Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo

During priming, naive CD4(+) Th cells differentiate into cells that produce either IFN-gamma or IL-4. Even though the cascade of pathways that induces IL-4-producing Th2 cells has been determined in vitro, the signals promoting Th2 differentiation under physiological conditions remain enigmatic, especially the natural role of the single most important Th2-inducing signal,IL-4. Using Th2 and naive Th cells, each expressing a distinct transgenic TCR, here we show that Th2 cells migrate with the same dynamics as naive Th cells in draining lymph nodes and bind to the same DC, when driven by antigen in complete Freund's adjuvant (CFA). Th2-cell-derived IL-4 deviates CFA-induced Th1 development toward a Th2 phenotype, if both cell populations co-localize in the same T cell area, and are activated simultaneously. Thus, intranodal Th2 cells directly influence Th cell differentiation in vivo, but only under restricted conditions. These findings have implications for the design of cytokine-based therapies and explain the spreading of Th2 responses to multiple aeroallergens in allergic asthma, where naive Th and Th2 cells co-localize in lung-draining lymph nodes.     

Increased Th1 and Th2 allergen-induced cytokine responses in children with atopic disease

BACKGROUND: Polyclonal cytokine responses following stimulation of T cells with mitogens or superantigens provides information on cytokine production from a wide range of T cells. Alternatively allergen-induced T cell responses can provide information on cytokine production by allergen-reactive T cells. While there is evidence of increased Th2 and reduced Th1 cytokine production following T cell stimulation with non-specific mitogens and superantigens, the evidence that Th1 cytokine production to allergens is decreased in line with a postulated imbalance in Th1/Th2 responses is unclear, with studies finding decreased, no difference or increased IFN-gamma responses to allergens in atopic subjects. OBJECTIVE: To examine childhood polyclonal and allergen-induced cytokine responses in parallel to evaluate cytokine imbalances in childhood atopic disease. METHODS: PBMC cytokine responses were examined in response to a polyclonal stimulus, staphylococcal superantigen (SEB), in parallel with two inhalant allergens, house dust mite (HDM) and rye grass pollen (RYE), and an ingested allergen, ovalbumin (OVA), in (a) 35 healthy children (non-atopic) and (b) 36 children with atopic disease (asthma, eczema and/or rhinitis) (atopic). RESULTS: Atopic children had significantly reduced IFN-gamma and increased IL-4 and IL-5 but not IL13 production to SEB superantigen stimulation when compared with non-atopic children. HDM and RYE allergens stimulated significantly increased IFN-gamma, IL-5 and IL-13, while OVA stimulated significantly increased IFN-gamma production in atopic children. CONCLUSION: We show that a polyclonal stimulus induces a reduced Th1 (IFN-gamma) and increased Th2 (IL-4 and IL-5) cytokine pattern. In contrast, the allergen-induced cytokine responses in atopic children were associated with both increased Th1 (INF-gamma) and Th2 (IL-5 and IL-13) cytokine production. The increased Th1 response to allergen is likely to reflect prior sensitization and indicates that increases in both Th1 and Th2 cytokine production to allergens exists concomitantly with a decreased Th1 response to a polyclonal stimulus in atopic children.     

Activation of human neonatal monocyte-derived dendritic cells by lipopolysaccharide down-regulates birch allergen-induced Th2 differentiation

Epidemiological studies describe an inverse association between the level of environmental endotoxin exposure during infancy and the prevalence of allergic disease in children. To study the effect of lipopolysaccharide (LPS) and lipopeptide Pam3Cys signaling via Toll-like receptor (TLR)4 and TLR2 on dendritic cells (DC), respectively, on birch allergen-induced T cell differentiation, cord blood monocyte-derived DC were exposed to birch allergen extract alone or in combination with LPS or Pam3Cys and thereafter co-cultured with naive autologous T cells. We demonstrate that birch allergen alone induced high levels of IL-13 from neonatal T cells, whereas the production of IL-5 and IFN-gamma was modest. Stimulation of DC with birch allergen together with LPS but not Pam3Cys resulted in a decreased IL-13 production by T cells compared to birch allergen alone. Furthermore, birch allergen together with LPS induced increased up-regulation of activation markers expressed on the surface and production of cytokines from DC relative to stimulation with birch allergen alone. Finally, birch allergen partially suppressed both LPS- and Pam3Cys-induced DC maturation. Our results indicate that concomitant TLR4 stimulation during the initial phase of immune activation to birch allergen in infants may inhibit the development of a T helper 2-type response.     

