Regulation of dendritic cell function by microbial stimuli

Dendritic cells (DC) initiate T cell responses and produce cytokines and other molecules that can regulate the class adaptive immunity. It is increasingly clear that DC in vivo are in a "resting" state and require exogenous signals to transit into an "effector" state in which they can prime T cells. Much of this DC activation process appears to be regulated by infection. Exposure of murine DC to certain pathogens or their products triggers DC migration to T cell areas of secondary lymphoid tissues, improves MHC presentation and increases DC co-stimulatory potential. Pathogen recognition can also initiate cytokine production and/or condition DC to produce cytokines in response to subsequent T cell feedback signals delivered via CD40 and similar receptors. Recognition of pathogens by DC is largely dependent on Toll-like receptors (TLRs). Interestingly, mouse splenic CD8alpha+ and CDalpha-CD4- DC have the ability to produce either IL-12 p70 or IL-10 depending on the nature of the pathogen encountered. In contrast, CD4+ DC seem incapable of producing IL-12 p70. Thus, the nature of the pathogen can dictate the type of cytokine that is made by some DC subsets, allowing them to prime distinct types of immune responses. Overall, DC display significant plasticity in their ability to respond to infection and direct adaptive immunity.     

Inflammatory stimuli recruit cathepsin activity to late endosomal compartments in human dendritic cells

Proteolysis by endocytic cysteine proteases is a central element of the antigen-presentation machinery in dendritic cells (DC). It controls the generation of immunogenic peptides, guides the transit of both MHC class II and MHC-like molecules through the endocytic compartment and converts class II into a peptide-receptive state - features closely linked to DC maturation. Differential activity of endocytic proteases, in particular cathepsins, in subcellular compartments has been implicated as a key regulatory element in controlling this machinery in murine DC. We analyzed the expression and subcellular distribution of the major endocytic cysteine proteases (cathepsins S, B, L and H) along with their major endogenous inhibitor, Cystatin C, in resting and stimulated human DC. Although the majority of cathepsin activity was restricted to lysosomes in resting DC, cathepsins selectively accumulated in late endosomes after LPS-induced stimulation. Surprisingly, expression and distribution of Cystatin C was unaffected by DC maturation. Thus, late endosomes represent a specialized compartment where proteolytic activity is developmentally regulated in DC. This could facilitate the conversion of exogenous protein into MHC class II-peptide complexes.     

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.     

Antigen delivery by dendritic cells

Dendritic cells (DC) link the innate and adaptive arms of the immune system and thus orchestrate the immune response to pathogens. A novel immune intervention strategy to control infectious diseases is based on the use of the potent immunostimulatory properties of DC for vaccination and immunotherapy. Recent advances in our understanding of DC biology and the molecular mechanisms by which DC instruct the development of an appropriate immune response to microorganisms provide means for DC-based approaches to manipulate the immune system. In experimental systems, DC vaccination has been documented to mediate protection against a wide spectrum of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens. The protocols for the generation, stimulation and antigen loading of DC are being optimized, and methods for DC targeting in situ are likely to become available soon, thus paving the way for clinical applications of DC-based vaccines.     

Cancer immunotherapy by dendritic cells

Cancerous lesions promote tumor growth, motility, invasion, and angiogenesis via oncogene-driven immunosuppressive leukocyte infiltrates, mainly myeloid-derived suppressor cells, tumor-associated macrophages, and immature dendritic cells (DCs). In addition, many tumors express or induce immunosuppressive cytokines such as TGF-beta and IL-10. As a result, tumor-antigen crosspresentation by DCs induces T cell anergy or deletion and regulatory T cells instead of antitumor immunity. Tumoricidal effector cells can be generated after vigorous DC activation by Toll-like receptor ligands or CD40 agonists. However, no single immunotherapeutic modality is effective in established cancer. Rather, chemotherapies, causing DC activation, enhanced crosspresentation, lymphodepletion, and reduction of immunosuppressive leukocytes, act synergistically with vaccines or adoptive T cell transfer. Here, I discuss the considerations for generating promising therapeutic antitumor vaccines that use DCs.     

Cross-presentation of antigens by dendritic cells

T lymphocytes recognize antigen presented on the surface of antigen-presenting cells byMHC class I and class II molecules. Classically, MHC class I molecules present self- or pathogen-derived antigens that are synthesized within the cell, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. It is becoming increasingly clear that some dendritic cells are also specialized to process exogenous antigens into the MHC class I pathway for presentation to CD8+ T cells. This process is known as cross-presentation. It provides a mechanism that can drive dendritic cells to generate either tolerance to self-antigens or immunity to pathogens. The cells responsible for, and mechanisms underlying, this decision between tolerance and immunity via cross-presentation has become the focus of intense study to determine how various dendritic cell subsets effect the different outcomes.     

