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Interleukin (IL)-6 is a pleiotropic cytokine involved in the regulation of hematological and immune responses. IL-6 is secreted chiefly by stromal cells, but little is known about its precise role in the homeostasis of human mesenchymal stromal cells (hMSCs) and the role it may play in hMSC-mediated immunoregulation. We studied the role of IL-6 in the biology of bone marrow derived hMSC in vitro by silencing its expression using short hairpin RNA targeting. Our results show that IL-6 is involved in immunosuppression triggered by hMSCs. Cells silenced for IL-6 showed a reduced capacity to suppress activated T-cell proliferation. Moreover, silencing of IL-6 significantly blocked the capacity of hMSCs to proliferate. Notably, increasing the intracellular level of IL-6 but not recovering the extracellular level could restore the proliferative impairment observed in IL-6-silenced hMSC. Our data indicate that IL-6 signals in hMSCs by a previously undescribed intracellular mechanism.

We demonstrate a straightforward method to encapsulate siRNA into naturally available and unmodified human apoferritin. The encapsulation into apoferritin is independent of the sequence of the siRNA and provides superior protection for those sensitive molecules. High efficiency in transfection can be achieved in human tumorigenic cells, human primary mesenchymal stem cells (hMSC) and peripheral blood mononuclear cells (PBMCs). In contrast to Lipofectamine, highly effective gene silencing can be achieved with ferritin as the delivery agent in both tumor cells and PBMCs at low siRNA concentrations (10 nM). As an endogenous delivery agent, apoferritin does not induce immune activation of T- and B-cells in human PBMCs. Apoferritin shows intrinsic anti-inflammatory effects and apoferritin-mediated delivery shows a preference for immune-activated T- and B-cells, a natural selectivity which may turn useful for drug delivery in case of infections or inflammatory diseases.

γδ T cells play a role in a wide range of diseases such as autoimmunity and cancer. The majority of circulating human γδ T lymphocytes express a Vγ9Vδ2+ (Vδ2+) T cell receptor (TCR) and following activation release pro-inflammatory cytokines. In this study, we show that IFNγ, produced by Vδ2+ cells, activates mesenchymal stem cell (MSC)-mediated immunosupression, which in turn exerts a negative feedback mechanism on γδ T cell function ranging from cytokine production to proliferation. Importantly, this modulatory effect is limited to a short period of time (

Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently used cell type for cell-based therapeutics. As for other cell types intended for research and translational use, it is important to establish correctly typed cell lines from human tissue donations. Here, we describe methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue donation. The methods have been used in the context of a biobank, prepared as standard operating procedures (SOPs), ensuring traceability and reproducibility of cell production.

Mesenchymal stem cells are often transplanted into inflammatory environments where they are able to survive and modulate host immune responses through a poorly understood mechanism. In this paper we analyzed the responses of MSC to IL-1β: a representative inflammatory mediator. Microarray analysis of MSC treated with IL-1β revealed that this cytokine activateds a set of genes related to biological processes such as cell survival, cell migration, cell adhesion, chemokine production, induction of angiogenesis and modulation of the immune response. Further more detailed analysis by real-time PCR and functional assays revealed that IL-1β mainly increaseds the production of chemokines such as CCL5, CCL20, CXCL1, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11 and CX3CL1, interleukins IL-6, IL-8, IL23A, IL32, Toll-like receptors TLR2, TLR4, CLDN1, metalloproteins MMP1 and MMP3, growth factors CSF2 and TNF-α, together with adhesion molecules ICAM1 and ICAM4. Functional analysis of MSC proliferation, migration and adhesion to extracellular matrix components revealed that IL-1β did not affect proliferation but also served to induce the secretion of trophic factors and adhesion to ECM components such as collagen and laminin. IL-1β treatment enhanced the ability of MSC to recruit monocytes and granulocytes in vitro. Blockade of NF-κβ transcription factor activation with IκB kinase beta (IKKβ) shRNA impaired MSC migration, adhesion and leucocyte recruitment, induced by IL-1β demonstrating that NF-κB pathway is an important downstream regulator of these responses. These findings are relevant to understanding the biological responses of MSC to inflammatory environments. Electronic supplementary material The online version of this article (doi:10.1007/s12015-012-9364-9) contains supplementary material, which is available to authorized users.