Langerin+ dendritic cells are responsible for LPS-induced reactivation of allergen-specific Th2 responses in postasthmatic mice

Allergic asthma is a T cell-dependent inflammatory lung disease that results from complex interactions between genetic predisposition and environmental factors, including exposure to lipopolysaccharide (LPS). In this study, we have shown that airway LPS exposure was sufficient to induce airway hyperreactivity (AHR) and eosinophil recruitment in mice that had previously experienced an acute episode of allergic asthma. LPS-induced disease reactivation depended on the activation of allergen-specific CD4(+) T cells by a subset of lung langerin(+) dendritic cells (DCs) that retained the allergen. Upon LPS exposure, migration of langerin(+) DCs from lungs to draining lymph nodes increased and LPS-exposed langerin(+) DCs instructed CD4(+) T cells toward a T helper (Th) 2 response. Selective depletion of langerin(+) DCs prevented LPS-induced eosinophil recruitment and T-cell activation, further demonstrating a critical role for langerin(+) DCs in disease reactivation. This finding provides a possible explanation for the subclinical worsening of asthmatics following exposure to low-dose LPS.     

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice

BACKGROUND: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). METHODS: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. RESULTS: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. CONCLUSIONS: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.     

Natural killer dendritic cells are an intermediate of developing dendritic cells

NK dendritic cells (DCs; NKDCs) appear to emerge as a distinct DC subset in humans and rodents, which have the functions of NK cells and DCs. However, the developmental relationship of NKDCs (CD11c(+)NK1.1(+)) to CD11c(+)NK1.1(-) DCs has not been addressed. Herein, we show that NKDCs exist exclusively in the compartment of CD11c(+)MHC II(-) cells in the steady state and express variable levels of DC subset markers, such as the IFN-producing killer DC marker B220, in a tissue-dependent manner. They can differentiate into NK1.1(-) DCs, which is accompanied by the up-regulation of MHC Class II molecules and down-regulation of NK1.1 upon adoptive transfer. However, NK cells (NK(+)CD11c(-)) did not differentiate into NK1.1(+)CD11c(+) cells upon adoptive transfer. Bone marrow-derived Ly6C(+) monocytes can be a potential progenitor of NKDCs, as some of them can differentiate into CD11c(+)NK1.1(+) as well as CD11c(+)NK1.1(-) cells in vivo. The steady-state NKDCs have a great capacity to lyse tumor cells but little capability to present antigens. Our studies suggest that NKDCs are an intermediate of developing DCs. These cells appear to bear the unique surface phenotype of CD11c(+)NK1.1(+)MHC II(-) and possess strong cytotoxic function yet show a poor ability to present antigen in the steady state. These findings suggest that NKDCs may play a critical role in linking innate and adaptive immunity.     

Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens

Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103(+) DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103(+) DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11b(hi) DCs primed Th1 differentiation. Moreover, mice lacking CD103(+) DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103(+) DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103(+) DCs have a significant role in priming Th2 responses to inhaled allergens.     

Surface molecules on stimulated plasmacytoid dendritic cells are sufficient to cross-activate resting myeloid dendritic cells

Human plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are 2 types of antigen-presenting cells that exert complementary roles in innate immune responses. We demonstrated previously that in the presence of suboptimal stimulation or when only 1 dendritic cell type is directly stimulated, contact-dependent crosstalk between mDCs and pDCs leads to the activation of both cell types and thus provides them with the ability to induce an optimal T-cell response. The precise mechanism is currently unknown. Here we demonstrate that pDCs, unable to secrete soluble factors because of previous stimulation, induce optimal mDC maturation, indicating that resting immature mDCs are fully competent to respond to Toll-like receptor-9-engaged pDCs in the absence of soluble factors. Thus, we conclude that immature mDCs already express receptors recognized by ligands that are upregulated on the surface of activated pDCs. Intercellular adhesion molecule-1 upregulated by activated pDCs may play a role in a donor-dependent manner.     

