The fates of dendritic cells (DCs) after antigen presentation have been studied extensively, but the influence of lymphoid microenvironments on DCs is mostly unknown. Here, using splenic stromal cells to mimic the immune microenvironment, we show that contact with stromal cells promoted mature DCs to proliferate in a fibronectin-dependent way and that both stromal cell contact and stromal cell-derived transforming growth factor-beta induced their differentiation into a new regulatory DC subset. We have identified an in vivo counterpart in the spleen with similar phenotype and functions. These differentiated DCs secreted nitric oxide, which mediated the suppression of T cell proliferation in response to antigen presentation by mature DCs. Thus, our findings identify an important mechanism by which the microenvironment regulates immune responses.

MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca2+), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKIIα could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKIIα. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKIIα expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-α, and IFN-β upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKIIα. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.

The liver has been generally considered an organ prone to tolerance induction and maintenance. However, whether and how the unique liver microenvironment contributes to tolerance maintenance is largely unknown. Here, we used liver fibroblastic stromal cells to mimic the liver microenvironment and found that liver stroma could induce Lin ؊ CD117 ؉ progenitors to differentiate into dendritic cells (DCs) with low CD11c, MHC II but high CD11b expression, high IL-10, but low IL-12 secretion. Such regulatory DCs could inhibit T-cell proliferation in vitro and in vivo, induce apoptosis of the activated T cells, and alleviate the damage of autoimmune hepatitis. Furthermore, liver stroma-derived macrophage colonystimulating factor (M-CSF) was found to contribute to the generation of such regulatory DCs. Regulatory DC-derived PGE2 and T cell-derived IFN-gamma were responsible for the regulatory function. The natural counterpart of regulatory DCs was phenotypically and functionally identified in the liver. Importantly, Lin ؊ CD117 ؉ progenitors could be differentiated into regulatory DCs in the liver once transferred into the liver. Infusion with liver regulatory DCs alleviated experimental autoimmune hepatitis. Therefore, we demonstrate that the liver microenvironment is highly important to program progenitors to differentiate into regulatory DCs in situ, which contributes to the maintenance of liver tolerance. (Blood. 2008; 112:3175-3185) IntroductionThe liver is a unique organ in which induction of tolerance may be favored over induction of immunity. There is a great deal of experimental and clinical evidence that support such an observation. For example, administration of antigens via the portal vein was found to induce immune tolerance, 1 and allogeneic liver transplantation could be established and maintained even without immunosuppression. 2 In addition, pathogens, such as hepatitis B virus, can cause chronic infection in the liver even after the initiation of immune response. 3,4 The phenomenon of "liver tolerance" has drawn much attention; however, the underlying mechanisms are not fully understood.Up to now, most studies on the mechanisms of liver tolerance mainly focused on the behavior of lymphocytes and antigenpresenting cells (APCs) in the liver. Natural killer (NK) and NKT cells rich in the liver can secrete chemokines to trap the activated T cells to undergo cell death in liver, 5-7 which was proposed as one reason for liver tolerance. Another explanation was that some DCs in the liver secrete IL-10, which in turn induces tolerance. [7][8][9] However, considering that such cells exist all over the body, we wonder why and how they can preferentially induce tolerance in the liver.As a heterogeneous population of APCs, DCs play pivotal roles in the initiation of immunity and induction of immunologic tolerance depending on their maturation state and subsets. [10][11][12] Recently, DCs with regulatory functions have attracted much attention because they can inhibit T-cell response and inflammation. On the basis ...

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-␤, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. IntroductionDendritic cells (DCs) play crucial roles in the initiation and regulation of immune responses. 1,2 The ability of DCs to initiate immune responses or induce tolerance is strictly dependent on their maturation state or subsets. It has been reported that immature DCs that are deficient of costimulatory molecules can induce T-cell anergy, generate regulatory T (Treg) cells, and promote alloantigen-specific tolerance. Several types of DCs with negative regulatory functions have been reported. 3 Most regulatory DCs are prepared in vitro using immunosuppressive cytokines, such as IL-10 and TGF-␤. [4][5][6] However, this may not reflect the real differentiation of regulatory DCs in the immune microenvironment in vivo.The development of hematopoietic cells in vivo occurs in the context of microenvironment or niche, 7 which consists of many types of stromal cells, such as fibroblasts, macrophages, endothelium cells, and adipose cells. The microenvironment provides various signals for hematopoietic cell development. 7 Different microenvironments support different types of cell differentiation. The spleen is an important lymphoid organ, and the mouse spleen maintains hematopoietic function throughout life. 8 Furthermore, hematopoietic stem cells (HSCs) are found in spleens of adult mice. 8 Therefore, it is conceivable that HSCs in the spleen may differentiate into different immune cells in situ.Splenic stromal cells cultured in vitro could mimic the splenic microenvironment in vivo to some extent, despite their differences in some constituents. There is evidence that long-term cultured splenic stromal cells can support the development of dendritic-like cells in the absence of exogenous cytokines, and the dendritic-like cells have the phenotype and function of DCs, 9-13 strongly suggesting that splenic microenvironment could induce hematopoietic progenitors to differentiate directly into DCs. Stromal cells cultured in vitro consist of multiple components. Purification of the various components will help to study the role of specific cell type in the induction of DCs. We established the method of preparing endothelial splenic stroma cells (ESSCs) and i...

CD8α(+) dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α(+) DCs, and Siglec-G deficient (Siglecg(-/-)) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I-peptide complexes were more abundant on Siglecg(-/-) CD8α(+) DCs than on Siglecg(+/+) CD8α(+) DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47(phox) and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I-peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity.