Infection of dendritic cells by enterobacteriaceae

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in initiation and modulation of specific immune responses. Various pathogens like viruses or bacteria are able to persist inside DC. In this study we investigated the ability of the Gram-negative bacteria Salmonella typhimurium and Escherichia coli to infect DC. DC isolated from peripheral blood of healthy donors were infected with wild-type S. typhimurium and a nonpathogenic E. coli stool isolate. Association of bacteria with DC was assessed by labeling of the bacteria with green fluorescent protein. Both Gram-negative bacteria were associated with DC as evidenced by microscopy and flow cytometry. The intracellular location could be confirmed by lysis of DC and subsequent determination of colony-forming units on agar plates, which showed a rapid decline in viable Gram-negative bacteria 6 h after infection, being by far more pronounced for E. coli than for S. typhimurium. Testing the stimulation of T cells by infected versus uninfected but otherwise identically treated human immature DC in a mitogen-dependent T cell proliferation assay, we found that S. typhimurium, but not E. coli exhibited a suppressive effect on T cell stimulation, being most significant on days 3–5 after infection. Thus, suppression of dendritic cell function was associated with an enteropathogenic bacterium, S. typhimurium, which can cause severe forms of enteritis. The bacteria with normally mild or no gastric symptoms, E. coli, had no influence on stimulation of T cells by DC. Received: 12 January 2000     

Recognition of autologous dendritic cells by human NK cells

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK-target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40-transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co-stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL-2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF-alpha of the cultures stimulated the development of a more mature DC phenotype, while addition of IL-10 resulted in a less mature phenotype, with lower expression of CD40 and other co-stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.     

Modulation of antitumor responses by dendritic cells

The discovery that dendritic cells (DC) play a key role in regulating antitumor immunity has prompted considerable efforts in developing DC-based cancer vaccines for use in clinical oncology. Early translational trials using antigen-loaded DC have established clear evidence of vaccine safety, and demonstrated bioactivity by stimulating immunological and even clinical responses in selected subjects. Despite these encouraging results, the vaccine-induced immune responses achieved to date are not yet sufficient to attain a robust and durable therapeutic effect in the cancer patient. Therefore, further improvements are required to enhance vaccine potency and optimize the potential for clinical success. This article presents a set of emerging concepts that, together, form a framework for a multi-pronged approach that will further enhance the efficacy of DC-based vaccination by either directly improving DC-mediated T cell activation or by inhibiting mechanisms that suppress the induction of an effective antitumor response. The clinical translation of these concepts will result in new opportunities to successfully modulate immune responses in clinical settings.     

Production of interleukin (IL)-10 and IL-12 by murine colonic dendritic cells in response to microbial stimuli

Intestinal dendritic cells (DC) are likely to regulate immunity to gut microflora, but little is known about their responses to bacterial antigens. Therefore, DC from normal murine colon were characterized and their cytokine responses to components of Gram-negative and/or Gram-positive bacteria assessed. Cells were obtained by digestion of colonic tissue and contained DC that were identified by flow cytometry as CD11c(+) major histocompatibility complex (MHC) class II(+) cells. Purified DC were obtained by immunomagnetic separation plus cell sorting. DC had the morphology of immature myeloid cells, were endocytically active, expressed low levels of co-stimulatory molecules and stimulated a weak allogeneic mixed leucocyte reaction. Analysis of flow cytometry data by a sensitive subtraction method allowed measurement of production of interleukin (IL)-12 and IL-10 by small numbers of gut DC by intracellular staining. Fewer than 5% of unstimulated DC produced either IL-10 or IL-12. IL-10 production was significantly up-regulated following stimulation with Bifidobacteria longum, but not after exposure to lipopolysaccharide (LPS) or Streptococcus faecium. In contrast, colonic DC produced IL-12 in response to both LPS and B.longum. Thus, colonic DC can produce both IL-12 and IL-10 following bacterial stimulation. Cell wall components from different bacteria stimulate distinct responses and may direct immune responses differentially in the gut.     

两步法从CD34+造血干细胞诱导树突状细胞

目的：研究如何从有限的造血干细胞获得大量的树突状细胞（DC），为进一步研究树突状细胞的生化特性及临床应用提供技术支持。方法：利用免疫磁珠法（MACS）富集CD34^ 细胞，先在培养体系中加入FLT3配体（FL）、血小板生成素（TPO）及干细胞因子（SCF）等细胞因子，然后对富集的细胞进行扩增，扩增后的细胞在GM－CSF和IL－4的作用下诱导7d，诱导后的细胞用流式细胞仪分析树突状细胞特征性表面标记CD1a。结果：两步法能够获得大量的DC，在第一步中用TPO／FL／SCF／IL－3／IL－6来扩增CD34^ 细胞能获得最多的DC。在相同的培养时间下（3周），两步法能扩增出274倍的DC，大大的超过了直接诱导的扩增倍数（24倍）。并且，随着培养时间的增长，DC的扩增倍数不断上升。结论：两步法能从少量的脐血CD34^ 前体细胞诱导扩增出大量的DC，为从病人动员的CD34^ 细胞诱导扩增DC用于临床提供了实验依据。     

Prolonged exposure of dendritic cells to maturation stimuli favors the induction of type-2 cytotoxic T lymphocytes

