Background Different subsets of dendritic cells (DCs), identified in mouse spleen by their differential expression of CD8alpha, can induce different T-helper (Th) responses after systemic administration. CD8alpha(-) DCs have been shown to preferentially induce Th type 2 (Th2) responses whereas CD8alpha(+) DCs induce Th1 responses.Objective To study if these DC subsets can still induce different Th responses in the Th2-prone milieu of the lung and differentially prime for eosinophilic airway inflammation. typical of asthma.Methods Donor mice first received daily Flt3L injections to expand DC numbers. Purified CD8alpha(+) or CD8alpha(-) splenic DCs were pulsed with ovalbumin (OVA) or phosphate-buffered saline and injected intratracheally into recipient mice in which carboxyfluorescein diacetate succinimidyl ester-labelled OVA-specific T cell receptor transgenic T cells had been injected intravenously 2 days earlier. T cell proliferation and cytokine production of Ag-specific T cells were evaluated in the mediastinal lymph nodes (MLNs) 4 days later. The capacity of both subsets of DCs. to prime for eosinophilic airway inflammation was determined by challenging the mice with OVA aerosol 10 days later.Results CD8alpha(-) DCs migrated to the MLN and induced a vigorous proliferative T cell response accompanied by high-level production of IL-4, IL-5, IL-10 and also IFN-gamma during the primary response and during challenge with aerosol, leading to eosinophilic airway inflammation. In the absence of migration to the MLN, CD8alpha(+) DCs still induced a proliferative response with identical levels of IFN-gamma but reduced Th2 cytokines compared with CD8alpha(-) DCs, which led to weak eosinophilic airway inflammation upon OVA aerosol challenge. Unpulsed DCs did not induce proliferation or cytokine production in Ag-specific T cells.Conclusion CD8alpha(-) DCs are superior compared with CD8alpha(+) DCs in inducing Th2 responses and eosinophilic airway inflammation in the Th2-prone environment of the lung.

The adaptive immune system can generate distinct classes of responses, but the mechanisms that determine this are poorly understood. In this study, we demonstrate that different Toll-like receptor (TLR) ligands induce distinct dendritic cell (DC) activation and immune responses in vivo. Thus, Escherichia coli LPS (TLR-4 stimulus), activates DCs to produce abundant IL-12(p70), but little IL-10, and stimulates Th1 and Tc1 responses. In contrast, Pam-3-cys (TLR-2 stimulus) elicits less IL-12(p70), but abundant IL-10, and favors Th2 and T cytotoxic 2 (Tc2) responses. These distinct responses likely occur via differences in extracellular signal-regulated kinase signaling in DCs. Thus, Pam-3-cys induces enhanced extracellular signal-regulated kinase signaling, compared with LPS, resulting in suppressed IL-12(p70) and enhanced IL-10 production, as well as enhanced induction of the transcription factor, c-Fos. Interestingly, DCs from c-fos(-/-) mice produce more IL-12(p70), but less IL-10, compared with control DCs. Therefore, different TLR ligands induce distinct cytokines and signaling in DCs, and differentially bias Th responses in vivo.

There is increasing evidence that dendritic cells (DC) display opposite functions in the immune system, as they may induce immunity or tolerance depending on intrinsic and environmental factors. In mice, adoptive transfer of mature DC pulsed extracorporeally with antigen induces the development of antigen‐specific Th1‐ and Th2‐type CD4+ cells. In this work, we compared the adjuvant properties of immature (freshly isolated) and mature (cultured) splenic DC in vivo. Our data show that injection of either cell population induces the clonal expansion of CD4+ T cells but that only mature DC trigger their differentiation into effector cells producing IFN‐γ. In contrast, transfer of immature DC provokes the development of intermediates in the differentiation process, similar to the central memory cells. These observations, together with data in the literature, suggest that DC may induce tolerance, memory, or immunity depending on their maturationstate.

Previous work has established that CD24 is a costimulatory molecule for T-cell clonal expansion. Studies using CD24 -/- mice demonstrated that CD24 plays a critical role in the CD28-independent immune response against virus and soluble antigens. The role of CD24 on dendritic cells (DCs) has not been reported. Here, we compare the CD24(+/+) and CD24(-/-) DCs in the induction of initial clonal expansion and elicitation of memory CD4(+) T cells in vivo. Our results demonstrate that the CD24 expressed on DCs is essential neither for the induction of initial T-cell clonal expansion nor for elicitation of memory activity of primed T cells in vivo.

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4+ and CD8+ T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-γ−/− mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-γ during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4+ T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-γ secretion, and cytotoxic activity of Ag-specific CD8+ T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-γ and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1β, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34+-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1β is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34+-derived DC nor peripheral blood DC produce IL-1β. Finally, DC activated with CD40L and IL-1β induce higher levels of IFN-γ secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1β is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1β highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, 11,4 0, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia cola or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC: The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.

Different subsets of dendritic cells (DCs) appear to play a role in determining the specific cytokines secreted by T helper (Th) cells. A model is proposed that links together factors such as the pathogen, microenvironment, DCs and T cells in a mechanism that results in a flexible determination of T-cell polarization.

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8alpha(-) DC induces a Th2-type response, whereas injection of CD8alpha(+) DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-gamma, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-gamma are required for Th1 priming by CD8alpha(+) DC, whereas IL-10 is required for optimal development of Th2 cells by CD8alpha(-) DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8alpha(+) DC. Conversely, incubation with IFN-gamma down-regulates the Th2-promoting capacities of CD8alpha(-) DC and increases the Th1-skewing properties of both subsets.

Cancer immunotherapy might be based on the administration to cancer patients of dendritic cells (DC) ‘pulsed’ with tumor-specific antigens. An alternative approach is to directly expand and/or activate DC in vivo using the cytokine Flt3 Ligand (FL). In mice, FL can drive large expansion of both lymphoid-related DC that appears to selectively enhance Th1-like immune responses and myeloid-related DC that enhances a more mixed Th phenotype. Immunization of FL-treated mice with a protein antigen leads to increased production of antibodies specific for that protein as well as to antigen-specific helper T cell responses. Studies of mouse tumor models have demonstrated that FL administration leads to the generation of protective anti-tumor immune responses, these effects being mediated by CTL and/or NK cells. When, FL has only minor or short term effects, the anti-tumor response can be significantly amplified by adding other cytokines, known to act at the T cell level, such as CD40 ligand (CD40L) or 4-1BBL, a TNF family member. Thus, combination of FL + CD40L or FL + 4-1BBL have superior anti-tumor effects vs. either cytokine alone. In conclusion, cytokines offer a variety of novel approaches for the treatment of cancer, infectious or auto-immune diseases.

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II+ epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8+ CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-γ-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-γ-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8+ CTL.

ABSTRACT Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8+T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8+ T-cell responses in vivo.

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8α− DC induces a Th2-type response, whereas injection of CD8α+ DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-γ, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-γ are required for Th1 priming by CD8α+ DC, whereas IL-10 is required for optimal development of Th2 cells by CD8α− DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8α+ DC. Conversely, incubation with IFN-γ down-regulates the Th2-promoting capacities of CD8α− DC and increases the Th1-skewing properties of both subsets.

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of naïve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II 1 epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8 1 CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN- g -producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN- g -producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8 1 CTL. The Journal of Immunology, 2001, 167: 384–391.