Objectives The purpose of this study was to compare the ability of human CD34(+) hematopoietic stem cells and bone marrow mesenchymal stem cells (MSC) to treat myocardial infarction (MI) in a model of permanent left descendent coronary artery (LDA) ligation in nude rats. Background Transplantation of human CD34(+) cells and MSC has been proved to be effective in treating MI, but no comparative studies have been performed to elucidate which treatment prevents left ventricular (LV) remodelling more efficiently. Methods Human bone marrow MSC or freshly isolated CD34(+) cells from umbilical cord blood were injected intramyocardially in infarcted nude rats. Cardiac function was analyzed by echocardiography. Ventricular remodelling was evaluated by tissue histology and electron microscopy, and neo-formed vessels were quantified by immunohistochemistry. Chronic local inflammatory infiltrates were evaluated in LV wall by hematoxylin-eosin staining. Apoptosis of infarcted tissue was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results Both cell types induced an improvement in LV cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, MSC were more effective for the reduction of infarct size and prevention of ventricular remodelling. Scar tissue was 17.48 +/- 1.29% in the CD34 group and 10.36 +/- 1.07% in the MSC group (p < 0.001 in MSC vs. CD34). Moreover, unlike MSC, CD34(+)-treated animals showed local inflammatory infiltrates in LV wall that persisted 4 weeks after transplantation. Conclusions Mesenchymal stem cells might be more effective than CD34(+) cells for the healing of the infarct. This study contributes to elucidate the mechanisms by which these cell types operate in the course of MI treatment. (J Am Coll Cardiol 2010;55:2244-53) (C) 2010 by the American College of Cardiology Foundation

Although marrow adipocytes and osteoblasts derive from a common bone marrow stromal cells (BMSCs), the mechanisms that underlie osteoporosis-associated bone loss and marrow adipogenesis during prolonged steroid treatment are unclear. We show in human BMSCs (hBMSCs) that glucocorticoid receptor (GR) signaling in response to high concentrations of glucocorticoid (GC) supports adipogenesis but inhibits osteogenesis by reducing c-Jun expression and hBMSC proliferation. Conversely, significantly lower concentrations of GC, which permit hBMSC proliferation, are necessary for normal bone mineralization. In contrast, platelet-derived growth factor (PDGF) signaling increases both JNK/c-Jun activity and hBMSC expansion, favoring osteogenic differentiation instead of adipogenesis. Indeed, PDGF antagonizes the proadipogenic qualities of GC/GR signaling. Thus our results reveal a novel c-Jun-centered regulatory network of signaling pathways in differentiating hBMSCs that controls the proliferation-dependent balance between osteogenesis and adipogenesis.

Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.

SummaryVav1 is a guanine nucleotide exchange factor (GEF) for Rho-family GTPases, which is activated by tyrosine phosphorylation following TCR stimulation. Vav1-deficient mice have defects in positive and negative selection of thymocytes as well as TCR-induced proliferation in mature T cells, demonstrating a critical role for Vav1 in transducing TCR signals. Binding of phospholipids to the PH domain of Vav1 has been proposed to regulate its GEF activity in vitro. To test this model in vivo, we have generated mice carrying a point mutation in the PH domain of Vav1, and we show that they have defects in T cell development and activation. We demonstrate that the mutation affects the function of Vav1 as a GEF and perturbs PI3K-dependent pathways downstream of Vav1. Unexpectedly, the mutation selectively affects TCR-induced proliferation of CD4+ but not CD8+ T cells, demonstrating differences in the wiring of TCR signaling pathways between the two lineages.

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.

