DC-T Cell Co-culture Research Articles

Th17 immunity to tuberculosis requires CD40-CD40L interaction and is enhanced by CD40 engagement on Mycobacterium tuberculosis-infected dendritic cells

Abstract Mycobacterium tuberculosis (Mtb) impairs dendritic (DC) cell functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper 17 (Th17) cells have emerged as important for protecting against tuberculosis (TB), but the mechanisms involved in generating antigen-specific Th17 responses during Mtb infection are not well defined. We previously reported that Mtb restricts Th17 responses and impairs CD40 expression on dendritic cells (DCs). We now demonstrate that generating IL-17 responses to Mtb requires CD40-dependent costimulation. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced generation of antigen specific IL-17 responses; cytokines were unable to compensate for this reduction. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that targeting the CD40-CD40L pathway represents a novel strategy to enhance early Th17 responses and improves adaptive immunity to TB.

DC-SIGN promotes allergen uptake and activation of dendritic cells in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD) is a common inflammatory skin disease, concomitant with allergic reactions to allergens. However, the exact mechanisms of allergen-induced immune responses in AD are not clear. The aim of this study is to explore the role of DC-SIGN in capturing and processing glycan-containing allergens and in the subsequent DC activation and T helper cell polarization in AD patients. MethodsDC-SIGN expression on DCs from AD patients was analysed by confocal microscopy and flow cytometry. DC-SIGN binding to common allergens was determined by ELISA. Activation of monocyte-derived dendritic cells (Mo-DCs) by allergens was analysed by evaluation of pro-inflammatory cytokines production, and their impact on T-cell responses was investigated by a DC-T cell coculture. ResultsDC-SIGN expression was higher on DCs in the lesional skin of AD patients compared with that of healthy controls and was correlated with disease severity. DC-SIGN could bind to many common allergens including house dust mite allergen (Der p2) and egg white allergen (Gal d2). Mo-DCs showed measurable expression of DC-SIGN and a concentration-dependent uptake of Der p2 and Gal d2, which was inhibited by mannan and anti-DC-SIGN Abs. Der p2 and Gal d2 induced the production of pro-inflammatory cytokines, including TNF-α and IL-6, by DCs from AD patients and facilitated Th2 and Th22 cell polarization. ConclusionsBinding of common allergens by DC-SIGN on DCs may initiate allergen sensitization of AD or provoke the relapse of AD. Regulating the allergen-DC-SIGN interaction might be a promising strategy to prevent or intervene in the progress of AD.

Differential Effects of Viscum album Preparations on the Maturation and Activation of Human Dendritic Cells and CD4⁺ T Cell Responses.

Extracts of Viscum album (VA); a semi-parasitic plant, are frequently used in the complementary therapy of cancer and other immunological disorders. Various reports show that VA modulates immune system and exerts immune-adjuvant activities that might influence tumor regression. Currently, several therapeutic preparations of VA are available and hence an insight into the mechanisms of action of different VA preparations is necessary. In the present study, we performed a comparative study of five different preparations of VA on maturation and activation of human dendritic cells (DCs) and ensuing CD4+ T cell responses. Monocyte-derived human DCs were treated with VA Qu Spez, VA Qu Frf, VA M Spez, VA P and VA A. Among the five VA preparations tested VA Qu Spez, a fermented extract with a high level of lectins, significantly induced DC maturation markers CD83, CD40, HLA-DR and CD86, and secretion of pro-inflammatory cytokines such as IL-6, IL-8, IL-12 and TNF-α. Furthermore, analysis of T cell cytokines in DC-T cell co-culture revealed that VA Qu Spez significantly stimulated IFN-γ secretion without modulating regulatory T cells and other CD4+ T cytokines IL-4, IL-13 and IL-17A. Our study thus delineates differential effects of VA preparations on DC maturation; function and T cell responses.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells.

PDGF upregulates CLEC-2 to induce T regulatory cells.

