TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.     

Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells

We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer's patch. CD11b(+)/CD8alpha(-) (myeloid) DCs are localized in the subepithelial dome, CD11b(-)/CD8alpha(+) (lymphoid) DCs in the interfollicular regions, and CD11b(-)/CD8alpha(-) (double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand(-) trimer, or Staphylococcus aureus and IFN-gamma. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-gamma production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.     

Activation of invariant NKT cells by the helminth parasite schistosoma mansoni

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-gamma and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-gamma and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.     

Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.     

Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation

Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.     

IL-12-impaired and IL-12-secreting dendritic cells produce IL-23 upon CD154 restimulation

Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p < or = 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-gamma, TNF, and IL-17, and transiently expanded FOXP3(+) regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.     

Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.     

Oncostatin M production by human dendritic cells in response to bacterial products

Oncostatin M (OSM) is a pleiomorphic cytokine that belongs to the IL-6 cytokine family. It is produced by activated T cells and monocytes/macrophages and plays an important role in the process of inflammatory responses. Although dendritic cells (DCs) have been shown to secrete a variety of cytokines, it is not elucidated whether DCs are able to produce OSM. To clarify this, using human DCs derived from peripheral blood cells, we measured the protein levels of OSM in the supernatants of DC cultures by ELISA and examined the expression of OSM mRNA by RT-PCR after stimulation with lipopolysaccharide (LPS) or fixed Staphylococcus aureus (SACS). Upon stimulation with bacterial products, DCs secreted a large amount of OSM protein in a dose- and time-dependent manner. Concomitantly, the expression of OSM mRNA by DCs was markedly up-regulated. Compared the ability of DCs to produce OSM with that of monocytes, which are major producers of OSM, DCs released significantly higher amounts of OSM protein in the culture supernatants than monocytes. These findings indicate for the first time that human monocyte-derived DCs can synthesize and secrete large amounts of OSM in response to bacterial products, suggesting that OSM produced by DCs at infectious sites may play a role in modulating inflammatory responses.     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

CD47 engagement inhibits cytokine production and maturation of human dendritic cells

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab')2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-alpha, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-alpha is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-gamma production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-gamma, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.     

TLR-dependent activation stimuli associated with Th1 responses confer NK cell stimulatory capacity to mouse dendritic cells

Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated DCs to elicit IFN-gamma production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas stimuli associated with Th2 responses do not have this property.     

Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimulated dendritic cells to differentiate into Th2 cells

Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.     

IL-10-producing B220+CD11c- APC in mouse spleen

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220(+)CD11c(-) populations with those of CD11c(+) spleen DC subsets. Low-density B220(+) cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220(+) cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2(-/-) mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220(+)CD11c(-) low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220(+) APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.     

TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development

During infection, CD4(+) Th cell responses polarize to become primarily Th1 or Th2. Th1 cells, which make IFN-gamma, are crucial for immunity to many bacterial and protozoal infections, whereas Th2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to helminth infections. Polarized Th1 responses are induced by dendritic cells (DCs), which respond to pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in Th1 cell outgrowth, and coordinately process and present Ag in the context of MHC class II to activate naive Th cells. In this study we show that in addition to providing positive signals for Th1 cell development, mouse DCs activated by TLR engagement can also provide a potent negative signal that prevents the development of Th2 cells. Production of this signal, which is not IL-12, IL-18, IL-23, IL-27, or IFN-gamma and is not provided via Th1 cells, is dependent upon a MyD88-dependent, TNF receptor-associated factor-6-independent signaling pathway in DCs. The signal is released from DCs in response to activation via TLR ligands and exerts an effect directly on Th cells rather than through a third-party cell. Our findings indicate that DCs can provide potent negative as well as positive instruction for Th response polarization, and that these instructional signals are distinct and independent.     

Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

产白介素-17T细胞在肺部细菌感染中的研究进展

目前产白介素-17(IL-17)T细胞主要包括了产IL-17 CD4+T细胞即Th17细胞、产IL-17γδT细胞和产IL-17 CD8+T细胞等。随着对Th17这一系细胞功能研究的不断深入,发现它们能通过产生IL-17和IL-22等细胞因子参与炎症反应。该文将对产IL-17 T细胞在肺部细菌感染中所起作用的最近研究进展进行综述。