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Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082–92. ©2018 AACR.

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BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

TGF-β–activated kinase 1 (TAK1) is known to play vital roles for innate and adaptive immunity; however, little is known about its potential role in limiting biological responses such as inflammation. In this study, we report that macrophage TAK1 participates in negatively regulating inflammation by restraining proinflammatory cell death. Macrophages from TAK1-deficient mice underwent cell death in response to LPS and poly(I:C), which took place in a manner dependent on TLR/TRIF-induced active Caspase8-mediated cleavage of gasdermin D, known as an executioner of pyroptosis. Likewise, TNF-α induced Caspase8-dependent gasdermin D processing following cell death in TAK1-deficient macrophages. Importantly, we demonstrated that this type of proinflammatory macrophage death is linked to susceptibility to septic shock in mice lacking TAK1 in macrophages in a TNF-α–independent fashion. Taken together, our data revealed that TAK1 acts as a signaling checkpoint to protect macrophages from unique proinflammatory cell death, ensuring the maintenance of innate immune homeostasis.

Atopic dermatitis is a chronic inflammatory skin disease with persistent pruritus. To clarify its molecular mechanism, it is important to establish a mouse model similar to the phenotypes of atopic dermatitis patients, particularly in exhibiting scratching behavior. Ikk2, a component of the IκB kinase complex, exerts pro-inflammatory responses, whereas its deficiency in keratinocytes paradoxically causes skin inflammation. In this study, we sought to generate a mouse model exhibiting skin inflammation by which dermal fibroblasts lack Ikk2 expression and evaluate whether cutaneous inflammatory phenotypes are similar to those of atopic dermatitis patients. To generate Ikk2-deficient mice (Nestincre;Ikk2FL/FL) in which Ikk2 is deleted in dermal fibroblasts, we crossed female Ikk2FL/FL mice to male Nestincre;Ikk2FL/+mice. These mice spontaneously developed skin inflammation limited to the face, with the appearance of Ikk2-deficient fibroblasts in the facial skin. These mice showed phenotypes similar to those of atopic dermatitis patients, including scratching behaviors, which are resistant to immunosuppressive or molecularly targeted drugs. These findings suggest that the Nestincre;Ikk2FL/FL mouse is an atopic dermatitis model that will be useful in clarifying atopic dermatitis pathogenesis and in developing a novel therapeutic agent for atopic dermatitis symptoms.

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3′-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3–dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non–gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082–92. ©2018 AACR.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models1. Although IFNγ can impede tumour growth by acting directly on cancer cells2,3, it must also act on the tumour stroma for effective rejection of large, established tumours4,5. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ–GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries6–8. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

In normal conditions gut homeostasis is maintained by the suppressive activity of regulatory T cells (Tregs), characterized by the expression of the transcription factor FoxP3. In human inflammatory bowel disease, which is believed to be the consequence of the loss of tolerance toward antigens normally contained in the gut lumen, Tregs have been found to be increased and functionally active, thus pointing against their possible role in the pathogenesis of this immune-mediated disease. Though, in inflammatory conditions, Tregs have been shown to upregulate the T helper (Th) type 1-related transcription factor Tbet and to express the pro-inflammatory cytokine IFN gamma, thus suggesting that at a certain point of the inflammatory process, Tregs might contribute to inflammation rather than suppress it. Starting from the observation that Tregs isolated from the lamina propria of active but not inactive IBD patients or uninflamed controls express Tbet and IFN gamma, we investigated the functional role of Th1-like Tregs in the dextran sulfate model of colitis. As observed in human IBD, Th1-like Tregs were upregulated in the inflamed lamina propria of treated mice and the expression of Tbet and IFN gamma in Tregs preceded the accumulation of conventional Th1 cells. By using a Treg-specific Tbet conditional knockout, we demonstrated that Tbet expression in Tregs is required for the development of colitis. Indeed, Tbet knockout mice developed milder colitis and showed an impaired Th1 immune response. In these mice not only the Tbet deficient Tregs but also the Tbet proficient conventional T cells showed reduced IFN gamma expression. However, Tbet deficiency did not affect the Tregs suppressive capacity in vitro and in vivo in the adoptive transfer model of colitis. In conclusion here we show that Tbet expression by Tregs sustains the early phase of the Th1-mediated inflammatory response in the gut.