The effect of platelet derived growth factor (PDGF) on immune cells is not elucidated. Here, we demonstrate PDGF inhibited the maturation of human DCs and induced IL-10 secretion. Culture of PDGF-DCs with T cells induced the polarization of T cells towards FoxP3 expressing T regulatory cells that secreted IL-10. Gene expression studies revealed that PDGF induced the expression of C-type lectin like receptor member 2, (CLEC-2) receptor on DCs. Furthermore, DCs transfected with CLEC-2 induced T regulatory cells in DC-T cell co-culture. CLEC-2 is naturally expressed on platelets. Therefore, to confirm whether CLEC-2 is responsible for inducing the T regulatory cells, T cells were cultured with either CLEC-2 expressing platelets or soluble CLEC-2. Both conditions resulted in the induction of regulatory T cells. The generation of T regulatory cells was probably due to the binding of CLEC-2 with its ligand podoplanin on T cells, since crosslinking of podoplanin on the T cells also resulted in the induction of T regulatory cells. These data demonstrate that PDGF upregulates the expression of CLEC-2 on cells to induce T regulatory cells.

MicroRNA-155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury.

Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Mir-155 has been reported to be involved in both innate and adaptive immune responses. But the role of Mir-155 in spinal cord injury is still unknown. In our current study, Mir-155 deficiency displays increased myelin sparring and enhanced SC repair process. The number of T cells, B cells and neutrophils are all significantly lower in Mir-155(-/-) group than that in WT group after SCI. IL-17A-producing cells and the expression of IL-17A are markedly lower in Mir-155(-/-) mice than that in WT mice. We also found higher production of IL-17 by WT CD4(+) T cells than Mir-155(-/-) CD4(+) T cells in vitro. In our further DC-T cell coculture system, Mir-155 deficiency in DCs results in significantly less IL-17 production from T cells. Furthermore, the inhibited Th17 differentiation induced by Mir-155 deficiency is partly dependent on increased expression of SOCS1. In conclusion, our present work provides evidence to support the concept that Mir-155 deficiency suppresses Th17 cell differentiation and improves locomotor recovery after SCI.

Transcriptional control of tolerance associated gene TCAIM is CREB, NFATc1 and C/EBPβ dependent resulting in generation of tolerogenic dendritic cells (P2152)

Abstract Cell therapy by tolerogenic dendritic cells has become an attractive therapeutic option in transplantation. We could recently show that high expression of TCAIM, formerly known as TOAG-1, in dendritic cells (DCs) is associated with stable tolerance following transplantation. Here, we investigated transcriptional control of TCAIM in murine bone marrow derived DCs (BMDCs) and human MUTZ-3 derived DCs (M3DCs). We have cloned the human TCAIM promoter to identify regulatory elements by site directed mutagenesis. In addition, we performed luciferase reporter as well as ChIP assays. Furthermore, BMDCs or M3DCs were pre-incubated with cAMP elevating drugs prior to LPS-mediated maturation. Phenotype and tolerogenic potential of DCs were investigated by qPCR, flow cytometry, CBA and MLR. Site-directed mutagenesis, reporter assays and ChIP defined TCAIM core promoter containing three CREB responsive elements. Co-transfections revealed positive regulation of promoter activity by NFATc1 and C/EBPβ. Furthermore, pre-incubation of M3DCs or BMDCs with cAMP elevating drugs leading to CREB activation resulted in high TCAIM expression, inhibited DC maturation, diminished pro-inflammatory cytokine release and induced high frequency of CD4+CD25+FoxP3+ Tregs in DC-T cell co-cultures. These results indicate that transcriptional activation of TCAIM is cAMP/CREB dependent and further amplified by NFATc1 and C/EBPβ, resulting in induction of tolerogenic potential in human M3DCs and murine BMDCs.

In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

BackgroundCoccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively.ResultsWe found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (TEM) and IFN-γ release.ConclusionsIn conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

CD8+ DC, but Not CD8−DC, Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

BackgroundTuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.Methodology/Principal Findings We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8α+ DC isolated from infected mice (iCD8+ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8+ DC recipients than sham-treated control mice. The adoptive transfer of the CD8α−DC from the infected mice (iCD8− DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8− DC produced significantly higher levels of IL-10 than iCD8+ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.Conclusions/SignificanceThe data indicate that in vivo BCG-primed CD8+ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases.