Background Efforts are continuously made to detect and investigate the pivotal processes and interplay between the response of sentinel lymph node and malignant cells from a primary tumor. Conversely, some frequently used tumor animal models, such as human cancer xenografts, rarely feature metastasis. Therefore, lymph node alterations are seldom assessed. We consider that studying lymph node response could contribute to the understanding of host reaction to cancer. In the present study, we explored the presence of regional lymph node alterations in parallel with tumor growth using a pancreatic tumor xenograft model which does not develop metastasis. Methods and findings We established an animal cancer model by the subcutaneous inoculation of PANC-1 (a metastatic human pancreatic cancer cell line) in the left upper flank of athymic nude mice. Tumor animals, along with controls (n = 7 / group) were subjected to Magnetic Resonance Imaging (MRI) in order to follow tumor growth and brachial and axillary lymph nodes alterations over several weeks. Further histological analyses were performed at the end of the study. The individual average of the different lymph nodes sizes was 15–40% larger in the tumor animals compared to control animals at week 8 to week 20. The tumor size and lymph node size were not correlated. Histological analysis of the lymph nodes showed paracortical histiocytosis. No metastasis to lymph nodes could be detected by histology. In tumor bearing animals, histiocytosis was associated with isolated apoptotic bodies and migration of human tumoral cells was confirmed by specific immunostaining of human origin markers. Conclusions The lack of metastasis as well as the pathological manifestation of the lymph node alteration in this pre-clinical model established here parallels findings in patients with sinus histiocytosis that is correlated with improved survival.

T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha) chain. Such a critical developmental checkpoint, known as beta-selection, results in progression from CD4(-) CD8(-) double negative (DN) to CD4(+) CD8(+) double positive (DP) TCRalphabeta(-) thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4(+) CD8(-) intermediate stage. Here we show that the CD4(+) CD8(-) to CD4(+) CD8(+) transition involves the sequential acquisition of the alpha and beta chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha, but not CD8beta, is expressed in vivo in a minor subset of DP TCRalphabeta(-) thymocytes, referred to as CD4(+) CD8alphaalpha(+) pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta(ic)). In contrast, expression of CD8alphabeta heterodimers was selectively found on DP TCRalphabeta(-) thymocytes that express TCRbeta(ic) and are enriched for cycling cells. Interestingly, CD4(+) CD8alphaalpha(+) pre-T cells are shown to be functional intermediates between CD4(+) CD8(-) TCRbeta(ic)(-) and CD4(+) CD8alphabeta(+) TCRbeta(ic)(+) thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta(ic) expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4(+) CD8alphaalpha(+) to CD4(+) CD8alphabeta(+) transition marks the key control point of pre-TCR-mediated beta-selection in human T-cell development.

Owing to its autofluorescence properties, green fluorescent protein (GFP) has aroused increasing interest as a marker system for many research applications. In this study we investigated the suitability of the "enhanced" GFP (EGFP), a mutant version of GFP optimized for flow cytometry and microscopy detection, as a reporter gene for retroviral transduction protocols. EGFP was shown to display a bright and stably maintained emission pattern in transfected GP+envAm12 packaging cells. Stable fluorescent emission was observed as well after transduction in NIH 3T3 fibroblasts and in the human Jurkat T cell line, in which EGFP was shown to confer no deleterious effect or growth disadvantage on the expressing cells. Moreover, EGFP expression could be detected after short-term retroviral exposure, thus allowing a rapid and quantitative retroviral titering assay, alternative to the standard colony-formation procedure. Most importantly, we showed the feasibility of EGFP as a marker gene in retroviral-mediated transduction of primary lymphoid precursors. In particular, transduction of CD34+CD1- human thymocytes by short-term cocultivation yielded up to 30% of EGFP-expressing cells, while maintaining CD34 expression levels. Finally, when cultured under multicytokine-supported conditions, such transduced intrathymic progenitors were shown to efficiently generate lymphoid-related dendritic cells, which displayed a distinct EGFP expression. Therefore, because of its rapid and easy detectability and its nontoxic characteristics, EGFP proves itself to be a valuable reporter gene by allowing the transduction of multipotential progenitors and by being compatible with the developmental programs of lymphoid lineage generation.

