Genomics of TGF-β1 signaling in stem cell commitment and dendritic cell development

Dendritic cells are professional antigen presenting cells and central for establishing and maintaining immunity and immunological tolerance. They develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Dendritic cell development and function are regulated by specific cytokines, including transforming growth factor type β1 (TGF-β1). Our previous work demonstrated the importance of TGF-β1 signaling for dendritic cell development and subset specification. Here, we used genome-wide gene expression profiling with DNA microarrays to investigate the activity of TGF-β1 on gene expression in dendritic cell development. This study identified specific gene categories induced by TGF-β1 with an impact on dendritic cell biology.     

Cycling of human dendritic cell effector phenotypes in response to TNF-alpha: modification of the current 'maturation' paradigm and implications for in vivo immunoregulation.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.     

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple na?ve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.     

Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 muM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.     

HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages

Immature dendritic cells are among the first cells infected by retroviruses after mucosal exposure. We explored the effects of human immunodeficiency virus-1 (HIV-1) and its Tat transactivator on these primary antigen-presenting cells using DNA microarray analysis and functional assays. We found that HIV-1 infection or Tat expression induces interferon (IFN)-responsive gene expression in immature human dendritic cells without inducing maturation. Among the induced gene products are chemokines that recruit activated T cells and macrophages, the ultimate target cells for the virus. Dendritic cells in the lymph nodes of macaques infected with simian immunodeficiency virus (SIV) have elevated levels of monocyte chemoattractant protein 2 (MCP-2), demonstrating that chemokine induction also occurs during retroviral infection in vivo. These results show that HIV-1 Tat reprograms host dendritic cell gene expression to facilitate expansion of HIV-1 infection.     

Murine dendritic cell development: difficulties associated with subset analysis

Dendritic cells are bone marrow-derived professional antigen presenting cells that play major roles in both the induction of primary immune responses and tolerance. It has become clear that dendritic cells are a heterogeneous group of cells that vary in cell surface marker expression and function. Multiple dendritic cell subsets have now been defined in mouse lymphoid organs and peripheral tissues. A knowledge of the function and relationship between dendritic cell subsets will be essential for understanding the regulation of immune homeostasis, immune responses and tolerance. While an increasing number of dendritic cell progenitors are being identified, the pathways that connect them remain unclear. In addition, it is unclear whether the functional divisions reflect maturation status, subset specialization or functional plasticity in response to specific pathogen and environmental signals. This review summarizes the current knowledge about the function and lineage relationship of dendritic cell subsets. It also discusses some of the difficulties associated with dendritic cell subset analysis.     

DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents

Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a monocyte-derived dendritic cell (MDDC)-specific lectin which participates in dendritic cell (DC) migration and DC-T lymphocyte interactions at the initiation of immune responses and enhances trans-infection of T cells through its HIV gp120-binding ability. The generation of a DC-SIGN-specific mAb has allowed us to determine that the acquisition of DC-SIGN expression during the monocyte-DC differentiation pathway is primarily induced by IL-4, and that GM-CSF cooperates with IL-4 to generate a high level of DC-SIGN mRNA and cell surface expression on immature MDDC. IL-4 was capable of inducing DC-SIGN expression on monocytes without affecting the expression of other MDDC differentiation markers. By contrast, IFN-alpha, IFN-gamma, and TGF-beta were identified as negative regulators of DC-SIGN expression, as they prevented the IL-4-dependent induction of DC-SIGN mRNA on monocytes, and a similar inhibitory effect was exerted by dexamethasone, an inhibitor of the monocyte-MDDC differentiation pathway. The relevance of the inhibitory action of dexamethasone, IFN, and TGF-beta on DC-SIGN expression was emphasized by their ability to inhibit the DC-SIGN-dependent HIV-1 binding to differentiating MDDC. These results demonstrate that DC-SIGN, considered as a MDDC differentiation marker, is a molecule specifically expressed on IL-4-treated monocytes, and whose expression is subjected to a tight regulation by numerous cytokines and growth factors. This feature might help in the development of strategies to modulate the DC-SIGN-dependent cell surface attachment of HIV for therapeutic purposes.     

Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-beta 1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-beta 1 costimulation (basic cytokine combination GM-CSF plus TNF-alpha, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-beta 1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-beta 1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-beta 1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-beta 1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.     

