Dendritic Cell Antigen Uptake Research Articles

How B cells drive T-cell responses: A key role for cross-presentation of antibody-targeted antigens.

In this review we discuss an underexposed mechanism in the adaptive immune system where B cell and T cell immunity collaborate. The main function of B cell immunity is the generation of antibodies which are well known for their high affinity and antigen-specificity. Antibodies can bind antigens in soluble form making so-called immune complexes (ICs) or can opsonize antigen-exposing cells or particles for degradation. This leads to well-known effector mechanisms complement activation, antibody-dependent cytotoxicity and phagocytosis. What is less realized is that antibodies can play an important role in the targeting of antigen to dendritic cells (DCs) and thereby can drive T cell immunity. Here we summarize the studies that described this highly efficient process of antibody-mediated antigen uptake in DCs in vitro and in vivo. Only very low doses of antigen can be captured by circulating antibodies and subsequently trapped by DCs in vivo. We studied the handling of these ICs by DCs in subcellular detail. Upon immune complex engulfment DCs can sustain MHC class I and II antigen presentation for many days. Cell biological analysis showed that this function is causally related to intracellular antigen-storage compartments which are functional endolysosomal organelles present in DCs. We speculate that this function is immunologically very important as DCs require time to migrate from the site of infection to the draining lymph nodes to activate T cells. The implications of these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed.

A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that βγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of βγ-CAT. The cellular action of βγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of βγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

In Vivo Irradiation of Mice Induces Activation of Dendritic Cells.

It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens the possibility for combining radio- and immunotherapies in cancer treatment. The presence of functionally competent dendritic cells (DCs) is crucial in mounting a successful antitumor immune response. While it has been shown that DCs are relatively radioresistant, few and contradictory data are available on how ionizing radiation alters the functional integrity of these cells. Therefore, our objective was to investigate the effect of whole-body irradiation on the function of splenic DCs. C57Bl/6 mice were irradiated with 0.1, 0.25, and 2 Gy X-rays and changes in the phenotype of splenic DCs were compared to unirradiated controls. An increase was seen in DC surface markers influencing DC-T cell interactions. In vivo cytokine production was determined by direct intracellular cytokine staining. Irradiation with 2 Gy induced a 1.6-fold increase in IL-1α production, while the combination of irradiation and lipopolysaccharide (LPS) treatment induced a 3.9-fold increase, indicating a strong synergism between irradiation and LPS stimulation. Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction. While DCs from control animals induced stronger proliferation of regulatory T cells, DCs from animals irradiated with 2 Gy induced stronger proliferation of effector T cells. Antigen uptake and presentation was investigated by measuring the capacity of DCs to internalize and present ovalbumine (OVA)-derived peptides on their major histocompatibility complex (MHCI) molecules. Irradiation with 2 Gy did not influence antigen uptake or presentation, while low doses stimulated antigen uptake and reduced the level of antigen presentation. In conclusion, high-dose in vivo irradiation induced increased expression of T cell costimulatory markers, enhanced production of proinflammatory cytokines and a stronger stimulation of effector T cell proliferation than that of regulatory T cells. However, it did not influence DC antigen uptake or presentation. On the other hand, low-dose irradiation increased antigen uptake and lowered antigen presentation of DCs, indicating that low- and high-dose irradiation act on different pathways in DCs.

Critical role of integrin CD11c in splenic dendritic cell capture of missing-self CD47 cells to induce adaptive immunity

CD11c, also known as integrin alpha X, is the most widely used defining marker for dendritic cells (DCs). CD11c can bind complement iC3b and mediate phagocytosis in vitro, for which it is also referred to as complement receptor 4. However, the functions of this prominent marker protein in DCs, especially in vivo, remain poorly defined. Here, in the process of studying DC activation and immune responses induced by cells lacking self-CD47, we found that DC capture of CD47-deficient cells and DC activation was dependent on the integrin-signaling adaptor Talin1. Specifically, CD11c and its partner Itgb2 were required for DC capture of CD47-deficient cells. CD11b was not necessary for this process but could partially compensate in the absence of CD11c. Mice with DCs lacking Talin1, Itgb2, or CD11c were defective in supporting T-cell proliferation and differentiation induced by CD47-deficient cell associated antigen. These findings establish a critical role for CD11c in DC antigen uptake and activation in vivo. They may also contribute to understanding the functional mechanism of CD47-blockade therapies.

