Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells

To exploit a novel strategy to regulate T cell-mediated immunity, we established human and murine modified dendritic cells (DCs) with potent immunoregulatory properties (designed as regulatory DCs), which displayed moderately high expression levels of major histocompatibility complex (MHC) molecules and extremely low levels of costimulatory molecules compared with their normal counterparts. Unlike human normal DCs, which caused the activation of allogeneic CD4(+) and CD8(+) T cells, human regulatory DCs not only induced their anergic state but also generated CD4(+) or CD8(+) regulatory T (Tr) cells from their respective naive subsets in vitro. Although murine normal DCs activated human xenoreactive T cells in vitro, murine regulatory DCs induced their hyporesponsiveness. Furthermore, transplantation of the primed human T cells with murine normal DCs into severe combined immunodeficient (SCID) mice enhanced the lethality caused by xenogeneic graft-versus-host disease (XGVHD), whereas transplantation of the primed human T cells with murine regulatory DCs impaired their ability to cause XGVHD. In addition, a single injection of murine regulatory DCs following xenogeneic or allogeneic transplantation protected the recipients from the lethality caused by XGVHD as well as allogeneic acute GVHD. Thus, the modulation of T cell-mediated immunity by regulatory DCs provides a novel therapeutic approach for immunopathogenic diseases.     

Generation of functionally mature dendritic cells from the multipotential stem cell line FDCP-mix

Dendritic cells (DCs) are crucial components of the immune system because of their unique ability as antigen-presenting cells for the initiation of a primary immune response. DCs, macrophages (Ms) and granulocytes (Gs) are believed to originate from a common myeloid progenitor cell. However, little is known about the molecular mechanisms leading to DC sublineage commitment. To establish a cell system that allows the molecular and biochemical analysis of DC differentiation and activation, we used the murine non-leukaemic, multipotential stem cell line FDCP-mix. FDCP-mix cells were cultured in various amounts of GM-colony stimulating factor (CSF) and interleukin (IL)-4 for up to 16 d and analysed for morphology, expression of CD34, c-kit, Gr-1, Mac-1, CD40, MHC-I, MHC-II and co-stimulatory molecules (CD80, CD86) using flow cytometry, and for their capacity to present foreign antigen to autologous T cells. Up to d 7, the majority of FDCP-mix cells consisted of cells differentiating along the G and M lineage. Thereafter, the number of dendritic cells increased until d 13. Differentiation along the DC lineage vs. the G and M lineage was favoured when FDCP-mix cells were cultured in high concentration GM-CSF (500 U/ml) throughout the culture and IL-4 from d 9 onwards. The dendritic cells generated from FDCP-mix cells were large, non-adherent cells with veiled processes and expressed MHC II, CD40, CD80 and CD86. After pulsing with a foreign antigen (keyhole limpet haemocyanin), FDCP-mix-derived dendritic cells stimulated [(3)H]-thymidine incorporation of naive T-cells in an autologous mixed lymphocyte reaction (MLR). Our results show that functionally mature dendritic cells are generated from the multipotential stem cell line FDCP-mix. This cell line thus provides the unique possibility of establishing multipotential transgenic cell lines capable of differentiation along the DC lineage. The experimental system described here should prove a valuable tool for studying DC differentiation and function.     

Mesenchymal stem cells and haematopoietic stem cell transplantation

The bone marrow serves as a reservoir for different classes of stem cells. In addition to haemopoietic stem cells, the bone marrow comprises a population of marrow stromal cells or mesenchymal stem cells (MSCs). These cells exhibit multilineage differentiation capacity, and are able to generate progenitors with restricted developmental potential, including fibroblasts, osteoblasts, adipocytes and chondrocyte progenitors. In addition, MSCs have been shown to possess immunosuppressive activity in vitro and in vivo. Clinical trials are underway to test whether MSCs are beneficial in patients undergoing allogeneic bone marrow transplantation. With the expanding role of stem cell transplants in different areas of medicine, including cardiology and orthopaedics, MSCs may become very important in the next few years.     

Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells

Dendritic cells (DC) are highly specialized antigen-presenting cells characterized by the ability to prime T-cell responses. Mesenchymal stem cells (MSC) are adult stromal progenitor cells displaying immunomodulatory activities including inhibition of DC maturation in vitro. However, the specific impact of MSC on DC functions, upon in vivo administration, has never been elucidated. Here we show that murine MSC impair Toll-like receptor-4 induced activation of DC resulting in the inhibition of cytokines secretion, down-regulation of molecules involved in the migration to the lymph nodes, antigen presentation to CD4(+) T cells, and cross-presentation to CD8(+) T cells. These effects are associated with the inhibition of phosphorylation of intracellular mitogen-activated protein kinases. Intravenous administration of MSC decreased the number of CCR7 and CD49dβ1 expressing CFSE-labeled DC in the draining lymph nodes and hindered local antigen priming of DO11.10 ovalbumin-specific CD4(+) T cells. Upon labeling of DC with technetium-99m hexamethylpropylene amine oxime to follow their in vivo biodistribution, we demonstrated that intravenous injection of MSC blocks, almost instantaneously, the migration of subcutaneously administered ovalbumin-pulsed DC to the draining lymph nodes. These findings indicate that MSC significantly affect DC ability to prime T cells in vivo because of their inability to home to the draining lymph nodes and further confirm MSC potentiality as therapy for immune-mediated diseases.     

Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11b(hi)Ia(lo) DCs secreting high levels of TGF-beta, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11b(hi)Ia(lo) DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11b(hi)Ia(lo) DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11b(hi)Ia(lo) DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.     

Virus-stimulated plasmacytoid dendritic cells induce CD4+ cytotoxic regulatory T cells

Immune responses to pathogens need to be maintained within appropriate levels to minimize tissue damage, whereas such controlled immunity may allow persistent infection of certain types of pathogens. Interleukin 10 (IL-10) plays an important role in such immune regulation. We previously showed that HSV-stimulated human plasmacytoid dendritic cells (pDCs) induced naive CD4+ T cells to differentiate into interferon gamma (IFN-gamma)/IL-10-producing T cells. Here we show that HSV-stimulated pDCs induce allogeneic naive CD4+ T cells to differentiate into cytotoxic regulatory T cells that poorly proliferate on restimulation and inhibit proliferation of coexisting naive CD4+ T cells. IL-3-stimulated pDCs or myeloid DCs did not induce such regulatory T cells. Both IFN-alpha and IL-10 were responsible for the induction of anergic and regulatory properties. High percentages of CD4+ T cells cocultured with HSV-stimulated pDCs, and to a lesser extent those cocultured with IL-3-stimulated pDCs, expressed granzyme B and perforin in an IL-10-dependent manner. CD4+ T cells cocultured with HSV-stimulated pDCs accordingly exhibited cytotoxic activity. The finding that virus-stimulated pDCs are capable of inducing CD4+ cytotoxic regulatory T cells suggests that this DC subset may play an important role in suppressing excessive inflammatory responses and also in inducing persistent viral infection.     

Immunostimulatory conventional dendritic cells evolve into regulatory macrophage-like cells

Dendritic cell (DC) homeostasis in peripheral tissues reflect a balance between DC generation, migration, and death. The current model of DC ontogeny indicates that pre-cDCs are committed to become terminal conventional DCs (cDCs). Here, we report the unexpected finding that proliferating immunostimulatory CD11c(+) MHC class II(+) cDCs derived from pre-cDCs can lose their DC identity and generate progeny that exhibit morphologic, phenotypic, and functional characteristics of regulatory macrophages. DC-derived-macrophages (DC-d-Ms) potently suppress T-cell responses through the production of immunosuppressive molecules including nitric oxide, arginase, and IL-10. Relative deficiency of granulocyte-macrophage colony stimulating factor (GM-CSF) provided a permissive signal for DC-d-M generation. Using a transgenic mouse model that allows tracking of CD11c(+) cells in vivo, we found that DC-d-M development occurs commonly in cancer, but not in lymphoid or nonlymphoid tissues under steady-state conditions. We propose that this developmental pathway serves as an alternative mechanism of regulating DC homeostasis during inflammatory processes.     

Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation

Double-negative (DN) regulatory T cells (Tregs) are specialized T lymphocytes involved in the down-modulation of immune responses, resulting in allotolerance after allogeneic haematopoietic stem cell transplantation (HSCT). Most of the properties of DN Tregs were identified in murine models, including the unique ability to suppress alloreactive syngeneic effector T cells in an antigen-specific manner via Fas/Fas-ligand interactions. We investigated the behaviour of DN Tregs following human allogeneic HSCT with regard to occurrence of graft-versus-host disease (GvHD) and restoration of T-cell receptor repertoire in a cohort of 40 patients. The frequency of DN Tregs and CD4/CD8 TCR repertoire was measured serially and at the time of diagnosis of GvHD by flow cytometry. Analysis demonstrated a positive correlation between degree of alloreactivity, as measured by grade of GvHD, and the number of variable beta chain (Vβ) family expansions in both T-cell populations. We also found that a deficiency of DN Tregs was associated with an increased number of Vβ family expansions, and most importantly, with the occurrence of GvHD. All individuals who demonstrated more than 1% DN Tregs did not develop GvHD, providing evidence that DN Tregs participate in peripheral tolerance to prevent GvHD when expanded after allogeneic HSCT.     