Summary: Direct bacterial recognition by innate receptors is crucial for bacterial clearance. Here, we show that the IgA receptor CD89 is a major innate receptor that directly binds bacteria independently of its cognate ligands IgA and c-reactive protein (CRP). This binding is only partially inhibited by serum IgA and induces bacterial phagocytosis by CD11c+ dendritic cells and monocytes and/or macrophages, suggesting a physiological role in innate host defense. Blood phagocytes from common variable immunodeficiency patients bind, internalize, and kill bacteria in a CD89-dependent manner, confirming the IgA independence of this mechanism. In vivo, CD89 transgenic mice are protected in two different models of sepsis: a model of pneumonia and the cecal ligation and puncture (CLP) polymicrobial model of infection. These data identify CD89 as a first-line innate receptor for bacterial clearance before adaptive responses can be mounted. Fc receptors may emerge as a class of innate receptors for various bacteria with pleiotropic roles. : de Tymowski et al. demonstrate that CD89 serves as an innate receptor during the early phase of infection. During the late phase, the receptor acts in both innate and adaptive immune responses through double interaction with IgA- or CRP-opsonized and non-opsonized bacteria. Keywords: Fc receptor, innate receptor, sepsis, ITAM, host defense

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts. Keywords: Pluripotent stem cells, Chromatin, Histone deacetylase, Cell cycle, Oncogene, Dppa4, Oct4

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.Immunology and Cell Biology advance online publication, 23 December 2014; doi:10.1038/icb.2014.108. ispartof: Immunology and Cell Biology vol:93 issue:4 pages:417-23 ispartof: location:United States status: published

Haematopoietic stem and progenitor cells (HSPCs), which sustain lifelong production of all haematopoietic cell types, are known to be exquisitely sensitive to DNA-damaging events, like γ-irradiatio...

Mouse mutants with an impaired DNA damage response frequently exhibit a set of remarkably similar defects in the HSPC compartment that are of largely unknown molecular basis. Using Mixed-Lineage-Leukemia-5 (Mll5)-deficient mice as prototypical examples, we have identified a mechanistic pathway linking DNA damage and HSPC malfunction. We show that Mll5 deficiency results in accumulation of DNA damage and reactive oxygen species (ROS) in HSPCs. Reduction of ROS efficiently reverses hematopoietic defects, establishing ROS as a major cause of impaired HSPC function. The Ink4a/Arf locus also contributes to HSPC phenotypes, at least in part via promotion of ROS. Strikingly, toxic ROS levels in Mll5-/- mice are critically dependent on type 1 interferon (IFN-1) signaling, which triggers mitochondrial accumulation of full-length Bid. Genetic inactivation of Bid diminishes ROS levels and reverses HSPC defects in Mll5-/- mice. Overall, therefore, our findings highlight an unexpected IFN-1 > Bid > ROS pathway underlying DNA damage-associated HSPC malfunction.

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7–8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80high mTECs seen in such mice. Of interest, increased CD80high mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor , an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro . Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor , an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context–dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.

SummaryThe emerging notion of environment-induced reprogramming of Foxp3+ regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3+ T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3− (“exFoxp3”) Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such “latent” Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.

The efficient and reproducible generation of differentiated progenitors from pluripotent stem cells requires the recapitulation of appropriate developmental stages and pathways. Here, we have used the combination of activin A, BMP4 and VEGF under serum-free conditions to induce hematopoietic differentiation from both embryonic and induced pluripotent stem cells, with the aim of modeling the primary sites of embryonic hematopoiesis. We identified two distinct Flk1-positive hematopoietic populations that can be isolated based on temporal patterns of emergence. The earliest arising population displays characteristics of yolk sac hematopoiesis, whereas a late developing Flk1-positive population appears to reflect the para-aortic splanchnopleura hematopoietic program, as it has reduced primitive erythroid capacity and substantially enhanced myeloid and lymphoid potential compared with the earlier wave. These differences between the two populations are accompanied by differences in the expression of Sox17 and Hoxb4, as well as in the cell surface markers AA4.1 and CD41. Together, these findings support the interpretation that the two populations are representative of the early sites of mammalian hematopoiesis.

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.

T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαβ/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified functions.

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

The ability to generate a wide spectrum of differentiated cell types from ES cells in culture offers a powerful approach for studying lineage induction and specification and a promising source of progenitors for cell replacement therapy. Although significant efforts are being made to optimize culture conditions for the generation of different cell populations from ES cells, the identification and efficient isolation of specific progenitors for many lineages within these cultures remains a major challenge. By specifically tracking hematopoietic and cardiac development, we demonstrate here that these two lineages arise from distinct mesoderm subpopulations that develop in sequential waves from pre-mesoderm cells. Access to these populations provides a unique approach to isolate and characterize the earliest progenitors of these lineages.