CD40 signalling induces IL‐10‐producing, tolerogenic dendritic cells

Dendritic cells (DC) are potent antigen-presenting cells capable to induce efficient antigen-specific T cell responses in vitro and in vivo. Herein, the maturation process is of great significance, as immature DC (iDC) are known to induce rather regulatory than effector T cell differentiation. This study was designed to characterize the role of the CD40-CD40L pathway for differentiation and function of human DC. Therefore, iDC were stimulated through CD40-CD40L interaction by transduction of DC with adenoviral vectors encoding for CD40L (Ad-CD40L). Resulting DC (CD40L-DC) were analysed concerning their phenotype, cytokine profile and T cell stimulatory capacity. Transduction induced a DC phenotype comparable to stimulation with proinflammatory cytokines as revealed by upregulation of CD83 and the costimulatory molecules CD80 and CD86. Additionally, Ad-CD40L-induced strong production of IL-12p70 not observed in cytokine-matured DC. Surprisingly, the T cell stimulatory capacity was markedly reduced in CD40L-DC. Furthermore, stimulation of CD8(+) T cells by peptide-loaded CD40L-DC resulted in a substantial reduction of antigen-specific IFN-gamma production. This phenomenon is due to an enhanced IL-10 production of CD40L-DC in DC-T cell coculture as well as a stabilization of the IL-10 receptor expression on activated T cells. These data demonstrate that DC stimulated through CD40-CD40L interaction differentiate into tolerogenic DC with immunomodulatory function.

IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function

Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17-neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-gamma production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function.

HIV‐1 induced generation of C5a attracts immature dendriticcells and promotes infection of autologous T cells

For the recruitment of dendritic cells (DC) to the site of infection, DC express several sensors for danger signals, such as receptors for C5a. This anaphylatoxin is generated upon complement activation. As HIV-1 triggers the complement cascade, we determined whether C5a is generated by the virus and tested the functional activity of C5a in migration and infection assays. The immature (i)DC responded in migration assays to recombinant C5a and native C5a, which was generated in situ upon activation of the complement system by HIV-1. In combined migration and infection assays, a C5a-dependent enhancement of HIV-1 infection in DC-T cell cocultures was observed. These results indicate that HIV induces generation of C5a and thereby attracts iDC, which in turn promote the productive infection of autologous primary T cells.

Artificial phosphatidylserine liposome mimics apoptotic cells in inhibiting maturation and immunostimulatory function of murine myeloid dendritic cells in response to 1-chloro-2,4-dinitrobenze in vitro

Phosphatidylserine (PS) exposed on the apoptotic cell surface inhibits inflammatory responses, implying that PS may regulate the function of dendritic cells (DCs) after being phagocytosed by the latter. Here we use PS liposomes to investigate the effects of PS on the maturation and immunostimulatory functions of DCs in response to the challenge of 1-chloro-2,4-dinitrobenze (DNCB) in vitro. We demonstrate that after treatment with PS, murine DCs display reduced expression of MHC II, CD80, CD86 and CD40, but increased programmed death ligand-1 (PD-L1 and PD-L2); and increased IL-10 and inhibited IL-12 cytokine production. PS-treated DCs exhibit normal endocytic function, but ability to stimulate allogeneic T cells is reduced, similar to immature dendritic cell (iDCs). Treatment of DCs with PS liposomes also suppressed DNCB induced CD4 + T cell proliferation and IFN-gamma production. Addition of exogenous IL-12p70 during the DC-T cell co-culture restored their IFN-gamma production. Furthermore, PS-treated DCs enhance the ratio of CD4(+) CD25(high)Foxp3(+) T cells to CD4(+) T cells and PD-1 expression on CD4(+) T cells. These data demonstrate that PS liposomes have therapeutic potential in allergic contact dermatitis (ACD).

IgG Opsonization of HIV Impedes Provirus Formation in and Infection of Dendritic Cells and Subsequent Long-Term Transfer to T Cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.

CCL19 and CCL21 Induce a Potent Proinflammatory Differentiation Program in Licensed Dendritic Cells

Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.

Defective dendritic cell function in HIV-infected patients receiving effective highly active antiretroviral therapy: neutralization of IL-10 production and depletion of CD4+CD25+ T cells restore high levels of HIV-specific CD4+ T cell responses induced by dendritic cells generated in the presence of IFN-alpha.