During thymocyte development, progression from T cell receptor (TCR)beta to TCRalpha rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRbeta chain paired with pre-TCRalpha (pTalpha). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTalpha is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3(-) thymocytes lacking surface pTalpha, but expressing cytoplasmic TCRbeta, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early alpha transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRalpha gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTalpha+ pre-T cells and TCRalpha/beta+ thymocytes. The results support a developmental model in which pre-TCR-expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRalpha gene expression.

Mesenchymal stromal cells (MSCs) are multipotent cells found in connective tissues that can differentiate into bone, cartilage, and adipose tissue. Interestingly, they can regulate immune responses in a paracrine way and allogeneic MSCs do not elicit immune response. These properties have encouraged a number of clinical trials in a broad range of regenerative therapies. Although these trials were first focused on their differentiation properties, in the last years, the immunosuppressive features have gained most of the attention. In this review, we will summarize the up-to-date knowledge about the immunosuppressive mechanisms of MSCs in vivo and in vitro and the most promising approaches in clinical investigation.

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF-κB) in human MSCs. This pathway is activated by TNF-α that is generated following TCR stimulation of T cells. Inhibition of NF-κB through silencing of IκB kinase β or the TNF-α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC-associated NF-κB activation primarily leads to inhibition of T-cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF-α/NF-κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF-κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.

Herpesvirus saimiri (HVS), an agent that can infect many human cell types, has been shown to immortalize selectively TCR alpha beta + CD3+ T lymphocytes. Human T cell precursors defined as CD34+CD3-CD4-CD8- were isolated from thymic samples and exposed to HVS in the presence of either IL-2 or IL-7. Cultures lacking the virus were non-viable by day 15. Test cultures, in contrast, showed a sustained proliferative activity lasting > 5 months, allowing the phenotypical and molecular analysis of the cellular progeny. In the presence of IL-7, TCR alpha beta + cells with three different phenotypes (mainly CD4+CD8-, but also CD4+CD8+ and CD4-CD8+) were immortalized, whereas no TCR gamma delta + cells were recovered. Kinetic studies showed that the expansion of immortalized TCR alpha beta + cells was preceded by a gradual loss of CD34+ cells followed by a transient accumulation of two distinct cell subsets: first CD1+CD4+CD3- cells and then CD4+CD8+ thymocytes. This resembles early phenotypic changes occurring during normal intrathymic T cell development. In the presence of IL-2, in contrast, only TCR gamma delta + cells were immortalized (mainly CD4-CD8+, but also CD4-CD8-). The results show that HVS can be used to read the CD3+ cellular outcome of T cell differentiation assays, including gamma delta + CD4-CD8+, gamma delta + CD4-CD8-, alpha beta + CD4+CD8-, alpha beta + CD4-CD8+ and alpha beta + CD4+CD8+ T cells. A clear role for different cytokines (IL-2 for gamma delta + cells, IL-7 for alpha beta + cells) in early T cell commitment was also apparent.

In murine T cell development, early thymocytes that productively rearrange the T cell receptor (TCR) beta locus are selected to continue maturation, before TCR alpha expression, by means of a pre-TCR alpha- (pT alpha-) TCR beta heterodimer (pre-TCR). The aim of this study was to identify equivalent stages in human thymocyte development. We show here that variable-diversity-joining region TCR beta rearrangement and the expression of full-length TCR beta transcripts have been initiated in some immature thymocytes at the TCR alpha/beta- CD4+CD8- stage, and become common in a downstream subset of TCR alpha/beta- CD4+CD8+ thymocytes that is highly enriched in large cycling cells. TCR beta chain expression was hardly detected in TCR alpha/beta- CD4+CD8- thymocytes, whereas cytoplasmic TCR beta chain was found in virtually all TCR alpha/beta- CD4+CD8+ blasts. In addition, a TCR beta complex distinct from the mature TCR alpha/beta heterodimer was immunoprecipitated only from the latter subset. cDNA derived from TCR alpha/beta- CD4+CD8+ blasts allowed us to identify and clone the gene encoding the human pT alpha chain, and to examine its expression at different stages of thymocyte development. Our results show that high pT alpha transcription occurs only in CD4+CD8- and CD4+CD8+ TCR alpha/beta- thymocytes, whereas it is weaker in earlier and later stages of development. Based on these results, we propose that the transition from TCR alpha/beta- CD4+CD8- to TCR alpha/beta- CD4+CD8+ thymocytes represents a critical developmental stage at which the successful expression of TCR beta promotes the clonal expansion and further maturation of human thymocytes, independent of TCR alpha.

