The advent of reprogramming technology has greatly advanced the field of stem cell biology and nurtured our hope to create patient specific renewable stem cell sources. While the number of reports of disease specific induced pluripotent stem cells is continuously rising, the field becomes increasingly more aware that induced pluripotent stem cells are not as similar to embryonic stem cells as initially assumed. Our state of the art understanding of human induced pluripotent stem cells, their capacity, their limitations and their promise as it pertains to the study and treatment of primary immunodeficiencies, is the content of this review.
Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.
thymus; T cell development; notch ligands; lymphopoiesis and lineage commitment; stromal cell lines
Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1.1+/CD117+/CD44+/CD25−). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver– derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow–derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.
Background and Objective
Severe thermal injury is associated with extreme and prolonged inflammatory and hypermetabolic responses, resulting in significant catabolism that delays recovery or even leads to multiple organ failure and death. Burned patients exhibit many symptoms of stress-induced diabetes, including hyperglycemia, hyperinsulinemia, and hyperlipidemia. Recently, the NLRP3 inflammasome has received much attention as the sensor of endogenous “danger signals” and mediator of “sterile inflammation” in type II diabetes. Therefore, we investigated whether the NLRP3 inflammasome is activated in the adipose tissue of burned patients, as we hypothesize that, similar to the scenario observed in chronic diabetes, the cytokines produced by the inflammasome mediate insulin resistance and metabolic dysfunction.
We enrolled 76 patients with burn sizes ranging from 1% to 70% total body surface area (TBSA). Severely burned patients all exhibited burn-induced insulin resistance and hyperglycemia.
Measurements and Main Results
We examined the adipose tissue of control and burned patients and found, via flow cytometry and gene expression studies, increased infiltration of leukocytes - especially macrophages - and evidence of inflammasome priming and activation. Furthermore, we observed increased levels of IL-1β in the plasma of burned patients when compared to controls.
In summary, our study is the first to show activation of the inflammasome in burned humans, and our results provide impetus for further investigation of the role of the inflammasome in burn-induced hypermetabolism and, potentially, developing novel therapies targeting this protein complex for the treatment of stress-induced diabetes.
burn; inflammasome; inflammation; hypermetabolism; morbidity; mortality
Numerous Locus Control Region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and “integration site-independence” would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells.
The study of hematopoiesis has been a focus for developmental biologists for over 100 years. What started as a series of microscopic observations in different animal model systems has since evolved into studies of gene expression and regulation, and subsequent protein–protein interactions, cell surface protein expression profiling, and functional mapping of cell fates. In this review, we will discuss the milestone discoveries that have been achieved in the field of hematopoietic development, as well as the techniques that have been employed. Finally, we look toward the future and consider unresolved questions. We also reflect on one of the earliest realizations made in this area of study: that hematopoiesis is evolutionarily conserved, and as a consequence we reflect on the impacts of early and current discoveries and their clinical implications. The future direction of the study of hematopoietic stem cells will probably make use of pluripotent stem cells to yield specific immune cell lineages and eventual clinical applications.
Differentiation of CD8 single-positive (SP) T-cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In the thymus and the OP9-DL1 system for T-cell development, Notch signals are required for progenitors to commit to the T-cell lineage, and necessary for their progression to the CD4+CD8+ (DP) stage of T-cell development. However, it remains unclear whether Notch is a prerequisite for the differentiation of DP cells to CD8 SP stage of development. Here, we demonstrate that Notch receptor-ligand interactions allow for efficient differentiation and selection of conventional CD8 T-cells from bone marrow (BM)-derived hematopoietic stem cells (HSCs). However, BM-HSCs isolated from Itk−/−
Rlk−/− mice gave rise to T-cells with decreased IFNγ production, while gained the ability to produce IL-17. We further reveal that positive- and negative-selection in vitro are constrained by peptide-MHC (pMHC) class I expressed on the OP9 cells. Finally, using an MHC class I-restricted T-cell receptor (TCR) transgenic model, we show that the commitment of DP precursors to the CD8 T-cell lineage is dependent on Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout T-cell differentiation, including the final step of CD8 SP selection.
CD73 expression is induced in response to TCR ligation and identifies a population of thymocytes that are committed to the γδ T cell fate.
Numerous studies indicate that γδ T cell receptor (γδTCR) expression alone does not reliably mark commitment of early thymic progenitors to the γδ fate. This raises the possibility that the γδTCR is unable to intrinsically specify fate and instead requires additional environmental factors, including TCR–ligand engagement. We use single cell progenitor assays to reveal that ligand acts instructionally to direct adoption of the γδ fate. Moreover, we identify CD73 as a TCR ligand-induced cell surface protein that distinguishes γδTCR-expressing CD4−CD8− progenitors that have committed to the γδ fate from those that have not yet done so. Indeed, unlike CD73− γδTCR+ progenitors, which largely adopt the αβ fate upon separation from the intrathymic selecting environment, those that express CD73 remain CD4−CD8− and committed to the γδ fate. CD73 is expressed by >90% of peripheral γδ cells, suggesting this is a common occurrence during development. Moreover, CD73 induction appears to mark a metastable intermediate stage before acquisition of effector function, suggesting that γδ lineage and effector fate are specified sequentially. These findings have important implications for the role of ligand in γδ lineage commitment and its relationship to the specification of effector fate.
Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.
Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1GFP/w line as a readout, we developed a reproducible protocol for generating FOXN1-GFP+ thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1+ TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1+ TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.
•FOXN1-GFP reporter hESC lines were generated•KGF promotes the proliferation of FOXN1-GFP+ cells•FOXN1-GFP+ cells express TEC-associated genes•ITGB4, HLA-DR, and EpCAM can be used to purify FOXN1+ TEC progenitors (219)
Stanley and colleagues generated human embryonic stem cell (hESC) reporter lines in which sequences encoding GFP were inserted into the FOXN1 locus, enabling the isolation of viable thymic progenitors from in vitro differentiation cultures. These reporter lines were used to identify integrin-β4, HLA-DR, and EpCAM as markers of human pluripotent stem cell-derived FOXN1+ thymic epithelial progenitors.
Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas1 and cytogenetically normal acute myeloid leukaemias (AML)2. These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG)3. It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.
Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1−/−) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1−/−, Tac4−/− and Tac1−/−/Tac4−/− mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.
A human thymic epithelial cell (TEC) line expressing human leukocyte antigen (HLA)-ABC and HLA-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs).
Materials and Methods
Cord blood (CB) or bone marrow (BM) HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4 or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis.
In co-cultures with CB or BM HPCs, TEC-Dl1 cells promote de novo generation of CD7posCD1apos T-lineage committed cells. Most CD7posCD1ahi cells are CD4posCD8pos double positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3hi CD4pos or CD3hi CD8pos single positive (SP) T cells from the CD4posCD8pos DP T cells; however, we detected CD3lo cells within the DP and SP CD4 and CD8 populations. The CD3lo SP cells expressed lower levels of IL-2Rα and IL-7Rα compared to CD3lo DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage.
We report for the first time a human TEC line that supports lymphopoiesis from CB and BM HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of HLA molecules and Notch ligands to T cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy.
lymphopoiesis; hematopoietic stem cell; stromal cells; umbilical cord blood
Human peripheral blood NK cells may be divided into two main subsets: CD56brightCD16− and CD56dimCD16+. Since TGF-β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34+Lin− hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF-β represses development of NK cells from CD34+ progenitors and inhibits differentiation of CD16+ NK cells. Moreover, TGF-β also results in conversion of a minor fraction of CD56brightCD16+ cells found in peripheral blood into CD56brightCD16− cells, highlighting a possible role of the former as a developmental intermediate and of TGF-β in influencing the genesis of NK subsets found in blood.
NK cells; Cell differentiation; Innate immunity
T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells.
HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule.
Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.
αβ and γδ 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 γδ T-cell receptor (γδTCR) play instructional roles in specifying the αβ and γδ T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify the fate and promote the development of these lineages remain poorly understood. Here we show that differential activation of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent IFNγ-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus.
We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for ‘off-the-shelf’ immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy.
Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias (T-ALL), making this receptor a promising target for drugs such as γ-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates PTEN expression and the activity of the PI3K-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with resistance to NOTCH1 inhibition in human T-ALL. Overall, these findings identify the transcriptional control of PTEN and the regulation of the PI3K/AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.
Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear if Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed co-culture system with Delta 1 transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in OP9-DL1 co-cultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR+ and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through DP and SP stages only when IL-7 concentrations were low and passages were infrequent. Lin− c-Kitlo GFP+ IL-7Rα+/− pro-lymphocytes were the most efficient of adult bone marrow cells in short term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. While marrow derived progenitors with immunoglobulin DH-JH rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with VH-DHJH rearrangement products.
The following disclaimer is a requirement of The Journal of Immunology: "This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.
Notch; stem cells; thymocyte; lymphocyte
Thymic precursors expressing the pre–T cell receptor (TCR), the γδTCR, or the αβTCR can all enter the CD4+8+ αβ lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into αβ lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of αβ T cells. In particular, in αβ lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas γδTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of αβ versus γδ lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of αβ T cell generation.
Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4− CD8− double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre–T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.
pre–T cell; cell death; development; thymus; mitosis
The first checkpoint in T cell development occurs between the CD4−CD8− and CD4+CD8+ stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal–regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.
thymus; thymocyte differentiation; cellular signaling; fetal thymic organ culture; pre-TCR
Information regarding the intracellular signaling processes that occur during the development
of T cells has largely been obtained with the use of transgenic mouse models, which although
providing invaluable information are time consuming and costly. To this end, we have developed
a novel system that facilitates the In Vivo analysis of signal transduction pathways during
T-lymphocyte development. This approach uses reporter-plasmids for the detection of
intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent
protein kinase. Reporter-plasmids are transfected into thymocytes in fetal thymic organ
culture by accelerated DNA/particle bombardment (gene gun), and the activation of a signaling
pathway is determined in the form of a standard luciferase assay. Importantly, this powerful
technique preserves the structural integrity of the thymus, and will provide an invaluable
tool to study how thymocytes respond to normal environmental stimuli encountered during
differentiation within the thymic milieu. Thus, this method allows for the monitoring of signals
that occur in a biological time frame, such as during differentiation, and within the natural
environment of differentiating cells.
Cellular signaling; FTOC; T-cell development; thymus; transfection