We previously demonstrated that GM-CSF/IFN-alpha combination allowed the differentiation of monocytes from HIV-infected patients into dendritic cells (DCs) exhibiting high CD8(+) T cell stimulating abilities. The present study was aimed at characterizing the ability of DCs generated in the presence of GM-CSF and IFN-alpha to induce CD4 T cell responses. DCs were generated from monocytes of HIV-infected patients in the presence of GM-CSF with either IFN-alpha (IFN-DCs) or IL-4 (IL-4-DCs) for 7 days. Eleven patients receiving highly active antiretroviral therapy and exhibiting CD4 cell counts above 400/mm(3) and plasma HIV-RNA <50 copies/ml for at least 1 year were included in the study. Both DC populations were found to be defective in inducing autologous (in response to tuberculin or HIV-p24) or allogeneic CD4 T cell proliferation. Neutralization of IL-10 during the differentiation of IFN-DCs, but not during the DC-T cell coculture, significantly increased their ability to stimulate autologous CD4 T cell proliferation in response to tuberculin and allogeneic CD4 T cell proliferation (4.1-fold and 3.0-fold increases, respectively, at the DC to T cell ratio of 1:10). Moreover, IL-10 neutralization and CD4(+)CD25(+) T cell depletion synergistically act to dramatically increase HIV-p24-specific CD4 T cell responses induced by IFN-DCs (31.7-fold increase) but not responses induced by IL-4-DCs. Taken together, our results indicate that IFN-DCs are more efficient than IL-4-DCs to stimulate CD4(+) T cell proliferation, further supporting their use for immune-based therapy in HIV infection.

Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism.

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.

Monophosphoryl lipid A activates both human dendritic cells and T cells.

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-kappaB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 microg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA(+) T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 microg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.

Helper T-cell responses elicited by Der p 1–pulsed dendritic cells and recombinant IL-12 in atopic and healthy subjects

Background: Environmental allergens, such as Dermatophagoides pteronyssinus group 1 antigen (Der p 1), induce TH2-type responses in atopic patients, whereas healthy individuals have TH1-type responses to the same antigens. Because of their efficient synthesis of IL-12, dendritic cells (DCs) are potent inducers of TH1-type immune responses. Objective: We sought to determine whether DCs would skew allergen-specific TH2-type responses from atopic individuals. Methods: Purified CD4+ T cells from healthy donors or atopic individuals were cultured in the absence or presence of recombinant (r)IL-12 with DCs derived from PBMCs and pulsed with Der p 1. Supernatants of DC-T cell cocultures were assayed by ELISA for IL-5 and IFN-γ. Results: A TH1-type response developed in purified CD4+ T cells from healthy donors in response to Der p 1–pulsed DCs, as indicated by high levels of IFN-γ in culture supernatants. In contrast, CD4+ T cells from atopic donors displayed a TH2-type profile characterized by high levels of IL-5 and low levels of IFN-γ. The addition of rIL-12 (10 ng/mL) to DC-T cell cocultures resulted in the induction of IFN-γ secretion by Der p 1–specific CD4+ T cells from atopic patients, whereas their production of IL-5 was not inhibited. Using flow cytometry after intracytoplasmic staining, we found that IFN-γ and IL-5 were secreted by distinct CD4+ T-cell subpopulations. Conclusion: The cytokine profile of Der p 1–specific TH2-like cells from atopic individuals is maintained when the allergen is presented by DCs, even in the presence of exogenous rIL-12. (J Allergy Clin Immunol 2000;105:346-52.)

Misoprostol Modulation of Dendritic Cell Function in Immune Responses.

Dendritic cells (DC) from blood and other tissues are potent accessory cells for primary immune responses. Because prostaglandins from monocytes and macrophages can suppress DC and T-cell function, we sought to investigate the binding properties of misoprostol (MPL), a prostaglandin E(1) analog, and its regulation of DC function. Results of mouse and human experiments have suggested 1) that MPL could significantly inhibit DC-induced T-cell proliferation in oxidative mitogenesis and allogeneic mixed leukocyte reactions by decreasing interleukin-2 production in DC-T cell cocultures, and 2) that MPL could bind to human peripheral blood mononuclear cells via specific high-affinity MPL binding sites as well as through nonspecific MPL uptake. Taken together, these data suggest that MPL can bind high-affinity and/or nonspecific cell surface receptors and subsequently regulate T-cell growth and cytokine production such as that induced by DC and associated with primary immune responses.