Cardiac progenitor cells (CPCs) from adult myocardium offer an alternative cell therapy approach for ischaemic heart disease. Improved clinical performance of CPCs in clinical trials requires a comprehensive definition of their biology and specific interactions with the environment. In this work we characterize specific human CPC surface markers and study some of their related functions. c-kit(pos) human CPCs (hCPCs) were characterized for cell surface marker expression, pluripotency, early and late cardiac differentiation markers and therapeutic activity in a rat model of acute myocardial infarction. The results indicate that hCPCs are a mesenchymal stem cell (MSC)-like population, with a similar immunoregulatory capacity. A partial hCPC membrane proteome was analysed by liquid chromatography-mass spectrometry/mass spectrometry and 36 proteins were identified. Several, including CD26, myoferlin and podocalyxin-like protein 1 (PODXL), have been previously described in other stem-cell systems. Suppression and overexpression analysis demonstrated that PODXL regulates hCPC activation, migration and differentiation; it also modulates their local immunoregulatory capacity. Therefore, hCPCs are a resident cardiac population that shares many features with hMSCs, including their capacity for local immunoregulation. Expression of PODXL appears to favour the immature state of hCPCs, while its downregulation facilitates their differentiation. Copyright © 2016 John WileySons, Ltd.

Epigenetic changes are regarded as emerging major players for hematopoietic stem cell (HSC) biology. Although some histone deacetylase (HDAC) inhibitors, such as valproic acid (VA), induce differentiation and apoptosis in a variety of leukemic cells in vitro, they produce a favorable effect on the expansion of normal HSCs. In this study, we have identified the VA target HDAC3 as a negative regulator of umbilical cord blood HSC expansion. We demonstrate that knockdown of the transcript dramatically improves CD34+ cell expansion, which correlates with a higher potential to generate colony-forming units in functional assays. We show that this effect is mediated at the level of primitive hematopoietic cells and that it is not due to negative effects on specific cell commitment or alterations in the cell cycle. HDAC3 inhibition does not block commitment to the monocytic lineage and the maturation of monocyte precursors, which are the main inhibited pathways in the presence of VA. Therefore, our results identify HDAC3 as a promising target for therapies aiming to expand HSCs.

12 p.-6 fig.-1 tab., Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor- 1 alpha (HIF-1a) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1a overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1a lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1a on MSC biology and suggests the stabilization of HIF-1a as a novel strategy for cellular therapies., This work was supported in part by grants from the Instituto de Salud Carlos III for the Regenerative Medicine Program of Valencian Community to Centro de Investigación Principe Felipe, KUTXA founding and from the FIS (PI07/784, CP08/80 and PI10/00743).

The pre-T cell receptor (pre-TCR) and IL-7 receptor (IL-7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre-TCR signaling directly maintains IL-7Ralpha expression as developing thymocytes undergo beta-selection. Inhibition of IL-7/IL-7R signaling in (CD44-CD25-) DN4 cells results in decreased generation of double-positive thymocytes due to increased death of rapidly proliferating beta-selected cells. Thus, we identify a mechanism by which pre-TCR signaling controls the selective survival of TCRbeta+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.