Selective maturation of dendritic cells by Nippostrongylus brasiliensis-secreted proteins drives Th2 immune responses

Helminth infections at mucosal and tissue sites strongly polarize towards Th2 immune responses, following pathways which have yet to be elucidated. We investigated whether dendritic cells (DC) exposed to gastrointestinal nematodes induce Th2 differentiation and, if so, whether this outcome reflects the absence of DC activation (the default hypothesis). We studied secreted proteins from the parasite Nippostrongylus brasiliensis, which induce Th2 development in vivo without live infection. Murine bone marrow-derived DC pulsed with N. brasiliensis excretory/secretory antigen (NES) can, on transfer to naive recipients, prime mice for Th2 responsiveness. Heat inactivation of NES abolishes both its ability to drive Th2 responses in vivo and its capacity to stimulate DC for Th2 induction. NES, but not heat-inactivated NES, up-regulates DC maturation markers associated with Th2 promotion (CD86 and OX40L), with little change to CD80 and MHC class II. Moreover, DC exposed to NES readily produce IL-6 and IL-12p40, but not IL-12p70. LPS induced high IL-12p70 levels, except in DC that had been pre-incubated with NES. These data contradict the default hypothesis, demonstrating that a helminth product (NES) actively matures DC, selectively up-regulating CD86 and OX40L together with IL-6 production, while blocking IL-12p70 responsiveness in a manner consistent with Th2 generation in vivo.     

Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation

Overproduction of cytokines by T helper 2 (Th2) cells in the lung is thought to be a cause of asthma. Here we report that innate lymphocytes termed lung natural helper (LNH) cells are a T cell-independent source of Th2 cell-type cytokines in protease allergen-treated lungs. LNH (Lin(-)Sca-1(+)c-kit(+/lo)CD25(+)CD127(+)) cells, when stimulated by IL-33 plus IL-2, IL-7, or thymic stroma lymphopoietin (TSLP), produced large amounts of IL-5 and IL-13. Intranasal administration of protease allergen papain induced eosinophil infiltration and mucus hyperproduction in the lung of wild-type and Rag1(-/-) mice, but not in Rag2(-/-)Il2rg(-/-) mice that lack LNH cells. LNH cell depletion inhibited papain-induced airway inflammation in Rag1(-/-) mice whereas adoptive transfer of LNH cells enabled Rag2(-/-)Il2rg(-/-) mice to respond to papain. Treatment of lung explants with papain induced IL-33 and TSLP production by stroma cells and IL-5 and IL-13 production by LNH cells. Thus, LNH cells are critical for protease allergen-induced airway inflammation.     

Resting B cells are not antigen-presenting cells in the induction of oral tolerance of specific Th2 immune responses in mice

BACKGROUND: It has been shown that antigen presentation by resting B cells can induce tolerance to intravenously administered protein antigens, but the role of resting B cells in the induction of oral tolerance is unclear. METHODS: Mice continuously treated since birth with rabbit anti-mouse IgM serum for 5 weeks were depleted of B cells. When 4 weeks old, B cell-depleted mice drank 10% chicken egg white (EW) for 5 days. Ten weeks later, they were immunized with 10 microgram of ovalbumin in alum and their T helper 2 (Th2) immune responses were tested. RESULTS: Th2 cell-mediated IgE and IgG1 antibody responses and spleen cell production of IL-4 and IL-5 were suppressed by prior EW feeding during anti-IgM treatment. When anti-IgM-treated spleen cells collected 1 week after EW ingestion were transferred to na?ve recipients, active suppression of Th2 immune responses was also demonstrated. CONCLUSIONS: Although resting small B cells aggregate in the mantle zone of follicles of intestinal Peyer's patches, the present data suggest that they are not antigen-presenting cells in the induction of oral tolerance of Th2 immune responses to oral antigens.     