Mesenchymal stem cells: heading into the clinic

In recent years, there has been an increasing interest in non-hematopoietic pluripotent progenitor cells that are found in the bone marrow. Mesenchymal stem cells (MSCs) are the first non-hematopoietic progenitors to be isolated from the bone marrow and extensively characterized. In addition to their ability to support hematopoiesis, MSCs can differentiate into osteocytes, chondrocytes, tenocytes, adipocytes and smooth muscle cells. This article will review our current understanding of bone marrow stroma and MSCs and their potential therapeutic role in the setting of hematopoietic stem cell transplantation.     

Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells

Aims/hypothesis  Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. Methods  Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4⁺FOXP3⁺ cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9–23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. Results  MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3⁺ regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3⁺ T cells. Conclusions/interpretation  MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Abnormal presence of semimature dendritic cells that induce regulatory T cells in HIV-infected subjects

Dendritic cells (DCs), because they orchestrate the immune response to microbes, represent an ideal target for pathogens attempting to evade the immune system. We hypothesized that interactions between human immunodeficiency virus (HIV) and DCs lead to the development of a semimature state, in which DCs migrate to lymph nodes but induce tolerance in T cells, rather than immunity. We found that lymph nodes from untreated HIV-infected subjects contained an abundance of semimature DCs, the disappearance of which correlated with the initiation of highly active antiretroviral therapy (HAART). Such lymph nodes also contained an abundance of T cells that had a regulatory phenotype and that persisted after HAART. Lymph node DCs from untreated HIV-infected subjects cultured with normal allogeneic T cells induced these T cells to adopt the phenotype of regulatory T cells, an ability that was lost after HAART. We conclude that HIV infection correlates with the presence of semimature DCs that stimulate T cell tolerance rather than immunity. These regulatory T cells may contribute to the lack of effective HIV immune responses.     

Mesenchymal stem cells efficiently inhibit the proinflammatory properties of 6-sulfo LacNAc dendritic cells

Mesenchymal stem cells emerged as a promising treatment modality for steroid-refractory graft-versus-host disease, which represents a major complication of allogeneic hematopoietic stem cell transplantation. Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses and play a crucial role in the pathogenesis of graft-versus-host disease. Here, we investigated the impact of mesenchymal stem cells on the proinflammatory capacity of 6-sulfo LacNAc (slan) dendritic cells, representing a major subpopulation of human blood dendritic cells. Mesenchymal stem cells markedly impair maturation of slanDCs and their ability to secrete proinflammatory cytokines, which was dependent on prostaglandin E₂. In contrast, the release of anti-inflammatory IL-10 was improved by mesenchymal stem cells. Furthermore, mesenchymal stem cells efficiently inhibit slanDC-induced proliferation of CD4⁺ and CD8⁺ T cells and polarization of naïve CD4⁺ T lymphocytes into Th1 cells. These results indicate that mesenchymal stem cells significantly impair the high proinflammatory capacity of slanDCs and further substantiate their potential for the treatment of graft-versus-host disease.     

Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo dramatic expansion during fetal liver development, but attempts to expand their numbers ex vivo have failed. We hypothesized that unidentified fetal liver cells produce growth factors that support HSC proliferation. Here we describe a novel population of CD3+ and Ter119- day-15 fetal liver cells that support HSC expansion in culture, as determined by limiting dilution mouse reconstitution analyses. DNA array experiments showed that, among other proteins, insulin-like growth factor 2 (IGF-2) is specifically expressed in fetal liver CD3+ cells but not in several cells that do not support HSCs. Treatment of fetal liver CD3+Ter119- cells with anti-IGF-2 abrogated their HSC supportive activity, suggesting that IGF-2 is the key molecule produced by these cells that stimulates HSC expansion. All mouse fetal liver and adult bone marrow HSCs express receptors for IGF-2. Indeed, when combined with other growth factors, IGF-2 supports a 2-fold expansion of day-15 fetal liver Lin-Sca-1+c-Kit+ long-term (LT)-HSC numbers. Thus, fetal liver CD3+Ter119- cells are a novel stromal population that is capable of supporting HSC expansion, and IGF-2, produced by these cells, is an important growth factor for fetal liver and, as we show, adult bone marrow HSCs.     