Photothermally Controlled MHC Class I Restricted CD8+ T-Cell Responses Elicited by Hyaluronic Acid Decorated Gold Nanoparticles as a Vaccine for Cancer Immunotherapy.

Cancer vaccines aim to induce a strong major histocompatibility complex class I (MHC-I)-restricted CD8+ cytotoxic T-cell response, which is an important prerequisite for successful cancer immunotherapy. Herein, a hyaluronic acid (HA) and antigen (ovalbumin, OVA)-decorated gold nanoparticle (AuNPs)-based (HA-OVA-AuNPs) vaccine is developed for photothermally controlled cytosolic antigen delivery using near-infrared (NIR) irradiation and is found to induce antigen-specific CD8+ T-cell responses. Chemical binding of thiolated HA and OVA to AuNPs facilitates antigen uptake of dendritic cells via receptor-mediated endocytosis. HA-OVA-AuNPs exhibit enhanced NIR absorption and thermal energy translation. Cytosolic antigen delivery is then permitted through the photothermally controlled process of local heat-mediated endo/lysosome disruption by laser irradiation along with reactive oxygen species generation, which helps to augment proteasome activity and downstream MHC I antigen presentation. Consequently, the HA-OVA-AuNPs nanovaccine can effectively evoke a potent anticancer immune response in mice under laser irradiation. This NIR-responsive nanovaccine is promising as a potent vaccination method for improving cancer vaccine efficacy.

Immune response effects of diverse vaccine antigen attachment ways based on the self-made nanoemulsion adjuvant in systemic MRSA infection.

Nanoemulsion adjuvants-based vaccines have potent induced immune responses against methicillin-resistant Staphylococcus aureus (MRSA) infection. However, the efficacies and immune responses of different antigen-attaching ways on self-made nanoemulsion adjuvants remain unknown. In this study, we designed three formulations of nanoemulsion adjuvants (encapsulation, mixture, and combination) to explore their immune response-enhancing effects and their underlying mechanism in a systemic infection model of MRSA. Our results showed that the three nanoemulsion-attachment ways formulated with a fusion antigen of MRSA (HlaH35LIsdB348–465) all improved humoral and cellular immune responses. When compared with the mixture and combination formulations, the nanoemulsion-encapsulation group effectively promoted the antigen uptake of dendritic cells (DCs) in vitro, the activation of DC in draining lymph nodes and the delayed release of antigen at injection sites in vivo. Moreover, the encapsulation group induced a more ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17 biased CD4+ T cell response when compared with the other two attachment ways. Our findings suggested that the encapsulated formulation of vaccine with nanoemulsion adjuvant is an effective attachment way to provide protective immunity against MRSA infection.

Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells.

The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.

4,4'-diaponeurosporene, a C30 carotenoid, effectively activates dendritic cells via CD36 and NF-κB signaling in a ROS independent manner.

Carotenoids could be divided into C30 carotenoids and C40 carotenoids. The immune functions of C40 carotenoids had been extensively researched, while those of C30 carotenoids still remain unclear. In this study, the immune functions of a biosynthetic C30 carotenoid, 4,4′-diaponeurosporene (Dia), were identified on dendritic cells (DCs). DCs treated with 1 μM Dia for 24 h showed morphologic and phenotypic characteristics of mature state and had an increased production of IL-6, IL-10, IL-12p70 and TNFα, while β-carotene had a suppressive effect on DCs maturation. Moreover, Dia promoted antigen uptake of DCs in vitro and increased the quantity of antigen loaded DCs in mesenteric lymph nodes (MLN). Dia-treated DCs also had an enhanced ability to stimulate T cell proliferation and Th1 polarization. Further researches showed that Dia activated DCs via CD36 as well as ERK, JNK, and NF-κB signals in a reactive oxygen species (ROS) independent manner.

β2-adrenoceptor signaling reduction in dendritic cells is involved in the inflammatory response in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is characterized by inflammation of the synovium, which leads to the progressive destruction of cartilage and bone. Adrenoreceptor (AR) signaling may play an important role in modulating dendritic cell (DC), which may be involved in the pathogenesis of RA. We examined the effect of the β-AR agonist isoprenaline (ISO) on DC function, the impact of the β2-AR agonist salbutamol on adjuvant-induced arthritic (AA) rats, and changes in β2-AR signaling in DCs during the course of AA. ISO inhibited the expression of the surface molecules CD86 and MHC-II, inhibited the stimulation of T lymphocyte proliferation by DC and TNF-α secretion, and promoted DC antigen uptake and IL-10 secretion. The effects of ISO on MHC-II expression, DC stimulation of T lymphocyte proliferation, and DC antigen uptake were mediated by β2-AR. Treatment with salbutamol ameliorated the severity of AA and histopathology of the joints and inhibited proliferation of thymus lymphocytes and FLS in vivo. β2-AR signaling was weaker in AA rats compared to the control. Elevated GRK2 and decreased β2-AR expression in DC cytomembranes were observed in AA and may have decreased the anti-inflammatory effect of β2-AR signaling. Decreased β2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on the function of bone marrow-derived dendritic cells and the effects of CP-25