Dendritic cell (DC) maturation influences the priming and polarization of T lymphocytes. We recently found that early activated DC (i.e. DC exposed to pro-maturation stimuli for 8 h) were more prone to prime in vivo a type-1 cytotoxic T cell (Tc1) response than DC exposed to pro-maturation stimuli for 48 h (48h-DC). We investigated whether 48h-DC, conversely, allowed the induction of Tc2 cells. Antigen-pulsed mouse bone-marrow-derived DC at any maturation stage, in the presence of exogenous IL-12, skewed in vitro naive CD8(+) T cells towards Tc1 cells, but 48h-DC most potently, in the presence of exogenous IL-4, favored the induction of Tc2 cells. In vivo, full maturation of DC promoted expansion of Tc2 and fall of Tc1 cells. Tc2 cells maintained a high cytolytic activity and produced significant amounts of IL-4, IL-5, IL-10 and TGF-beta. Our results indicate that polarization of naive CD8(+) T cells to Tc2 cells is dependent on the amount of time DC have been exposed to maturation stimuli, and might be favored in late and/or chronic phases of an immune response.     

Allergen uptake and presentation by dendritic cells

Allergic diseases such as atopic dermatitis, rhinitis and asthma are thought to result from a dysregulated immune response to commonly encountered antigens in genetically predisposed individuals. This response leads to chronic eosinophil-rich allergic inflammation and is controlled by Th2 lymphocytes. The first step in the allergic immune response is the uptake and presentation of allergen by professional antigen presenting cells such as dendritic cells, macrophages and B lymphocytes. Immature dendritic cells reside in the epithelia of the skin, upper and lower airways and gut and have the potential to sense foreign antigens and non-specific inflammatory tissue damage. Following recognition and uptake of Ag, mature dendritic cells migrate to the T-cell rich area of draining lymph nodes, display an array of Ag-derived peptides on the surface of major histocompatibility complex molecules and acquire the cellular specialization to select and activate naive Ag-specific T cells. By the nature of the signals they provide to naive T cells, mature dendritic cells are critical for polarizing Th0 helper cells into either Th1 or Th2 effector cells and for inducing long-lived memory Th cells. This article reviews recent information implying dendritic cells in the pathogenesis of allergic disease.     

Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets

The induction of antigen-specific tolerance is critical for maintaining immune homeostasis and preventing autoimmunity. Because the central tolerance that eliminates potentially harmful autoreactive T cells is incomplete, peripheral mechanisms for suppressing self-reactive T cells play an important role. Dendritic cells (DCs) are professional antigen-presenting cells, which have an extraordinary capacity to stimulate naïve T cells and initiate primary immune responses. Recent accumulating evidence indicates that several subsets of human DCs also play a critical role in the induction of peripheral tolerance by anergizing effector CD4⁺ and CD8⁺ T cells or by inducing the differentiation of naïve T cells into T-regulatory cells, which produce interleukin (IL)-10. Human DC subsets with the property of suppressing an antigen-specific T-cell response include plasmacytoid DCs, which are either in an immature state or in a mature state induced by CD40 ligand stimulation, and monocyte-derived DCs, which are either in an immature state or have had their state modulated by treatment with IL-10 or CD8⁺CD28⁻ T cells. These “tolerogenic” DCs may be relevant to therapeutic applications for autoimmune and allergic diseases as well as organ transplant rejection.     

Peripheral regulation of T cells by dendritic cells during infection

It is well accepted that T cell responses are integral in providing protection during pathogenic infections. In numerous tissues, T cell responses are generated to combat infection. Typically, these T cell responses are primed in draining lymph nodes (LN) by dendritic cells (DC) that have migrated from the infected tissue. Previously, it was thought that after the initial encounter between DC and T cells in the LN, the T cells underwent a programmed response. However, it has become increasingly clear that direct interactions between DCs and T cells in infected, peripheral tissues can modulate the activation, effector function, tissue residence, and memory responses of these T cells. This review will highlight the contribution of local, direct DC: T cell interactions to the regulation of T cell responses in various tissues during inflammation and infection .     

Mini-review: Presentation of pathogen-derived antigens in vivo

Most intracellular pathogens induce robust T cell responses upon infection of mammalian hosts. In most cases, these T cell responses are protective and result in pathogen clearance. It is therefore important to determine how T cells are primed and how they differentiate into cytokine-secreting and/or cytotoxic effector cells. In contrast to B cells, which recognize soluble Ag, CD8(+) and CD4(+) T cells react to Ag-derived peptides bound to MHC I or MHC II molecules, respectively. Therefore, elucidating the mechanisms by which pathogen-derived Ag become available for presentation is necessary to understand how pathogens trigger T cell responses in vivo. Although many excellent reviews have focused on the mechanisms involved in Ag processing, very few have pointed to the specificity of host-pathogen interactions. In this respect, it should be noticed that these interactions are very different from one pathogen to another, and may result in the involvement of different cells and molecules. Because of space limitations, we have decided to focus this review on two intracellular pathogens--vaccinia virus and Listeria monocytogenes. We have chosen these two pathogens because they both induce a strong CD8(+) T cell response and because they have been extensively studied by both microbiologists and immunologists.