Thomas Hieronymus

Transcriptional profiling identifies Id2 function in dendritic cell development

Dendritic cells (DCs) are potent antigen-presenting cells with a pivotal role in antigen-specific immune responses. Here, we found that the helix-loop-helix transcription factor Id2 is up-regulated during DC development in vitro and crucial for the development of distinct DC subsets in vivo. Id2−/− mice lack Langerhans cells (LCs), the cutaneous contingent of DCs, and the splenic CD8α+ DC subset is markedly reduced. Mice deficient for transforming growth factor (TGF)-β also lack LCs, and we demonstrate here that, in DCs, TGF-β induces Id2 expression. We also show that Id2 represses B cell genes in DCs. These findings reveal a TGF-β–Id2 signaling pathway in DCs and suggest a mechanism by which Id2 affects the lineage choice of B cell and DC progenitors.

CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

Two Distinct Types of Langerhans Cells Populate the Skin during Steady State and Inflammation

Langerhans cells (LCs), the dendritic cells (DCs) in skin epidermis, possess an exceptional life cycle and developmental origin. Here we identified two types of LCs, short-term and long-term LCs, which transiently or stably reconstitute the LC compartment, respectively. Short-term LCs developed from Gr-1(hi) monocytes under inflammatory conditions and occurred independently of the transcription factor Id2. Long-term LCs arose from bone marrow in steady state and were critically dependent on Id2. Surface marker and gene expression analysis positioned short-term LCs close to Gr-1(hi) monocytes, which is indicative of their monocytic origin. We also show that LC reconstitution after UV light exposure occurs in two waves: an initial fast and transient wave of Gr-1(hi) monocyte-derived short-term LCs is followed by a second wave of steady-state precursor-derived long-term LCs. Our data demonstrate the presence of two types of LCs that develop through different pathways in inflammation and steady state.

Pluripotency Associated Genes Are Reactivated by Chromatin‐Modifying Agents in Neurosphere Cells

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin‐modifying agents trichostatin A (TSA) and 5‐Aza‐2′‐deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long‐term, multilineage, and transplantable. Here, we have analyzed the TSA/AzaC‐induced changes in gene expression by global gene expression profiling. TSA/AzaC caused both up‐ and downregulation of genes, without increasing the total number of expressed genes. Chromosome analysis showed no hot spot of TSA/AzaC impact on a particular chromosome or chromosomal region. Hierarchical cluster analysis revealed common gene expression patterns among neurosphere cells treated with TSA/AzaC, embryonic stem (ES) cells, and hematopoietic stem cells. Furthermore, our analysis identified several stem cell genes and pluripotency‐associated genes that are induced by TSA/AzaC in neurosphere cells, including Cd34, Cd133, Oct4, Nanog, Klf4, Bex1, and the Dppa family members Dppa2, 3, 4, and 5. Sox2 and c‐Myc are constitutively expressed in neurosphere cells. We propose a model in which TSA/AzaC, by removal of epigenetic inhibition, induces the reactivation of several stem cell and pluripotency‐associated genes, and their coordinate expression enlarges the differentiation potential of somatic precursor cells.

Cord blood-hematopoietic stem cell expansion in 3D fibrin scaffolds with stromal support

Expansion of multipotent, undifferentiated and proliferating cord blood (CB)-hematopoietic stem cells (HSC) in vitro is limited and insufficient. Bone marrow (BM) engineering in vitro allows mimicking the main components of the hematopoietic niche compared to conventional expansion strategies. In this study, four different 3D biomaterial scaffolds (PCL, PLGA, fibrin and collagen) were tested for freshly isolated cord blood (CB)-CD34(+) cell expansion in presence of (i) efficient exogenous cytokine supplementation and (ii) umbilical cord (UC)-mesenchymal stem cells (MSC). Cell morphology, growth and proliferation were analyzed in vitro as well as multi-organ engraftment and multilineage differentiation in a murine transplantation model. All scaffolds, except 3D PLGA meshes, supported CB-CD34(+) cell expansion, which was additionally stimulated by UC-MSC support. CB-CD34(+) cells cultured on human-derived 3D fibrin scaffolds with UC-MSC support i) reached the highest overall growth (5 × 10(8)-fold expansion of total nuclear cells after fourteen days and 3 × 10(7)-fold expansion of CD34(+) cells after seven days, p < 0.001), ii) maintained a more primitive immunophenotype for more cell divisions, iii) exhibited superior morphological, migratory and adhesive properties, and iv) showed the significantly highest numbers of engraftment and multilineage differentiation (CD45, CD34, CD13, CD3 and CD19) in BM, spleen and peripheral blood in long-term transplanted NSG mice compared to the other 3D biomaterial scaffolds. Thus, the 3D fibrin scaffold based BM-mimicry strategy reveals optimal requirements for translation into clinical protocols for CB expansion and transplantation.

Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

Background Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context.

Synergistic effects of growth factors and mesenchymal stromal cells for expansion of hematopoietic stem and progenitor cells

OBJECTIVE The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. MATERIALS AND METHODS Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor-1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34(+) cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. RESULTS Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit-initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. CONCLUSIONS The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.

A responsive MRI contrast agent to monitor functional cell status

It has been shown that insoluble Gd chelates are suitable MRI contrast agents for conditional activation by intracellular lipases. The DTPA-based, insoluble, inactive contrast agent was internalized into dendritic cells by phagocytosis. Cleavage of long aliphatic side chains by intracellular lipase activity leads to the contrast agents solubility and hereby its activation depending on the enzyme expression. Uptake and activation of the contrast agent was much reduced in Flt3+ CD11b+ progenitor cells. Detectability limits in the T(1)-weighted MR images were estimated in phantoms and in vivo in the rat brain. Marginal toxic effects were only observed at very high concentrations of the contrast agent. The chelate can easily be modified to be targeted by enzymes expressed during specific change of cell status like activation or differentiation. Such a system is suitable for functional cellular in vivo MR imaging.

The impact of c-met/scatter factor receptor on dendritic cell migration.

Dendritic cells (DC) are professional antigen‐presenting cells that possess both migratory properties and potent T cell stimulatory activity, and that allow the uptake of antigenic material inperipheral tissues and its subsequent presentation in the T cell areas of lymphoid organs. Thus motility represents a central property that is required for DC function. Here we report on the expression of the receptor tyrosine kinase c‐met in DC. c‐Met is the high affinity receptor for scatter factor (SF)/hepatocyte growth factor, and ligand‐activated c‐met exhibits mitogenic, morphogenic andmotogenic activity in vivo and in vitro. c‐Met is signaling competent in DC since it is effectively tyrosine phosphorylated in response to SF ligand. It is demonstrated here that ligand‐activated c‐met regulates DC adhesion to the extracellular matrix component laminin but leaves antigen presenting function unaffected. Importantly, in ear sheet explant experiments activationof c‐met by ligand induces emigration of cutaneous DC (Langerhans cell, LC) from skin, but SF is not a chemoattractant factor for DC. Our results suggest an important role of the c‐met/SF system in DC/LC migration.

Progressive and Controlled Development of Mouse Dendritic Cells from Flt3+CD11b+ Progenitors In Vitro

Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3+CD11b+ progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3+CD11b+ progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3+CD11b+ progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3+CD11b+ progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3+CD11b+ progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin α6. Thus, Flt3+CD11b+ progenitors express several stem cell surface Ags and develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo, and thus into both of the main conventional DC subtypes.