Ryanodine receptor activation by Ca v 1.2 is involved in dendritic cell major histocompatibility complex class II surface expression

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.     

Suppression of the maturation and activation of the dendritic cell line DC2.4 by melanoma-derived factors

Dendritic cells play critical roles in both innate and adaptive immunity, and their numerous functions are tightly linked to their maturation and activation status. Here, we characterize the murine dendritic cell line DC2.4 as a model for studying dendritic cell maturation and activation, and we evaluate the influence of melanoma tumor cells on these processes. Exposure of DC2.4 cells to the Toll-like receptor ligand lipopolysaccharide induces both maturation and activation of these cells, characterized by upregulation of costimulatory molecule expression and proinflammatory cytokine/chemokine production. This maturation and activation is suppressed by soluble factors derived from both the highly tumorigenic B16-F1 and the poorly tumorigenic D5.1G4 murine melanoma cell lines. Interestingly, the extent of DC2.4 immunosuppression by these melanomas correlates with their tumorigenicity, suggesting a potentially vital role for dendritic cell/tumor cell interactions in the regulation of anti-tumor immunity and tumor outgrowth.     

Improvement of human dendritic cell culture for immunotoxicological investigations

A toxic injury such as a decrease in the number of immature dendritic cells caused by a cytotoxic effect or a disturbance in their maturation process can be responsible for immunodepression. There is a need to improve in vitro assays on human dendritic cells used to detect and evaluate adverse effects of xenobiotics. Two aspects were explored in this work: cytotoxic effects of xenobiotics on immature dendritic cells, and the interference of xenobiotics with dendritic cell maturation. Dendritic cells of two different origins were tested. Dendritic cells obtained either from umbilical cord blood CD34⁺ cells or, for the first time, from umbilical cord blood monocytes. The cytotoxicity assay on immature dendritic cells has been improved. For the study of the potential adverse effects of xenobiotics on the maturation process of dendritic cells, several parameters were selected such as expression of markers (CD86, CD83, HLA-DR), secretion of interleukins 10 and 12, and proliferation of autologous lymphocytes. The relevance and the efficiency of the protocol applied were tested using two mycotoxins, T-2 toxin and deoxynivalence, DON, which are known to be immunosuppressive, and one phycotoxin, domoic acid, which is known not to have any immunotoxic effect. Assays using umbilical cord monocyte dendritic cell cultures with the protocol defined in this work, which involves a cytotoxicity study followed by evaluation of several markers of adverse effects on the dendritic cell maturation process, revealed their usefulness for investigating xenobiotic immunotoxicity toward immune primary reactions.     

Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors

Dendritic cell (DC) specific transgenic mice are a most important model for investigating dendritic cell functions in vivo. Recently, lentivirus mediated gene transfer has become a powerful and convenient method for generation of transgenic mice. We cloned a 1.2 kb CD11c promoter and constructed a lentiviral vector, which efficiently drove DC-specific expression in vitro. After microinjection of purified virus into the perivitelline space of single-cell embryo, more than 80% newborn mice were transgenic and 7 F0 founders were rapidly generated in 2 months. GFP was strictly expressed in CD11c+ cells in spleens, thymus and lymph nodes of the transgenic mice. Importantly, the physiological characteristics and functions of DCs in the transgenic mice were not altered by the specific expression. These results indicate that this vector could be used to rapidly prepare DC-specific transgenic mice. Thus, this lentiviral vector system may provide a convenient and useful tool to study the properties of DCs in vivo.     

Dendritic cells presenting tumor antigen

 Since the first identification of dendritic cells by Steinman and Cohn in 1973, progress in understanding their biology has included the development of novel methods of cell culture, recognition of critical aspects of migration and maturation, and appreciation of their major role as antigen-presenting cells (APC), and how this activity is regulated by cytokines and expression of accessory molecules. Dendritic cells are the major APC involved in the initiation of the immune response and the development of tolerance. There is considerable evidence that they can acquire antigen in the peripheral tissues and process, transport, and present it to T cells in secondary lymphoid tissue. A number of studies show that, in vitro or in vivo, antigen-pulsed dendritic cells can directly sensitize T cells and stimulate the development of antigen-specific immune responses, including both protective and therapeutic antitumor responses. In this paper, several important aspects of dendritic cell biology are discussed and a number of studies confirming the role of these professional APC in antitumor immunity are reviewed. Received: 6 August 1996 / Accepted: 20 September 1996