Hypoxia Inducible Factor (HIF), consisting of HIF1alpha and ARNT (HIF1beta) subunits, activates multiple genes in response to oxygen (O(2)) deprivation. Arnt(-/-) mice exhibit substantial defects in blood cell and vessel development. We demonstrate that hypoxia accelerates the expression of Brachyury (a mesoderm-specific transcription factor), BMP4 (a mesoderm-promoting growth factor) and FLK1 (a marker of hemangioblasts, the bipotential progenitor of endothelial and hematopoietic cells) in differentiating ES cell cultures. Significantly, proliferation of embryonic hemangioblasts (BL-CFCs) is regulated by hypoxia, as Arnt(+/+) ES cells generate increased numbers of FLK1(+) cells, and BL-CFCs with accelerated kinetics in response to low O(2). This response is HIF-dependent as Arnt(-/-) ES cells produce fewer FLK1(+) cells and BL-CFCs, under both normoxic and hypoxic conditions. Interestingly, this defect is rescued when Arnt(-/-) ES cells are co-cultured with Arnt(+/+) ES cells. Vegf(+/-)or Vegf(-/-) ES cells generate proper numbers of FLK1(+) cells but fewer BL-CFCs, suggesting that additional factors regulated by HIF (other than VEGF) are involved in these early events. Thus, hypoxic responses are important for the establishment of various progenitor cells, including early mesoderm and its differentiation into hemangioblasts. Together these data suggest that ineffective responses to hypoxia in Arnt(-/-) embryos abrogate proper cardiovascular development during early embryogenesis, including the pathways controlling hemangioblast differentiation.

Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the central nervous system (CNS) promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high Foxp3 levels before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg cell-specific demethylated region in the Foxp3 locus down-regulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2:anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and may be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies, and give insights into the dynamics of the Treg cell network during auto-reactive CD4+ T cell effector responses in vivo.

Expression of a TCRγ transgene in RAG-1 −/− mice resulted in the development of a limited number of CD4 + CD8 + (DP) thymocytes. In vivo treatments with anti-TCRγ antibody enhanced the number of DP thymocytes, demonstrating that TCRγ chains were expressed on the cell surface in the absence of δ, α, or β chains. Mutations in pTα or CD3e genes abolished transgene-induced DP cell development, indicating that TCRγ can associate with pTα and CD3 to form a novel pre-TCR. With a transgene containing additional regulatory sequences, TCRγ expression was down-regulated in DP cells, and little DP cell development occurred. Thus, the function of the endogenous TCRγ/pTα is limited by the transcriptional down-regulation of TCRγ genes that normally accompanies DP cell development.

The development of pre-T-cells with productive T-cell receptor beta (TCR beta) rearrangements can be furthered by each of the pre-T-cell receptors (pre-TCR), the alpha beta TCR as well as the gamma delta TCR, albeit by distinct mechanisms. While the gamma delta TCR affects CD4-8- precursor cells irrespective of their TCR beta rearrangement status both the pre-TCR and the alpha beta TCR select only cells with productive TCR beta genes for expansion and maturation. The alpha beta TCR is much less effective than the pre-TCR because of the paucity of TCR alpha proteins in TCR beta positive precursors.

The clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a species-specific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.

Summary Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24 + ITGB4 + Notch hi cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC.

Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero–Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage- labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia., 慢性腎臓病の2大合併症である腎臓の線維化と腎性貧血のメカニズムの解明. 京都大学プレスリリース. 2011-09-13.

C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE(-/-) and LDLR(-/-) mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.

Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.

Changes in cell surface markers and patterns of gene expression are commonly used to construct sequences of events in hematopoiesis. However, the order may not be as rigid as once thought and it is unclear which changes represent the best milestones of differentiation. We developed a fate-mapping model where cells with a history of RAG-1 expression are permanently marked by red fluorescence. This approach is valuable for appreciating lymphoid-lineage relationships without need for irradiation and transplantation. Hematopoietic stem cells (HSC) as well as myeloid and dendritic cell progenitors were unlabeled. Also as expected, most previously identified RAG-1+ early lymphoid progenitors in bone marrow and all lymphoid-affiliated cells were marked. Of particular interest, there was heterogeneity among canonical common lymphoid progenitors (CLP) in bone marrow. Labeled CLP expressed slightly higher levels of IL-7Rα, displayed somewhat less c-Kit, and generated CD19+ lymphocytes faster than the unlabeled CLP. Furthermore, CLP with a history of RAG-1 expression were much less likely to generate dendritic and NK cells. The RAG-1-marked CLP were lineage stable even when exposed to LPS, while unlabeled CLP were redirected to become dendritic cells in response to this TLR4 ligand. These findings indicate that essential events in B lymphopoiesis are not tightly synchronized. Some progenitors with increased probability of becoming lymphocytes express RAG-1 while still part of the lineage marker-negative Sca-1+c-Kithigh (LSK) fraction. Other progenitors first activate this locus after c-Kit levels have diminished and cell surface IL-7 receptors are detectable.