The zinc-finger protein A20 is a key player in the negative feedback regulation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) pathway in response to multiple stimuli. Tumor necrosis factor alpha (TNFα), a cytokine with pleiotropic effects on cellular proliferation and differentiation, dramatically increases A20 expression in all tissues. As TNFα inhibits adipocyte differentiation, we have determined the contribution of A20 to the adipogenic capacity of human mesenchymal stromal cells (MSCs). Here we show that A20 is constitutively expressed in MSCs, which previously has been observed only in cells that are either tumor or immune cells (T/B lymphocytes). TNFα stimulation induced a rapid degradation of A20 protein mediated exclusively by the proteasome in MSCs and not by caspases. This degradation is concomitant to the induction of its own mRNA, which suggests that a tight regulation of NF-κB signaling in MSCs is fundamental. On one hand, we demonstrate that the knockdown of A20-mediated transcript dramatically decreases the adipogenic capacity of MSCs, which correlates with the phenotype observed in the presence of TNFα. On the other hand, A20 overexpression blocks NF-κB activation and drives to increased adipogenesis, even in the presence of TNFα treatment. In conclusion, our data demonstrate that the presence of A20 allows MSCs to differentiate into adipocytes by maintaining NF-κB signaling at a basal state.

In addition to T-lineage cells, a small proportion of hematopoietic non-T cells are present in the human postnatal thymus. However, the origin of this minor non-T cell thymic compartment is presently unknown. In this study we have analyzed the developmental potential of the earliest human intrathymic precursors, characterized as CD34+ cells expressing intermediate levels of CD44. We show that these CD34+CD44int thymocytes cultured with interleukin 7 were able to develop simultaneously into both T- and non-T (monocytes and dendritic cells) -lineage cells. Both developmental pathways progress through a CD1+CD4+ intermediate stage, currently believed to be the immediate precursor of double positive thymocytes. However, separate progenitors for either T or non-T cells could be characterized within CD1+CD4+ thymocytes by their opposite expression of CD44. Downregulated levels of CD44 identified CD1+CD4+ T-lineage precursors, whereas CD44 upregulation occurred on CD1+CD4+ intermediates that later differentiated into non-T cells. Therefore, commitment of human early intrathymic precursors to either T or non-T cell lineages can be traced by the differential expression of the CD44 receptor.

Accurate regulation of nuclear factor-κB (NF-κB) activity is crucial to prevent a variety of disorders including immune and inflammatory diseases. Active NF-κB promotes IκBα and A20 expression, important negative regulatory molecules that control the NF-κB response. In this study, using two-hybrid screening we identify the RING-type zinc-finger protein 114 (RNF114) as an A20-interacting factor. RNF114 interacts with A20 in T cells and modulates A20 ubiquitylation. RNF114 acts as negative regulator of NF-κB-dependent transcription, not only by stabilizing the A20 protein but also IκBα. Importantly, we demonstrate that in T cells, the effect of RNF114 is linked to the modulation of T-cell activation and apoptosis but is independent of cell cycle regulation. Altogether, our data indicate that RNF114 is a new partner of A2O involved in the regulation of NF-κB activity that contributes to the control of signaling pathways modulating T cell-mediated immune response.

Current data support the notion that the thymus is seeded by a yet uncommitted progenitor cell able to generate T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). We assess in this report the developmental relationship of DCs and NK cells derived from a small subset of CD34+ human postnatal thymocytes that, like the earliest precursors in the fetal thymus, display low CD33 surface expression. Culture of these isolated CD34+CD33lo thymic progenitors with a mixture of cytokines, including interleukin-7 (IL-7), IL-1α, IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, results in predominant generation of DCs. However, the addition of IL-2 to the cytokine mixture leads to the simultaneous development of DCs and NK cells. Both developmental pathways progress through a transient population of CD34+CD44brightCD5lo/−CD33+ large-sized cells, distinct from small-sized T-lineage precursors, that contain bipotential NK/DC progenitors. These data provide evidence of linked pathways of NK cell and DC development from intrathymic precursors and suggest that NK cells and DCs branch off the T lineage through a common intermediate progenitor.