/Ionomycin. Collectively, our work shows for the first time that HEB is required for the generation of IL-17 producing Vγ2 T cells for various lymphoid and mucosal tissues.

Early thymocytes possess multilineage potential, which is progressively restricted as cells transit through the double-negative stages of T-cell development. DN1 cells retain the ability to become natural killer cells, dendritic cells, B cells, and myeloid cells as well as T cells, but these options are lost by the DN3 stage. The Notch1 signaling pathway is indispensable for initiation of the T-cell lineage and inhibitory for the B-cell lineage, but the regulatory mechanisms by which the T-cell fate is locked in are largely undefined. Previously, we discovered that the E-protein transcription factor HEBAlt promoted T-cell specification. Here, we report that HEB(-/-) T-cell precursors have compromised Notch1 function and lose T-cell potential. Moreover, reconstituting HEB(-/-) precursors with Notch1 activity enforced fidelity to the T-cell fate. However, instead of becoming B cells, HEB(-/-) DN3 cells adopted a DN1-like phenotype and could be induced to differentiate into thymic NK cells. HEB(-/-) DN1-like cells retained GATA3 and Id2 expression but had lower levels of the Bcl11b gene, a Notch target gene. Therefore, our studies have revealed a new set of interactions between HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes.

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 β-selection checkpoint of T-cell development due to impaired expression of pTα and function of CD3e, 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 CD3e signaling defect in HEB(-/-) Rag-1(-/-) thymocytes. The HEBAlt transgene did not permit Rag-1(-/-) precursors to bypass β-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 β-selection checkpoint.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus , a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

PU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Rα expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects.

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.

T lymphocytes and B lymphocytes are present in jawed vertebrates, including cartilaginous fishes, but not in jawless vertebrates or invertebrates. The origins of these lineages may be understood in terms of evolutionary changes in the structure and regulation of transcription factors that control lymphocyte development, such as PU.1. The identification and characterization of three members of the PU.1 family of transcription factors in a cartilaginous fish, Raja eglanteria , are described here. Two of these genes are orthologs of mammalian PU.1 and Spi-C, respectively, whereas the third gene, Spi-D, is a different family member. In addition, a PU.1-like gene has been identified in a jawless vertebrate, Petromyzon marinus (sea lamprey). Both DNA-binding and transactivation domains are highly conserved between mammalian and skate PU.1, in marked contrast to lamprey Spi, in which similarity is evident only in the DNA-binding domain. Phylogenetic analysis of sequence data suggests that the appearance of Spi-C may predate the divergence of the jawed and jawless vertebrates and that Spi-D arose before the divergence of the cartilaginous fish from the lineage leading to the mammals. The tissue-specific expression patterns of skate PU.1 and Spi-C suggest that these genes share regulatory as well as structural properties with their mammalian orthologs.

Lymphocyte development provides an excellent model system for studying the evolutionary divergence of cell lineages. Based on their appearance in vertebrate phylogeny, the origins of lymphoid cell lineages are likely to lie in events which occurred during a defined range of time at least 450 million years ago. The dramatic emergence of both B cells and T cells in the cartilaginous fish (Litman et al. 1999) indicates the occurrence of at least one major event pivotal for the development of lymphocytes as we know them, after the divergence of the vertebrates from the other chordates but before the diversification of the jawed vertebrates. One such event may have been the acquisition, perhaps by horizontal transfer, of the recombination-activating genes (RAGs; Agrawal et al. 1998). The large-scale gene duplication events which are believed to have occurred at approximately this same time (Holland et al. 1994; Pebusque et al. 1998) may have provided another powerful mechanism for rapid evolutionary change. Lymphoid development is dependent upon networks of transcription factors, which serve not only to activate a series of temporally controlled gene batteries during differentiation but also to stabilize the mature phenotype. These transcription factors are generally members of multigene families whose origins are far more ancient than the lymphoid lineages in which they operate and provide a bridge across phylogenetic distances which have been thus far inaccessible to the study of rearranging antigen receptors. Furthermore, it is likely that duplication and/or divergence of both the cis-regulatory regions and the structural portions of transcription factor family members has contributed to the diversification of hematopoietic cell types in vertebrates.