The derivation of human embryonic stem (hES) cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of hES cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Here we report the identification of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-fluorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cell-derived lineages. Through recombinase-mediated cassette exchange, we show replacement of the tdRFP cDNA with other cDNAs, providing a cell line in which transgenes can be readily introduced into a broadly expressed locus.

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury+ population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury+cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

The development of pre-T cells with productive TCR-beta rearrangements can be mediated by each the pre-T cell receptor (pre-TCR), the TCR-alphabeta as well as the TCR-gammadelta, albeit by distinct mechanisms. Although the TCR-gammadelta affects CD4-8- precursor cells irrespective of their rearrangement status by TCR-beta mechanisms not involving TCR-beta selection, both the pre-TCR and the TCR-alphabeta select only cells with productive TCR-beta genes for expansion and maturation. The TCR-alphabeta appears to be much less effective than the pre-TCR because of the paucity of TCR-alpha proteins in TCR-beta-positive precursors since an early expressed transgenic TCR-alphabeta can largely substitute for the pre-TCR. Thus, the TCR-alphabeta can assume a role not only in the rescue from programmed cell death of CD4+8+ but also of CD4-8- thymocytes. In evolution this double function of the TCR-alphabeta may have been responsible for the maturation of alphabeta T cells before the advent of the pre-TCR-alpha chain.

Top of pageAbstract The ability of embryonic stem (ES) cells to generate diverse cell types in cultures offers outstanding opportunities to study the molecular events governing lineage specification as well as a source of cell populations and tissues for transplantation for cell based therapy. For this potential of the ES cell system to be realized, however, it is essential to first understand the processes that lead to the induction of the three primary germ layers, ectoderm, mesoderm and endoderm, that ultimately form the various tissues of the body. To begin to address these questions, we targeted the green fluorescent protein (GFP) cDNA to the mesodermal gene, brachyury (bry). Using these ES cells (GFP-Bry), it is possible to track the formation of mesoderm and isolate mesodermal cells by sorting GFP+ cells from the differentiation cultures. With this approach, we have shown that mesoderm that generates hematopoietic and vascular cells is distinct from mesoderm that gives rise to the cardiac lineage. This is an important observation as it demonstrates that cell fates are established early in development. In addition, it clearly shows that isolation of appropriate staged cells will provide access to large numbers of relatively pure progenitors of the hematopoietic/vascular and cardiac lineages. Together with the expected mesoderm derivatives, we have also shown that endoderm derivatives, including hepatocyte-like cells, lung cells and gut tissue also develop from a brachyury+ progenitor. These findings suggest that both mesoderm and endoderm may develop from a common mesendoderm progenitor and that the initial events involved in the induction of these lineages is regulated by the same molecules. Further specification to mesoderm and endoderm is likely mediated by distinct factors or sets of factors. Current experiments are aimed at defining these regulators as well as evaluating the function of the mesoderm- and endoderm-derived cell populations.

The intestinal immune system discriminates between tolerance toward the commensal microflora and robust responses to pathogens. Maintenance of this critical balance is attributed to mucosal dendritic cells (DCs) residing in organized lymphoid tissue and dispersed in the subepithelial lamina propria. In situ parameters of lamina propria DCs (lpDCs) remain poorly understood. Here, we combined conditional cell ablation and precursor-mediated in vivo reconstitution to establish that lpDC subsets have distinct origins and functions. CD103(+) CX(3)CR1(-) lpDCs arose from macrophage-DC precursors (MDPs) via DC-committed intermediates (pre-cDCs) through a Flt3L growth-factor-mediated pathway. CD11b(+) CD14(+) CX(3)CR1(+) lpDCs were derived from grafted Ly6C(hi) but not Ly6C(lo) monocytes under the control of GM-CSF. Mice reconstituted exclusively with CX(3)CR1(+) lpDCs when challenged in an innate colitis model developed severe intestinal inflammation that was driven by graft-derived TNF-alpha-secreting CX(3)CR1(+) lpDCs. Our results highlight the critical importance of the lpDC subset balance for robust gut homeostasis.

SummaryThe transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3+ T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.