Myeloid dendritic cells stimulate both Th1 and Th2 immune responses depending on the nature of the antigen.

It has been shown that different types of pathogens induce different immune responses. Recovery from intracellular bacterial and viral infection is dependent on the secretion of Th1 cytokines, such as interferon-gamma (IFN-gamma), and on the generation of cytotoxic T cells. In contrast, responses to some parasitic invaders are of the Th2 type, characterized by secretion of interleukin-4 (IL-4). At present, it is not clear what directs this choice, and the most prevalent hypotheses are based on the dendritic cells (DC). In this work, we studied the immune responses generated in mice to a number of antigens, both replicating and nonreplicating, using bone marrow-derived DC as vehicles for immunization. We demonstrate that DC infected with influenza virus prime for a pure Th1 response in vivo devoid of IL-4 induction. This immune response correlates with the induction of DC maturation by the virus. In contrast, nonreplicating antigens, such as fetal bovine serum (FBS), beta-galactosidase, or inactivated influenza virus, do not mature the DC and prime for responses characterized by the secretion of large amounts of IL-4. These data support the hypothesis that myeloid DC are capable of eliciting both types of responses depending on the nature of the antigen.     

Lung dendritic cell-epithelial cell crosstalk in Th2 responses to allergens

Dendritic cells (DC) have been shown to be responsible for the initiation and maintenance of adaptive Th2 responses in asthma. It is increasingly clear that DC functions are strongly influenced by crosstalk with neighboring cells like epithelial cells, which can release a number of innate cytokines promoting Th2 responses. Clinically relevant allergens often interfere directly or indirectly with the innate immune functions of airway epithelial cells and DC. A better understanding of these interactions might lead to a better prevention and ultimately to new treatments for asthma.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4⁺ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-γ secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1-induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-γ-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1-induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4⁺ T cell responses.     

Mast cells promote Th1 and Th17 responses by modulating dendritic cell maturation and function

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However, it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs, we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore, the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ, IL-2, IL-6 and TGF-β into coculture supernatants and increased the IL-12p70, IFN-γ, IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-"primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly, we observed that MC-primed DCs stimulated CD4+ T cells to release high levels of IFN-γ and IL-17, demonstrating that MCs promote Th1 and Th17 responses. Confirming our in vitro findings, we found that the enhanced disease progression of MC-deficient mice in Leishmania major infection is correlated with impaired induction of both Th1 and Th17 cells.     

Soluble CD14 and CD83 from human neonatal antigen-presenting cells are inducible by commensal bacteria and suppress allergen-induced human neonatal Th2 differentiation

CD14 is expressed on the cell surface of various antigen-presenting cells, and CD83 is a maturation marker for dendritic cells (DC). CD14 and CD83 are also present as soluble proteins, and both have immunoregulatory functions. We examined whether neonatal cord blood monocytes or DC released soluble CD14 (sCD14) or sCD83 when exposed to the commensal intestinal bacteria Clostridium perfringens, Staphylococcus aureus, Lactobacillus rhamnosus, Escherichia coli, and Bacteroides fragilis. We found that the gram-positive bacteria C. perfringens and S. aureus, but not gram-negative bacteria, induced the release of sCD14 from monocytes. DC, on the other hand, released sCD14 in response to both gram-positive and gram-negative bacteria. Moreover, the expression of the virulence factor staphylococcal protein A seemed to be important for S. aureus-induced sCD14 production from both monocytes and DC. Soluble CD83 was released from DC, but not from monocytes, when exposed to both gram-positive and gram-negative bacteria. Finally, to investigate whether sCD14 or sCD83 could modulate neonatal allergen-induced T-cell differentiation, DC were exposed to birch allergen alone or in the presence of sCD14 or sCD83 and then cocultured with autologous T cells. We demonstrate that sCD14 and sCD83 inhibited the birch allergen-induced Th2 differentiation by suppressing interleukin 13 production. Together, these results suggest that the commensal intestinal flora may be an important stimulus for the developing immune system by inducing the immunoregulatory proteins sCD14 and sCD83, which may be involved in preventing T-cell sensitization to allergens in infants.