The tissue microenvironment may affect the development and function of immune cells such as DC. Whether and how the pulmonary stromal microenvironment can affect the development and function of lung DC need to be investigated. Regulatory DC (DCreg) can regulate T-cell response. We wondered whether such regulatory DC exist in the lung and what is the effect of the pulmonary stromal microenvironment on the generation of DCreg. Here we demonstrate that murine pulmonary stromal cells can drive immature DC, which are regarded as being widely distributed in the lung, to proliferate and differentiate into a distinct subset of DCreg, which express high levels of CD11b but low levels of MHC class II (I-A), CD11c, secrete high amounts of IL-10, NO and prostaglandin E 2 (PGE 2 ) and suppress T-cell proliferation. The natural counterpart of DCreg in the lung with similar phenotype and regulatory function has been identified. Pulmonary stroma-derived TGF-b is responsible for the differentiation of immature DC to DCreg, and DCreg-derived PGE2 contributes to their suppression of T-cell proliferation. Moreover, DCreg can induce the generation of CD4 1 CD25 1 Foxp3 1 Treg. Importantly, infusion with DCreg attenuates T-cell-mediated eosinophilic airway inflammation in vivo. Therefore, the pulmonary microenvironment may drive the generation of DCreg, thus contributing to the maintenance of immune homoeostasis and the control of inflammation in the lung.Key words: DC . Immune regulation . Lung inflammation . Treg . Stromal cells Introduction DC are the most potent APC in the immune system, with unique capacity to activate naïve T cells and to initiate immune responses [1]. Now, increasing evidence demonstrates that, depending on the maturation state or subsets, DC can induce tolerance or downregulate immune responses [2,3]. Immature DC (imDC) have been shown to be able to induce Treg and promote alloantigen-specific tolerance. Recently, several types of DC with negative regulatory functions, designated as regulatory DC (DCreg), have been reported [3][4][5]. Up to now, regulatory DCreg were generated by culturing imDC in the presence of immunosuppressive cytokines such as IL-10 and TGF-b or immunomodulatory drugs [4][5][6], which does not represent the in vivo physiologic conditions of DCreg in the organ microenvironment. More and more data show that the microenvironment in certain tissues has the ability to induce DC development [7][8][9][10] and also affects the function of DC; for example, DC in brain and kidney display regulatory functions but not T-cell-activating functions [11,12]. Our previous studies demonstrate that endothelial splenic stromal cells, which are used to mimic the in vivo splenic microenvironment, can promote the proliferation of mature DC (maDC) [7] and hematopoietic stem cells [8], driving them to à These authors contributed equally to this work. The lung is one of the organs connected with the outside environment of the organism and interfaces a vast quantity of environmental antigens, some of which are dan...

Regulatory dendritic cells (DCs) play an important role in maintaining peripheral tolerance or immune homeostasis. Our previous study demonstrated that mature DCs could be driven by splenic stroma to proliferate and differentiate into a novel subset of regulatory DCs (diffDCs) displaying a Th2-biased cytokine profile. However, the underlying mechanisms for the unique cytokine profile of diffDCs and how diffDCs regulate the innate and adaptive immunity in response to toll-like receptor (TLR) agonists remain unclear. Here, we report that unlike immature DCs, diffDCs secrete more interleukin 10 (IL-10) but little IL-12p70 in response to lipopolysaccharide (LPS) or other TLR agonists. Up-regulation of extracellular signal-regulated kinase (ERK1/2) activation was shown to be responsible for IL-10 preferential production, and suppression of p38 activation was for impaired IL-12p70 production in diffDCs. Interestingly, LPS treatment could not reverse the inhibitory effect of diffDCs on the proliferation of antigen-specific CD4 ؉ T cells. However, diffDCs could activate natural killer (NK) cells through diffDC-derived IL-10, and even more markedly after stimulation of TLR agonists. These diffDC-activated NK cells could in turn kill surrounding diffDCs. Our results illuminate signal pathways for the unique cytokine profile of diffDCs, and diffDCs can exert their regulatory function even after inflammatory stimuli, thus reflecting one way for strict regulation of immune response. IntroductionDendritic cells (DCs) are the most potent professional antigenpresenting cells (APCs) that integrate a wide array of incoming signals and convey them to lymphocytes, directing the appropriate immune responses. 1 Induction of immune response or tolerance by DCs may be explained by DCs at different developmental stages with different functions, or existence of different subsets of DCs. Immature DCs (imDCs) have been shown to be able to induce tolerance. 2 However, upon inflammatory stimulation or uptake of pathogenic antigens, imDCs migrate to secondary lymph organs undergoing maturation. Mature DCs (maDCs) have a potent immune-stimulatory function by production of cytokines such as interleukin 12 (IL-12) p70, up-regulation of costimulatory molecules, and potent ability to induce Th1 response and activate an antigen-specific CD8 ϩ T-cell response. 3 Recently, subsets of regulatory DCs were identified and considered to be important in maintaining immune homeostasis. [4][5][6] Regulatory DCs negatively regulate immune response by inducing a generation of regulatory T (Treg) cells or a preferential Th2 response. [7][8][9] During investigation of the fate of maDCs after antigen presentation and T-cell activation, we surprisingly found that splenic stromal cells, mimicking the secondary lymph organ microenviroment where maDCs present antigen to lymphocytes, could drive maDCs to proliferate and differentiate into a novel subtype of regulatory DCs (diffDCs), which inhibit rather than activate antigen-specific T-cell proliferation. 10 Compared...

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