Antigen-bearing immature dendritic cells induce peptide-specific CD8(+) regulatory T cells in vivo in humans

Regulatory T cells (T(R)s) can suppress the function of other effector T cells in the setting of autoimmunity, transplantation, and resistance to tumors. The mechanism for the induction of T(R)s has not been defined. We previously reported that an injection of immature dendritic cells (DCs) pulsed with influenza matrix peptide (MP) led 7 days later to antigen-specific silencing of effector T-cell function in the blood of 2 healthy human subjects. Here, we found that interferon-gamma-producing effectors return by 6 months. Importantly, in mixing experiments, CD8(+) T cells from the sample obtained 7 days after injection could suppress MP-specific effectors obtained before injection and those in recovery samples. This suppression or regulation was specific for the immunizing peptide (MP) and cell-dose dependent, and it required contact between the 2 samples. These data show the capacity of immature DCs to induce antigen-specific regulatory CD8(+) T cells in humans.     

Bone marrow Schwann cells induce hematopoietic stem cell hibernation

Hematopoietic stem cells (HSCs) are clonogenic cells capable of both self-renewal and multilineage differentiation. In adult mouse bone marrow (BM), most HSCs remain in the non-dividing G₀-phase of cell cycle, in close contact with supporting cells known as the HSC “niche”. In the present study, we focused on signaling mechanisms that regulate stem cell dormancy in the BM niche. We show that TGF-β type II receptor deficiency causes reduced phosphorylation of Smad2/3 and impairs long-term repopulating activity in HSCs, suggesting a significant role for TGF-β/Smad signaling in hematopoiesis. Furthermore, we aimed at defining the candidate BM niche responsible for homeostasis of hematopoiesis, and revealed that non-myelinating Schwann cells sustain HSC hibernation by converting TGF-β from its latent to its active form.     

骨髓间充质干细胞在脑缺血再灌注大鼠脑内存活并分化为神经元样细胞的实验研究

目的观察骨髓间充质干细胞(MSCs)经静脉移植在大脑中动脉缺血再灌注(MCAO)大鼠脑内存活并分化为神经元样细胞。方法常规方法分离、培养大鼠MSCs,根据Aspey方法制成MCAO模型,经尾静脉注射3×106溴脱氧尿嘧啶(BrdU)标记的大鼠MSCs,28d后处死大鼠,取脑组织行免疫荧光检测。结果共聚焦显微镜下观察到MSCs移植后脑梗死灶边缘聚大量BrdU阳性细胞,少量神经元特异性烯醇化酶(NSE)和BrdU双染细胞。结论经静脉移植的MSCs可移行至MCAO脑梗死灶周围并分化为神经元样细胞。     

Partitioning of bone marrow into stem cell regulatory domains.

To examine the hypothesis that bone marrow consists of discrete stem cell regulatory volumes or domains, we studied spleen colony-forming unit (CFU-S) population growth kinetics in unirradiated WBB6F1-W/Wv mice receiving various doses of +/+ bone marrow cells. Assay of femoral marrow CFU-S content in the eight recipient dose groups revealed a family of growth curves having an initial dose-independent exponential phase and a subsequent dose-dependent deceleration phase. CFU-S content at the growth transition (inflection point) was not a simple linear function of inoculum dose but was shown rather to reflect a random distribution of initially seeded donor CFU-S in discrete volumes of recipient bone marrow. The inoculum dose resulting in a mean of 1 CFU-S per bone marrow sampling unit was estimated to be 17 x 10(6) bone marrow cells, corresponding to a total marrow uptake of approximately 5100 CFU-S (based on a seeding efficiency factor of 10%). If we assume single-hit kinetics, it follows that the recipient W/Wv bone marrow may contain approximately 5100 domains in which stem cell proliferation is geared to the density of the stem cell population. When the various inocula were corrected for multiple seeding in a given domain, the mean inflection point per domain was similar and indicative of five or so divisions before departure from exponential growth at approximately 20% of final CFU-S content 8 days after bone marrow injection. The partitioning of bone marrow into highly localized functional units is consistent with the putative regulatory role of short-range interactions between stem cells and essential stromal elements.