This study was to investigate PGE2 and TNF-alpha signaling pathway involving in the maturation and activation of bone marrow dendritic cells (DCs) and the effect of CP-25. Bone marrow DCs were isolated and stimulated by PGE2 and TNF-alpha respectively. The markers of maturation and activation expressed on DCs, such as CD40, CD80, CD83, CD86, MHC-II, and the ability of antigen uptake of DCs were analyzed by flow cytometry. The proliferation of T cells co-cultured with DCs, the signaling pathways of PGE2–EP4–cAMP and TNF-alpha–TRADD–TRAF2–NF-κB in DCs were analyzed. The results showed that both PGE2 and TNF-alpha up-regulated the expressions of CD40, CD80, CD83, CD86, and MHC-II, decreased the antigen uptake of DCs, and DCs stimulated by PGE2 or TNF-alpha could increase T cell proliferation. CP-25 (10−5, 10−6, and 10−7mol/l) decreased significantly the expressions of CD40, CD80, CD83, CD86 and MHC-II, increased the antigen uptake of DCs, and suppressed T cell proliferation induced by DCs. PGE2 increased the expressions of EP4, NF-κB and down-regulated cAMP level of DCs. TNF-alpha could also up-regulate TNFR1, TRADD, TRAF2, and NF-κB expression of DCs. CP-25 (10−5, 10−6, and 10−7mol/l) decreased the expressions of EP4 and NF-κB, increased cAMP level in DCs stimulated by PGE2. CP-25 (10−5, 10−6, and 10−7mol/l) also could down-regulate significantly TNFR1, TRADD, TRAF2, and NF-κB expression in DCs stimulated by TNF-alpha. These results demonstrate that PGE2 and TNF-alpha could enhance DCs functions by mediating PGE2–EP4–cAMP pathway, TNF-alpha–TNFR1–TRADD–TRAF2–NF-κB pathway respectively. CP-25 might inhibit the function of DCs through regulating PGE2–EP4–cAMP and TNF-alpha–TNFR1–TRADD–TRAF2–NF-κB pathways.

Measuring the properties of different forms of keyhole limpet hemocyan reveals important differences (VAC5P.1116)

Abstract KLH can exist as cylinders composed of 20 copies (didecamer) of subunits, or as separate subunits. It is a well-established immune stimulant and vaccine component. However, literature reports cite dramatically different immune responses to different preparations of KLH. We are developing functional assays to investigate the immunogenicity of different forms and preparations of KLH with the goal of improving the predictive outcome of its use as vaccine carrier or diagnostic agent for both research and clinical studies. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells, human PBMC, mouse DC line JAWSII, as well as human leukemia lines THP-1 and KG-1, were treated with different forms of KLH. To compare the efficiency of DCs to stimulate KLH-specific adaptive immune responses, KLH treated DCs were used to activate T cells extracted from spleens of mice that were immunized with KLH. DC antigen uptake efficiency, DC maturation, T cell proliferation and cytokine secretion were compared. Ovalbumin was conjugated to different forms of KLH. DCs derived from mouse bone marrow mononuclear cells were treated with the KLH-Ovalbumin conjugates for antigen uptake efficiency and ovalbumin-specific T cell activation studies. We found that didecameric KLH and subunit KLH have significantly different properties. We report here the results of these studies which suggest the need for methods to identify appropriate forms of KLH when used as hapten carrier or immunogen.

Chemokine programming dendritic cell antigen response: part II – programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency.

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine.

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.

Human sinonasal epithelial cells direct dendritic function and T‐cell T helper 1/T helper 2 skewing following Aspergillus exposure

In lower airway disease such as asthma, epithelial cells have been shown to be potent regulators of dendritic cell (DC) functions. However, it is unclear how human sinonasal epithelial cells (HSNECs) from patients with sinusitis regulate DC functions. Therefore, in these studies we investigated the ability of Aspergillus fumigatus exposed HSNECs to regulate DC antigen uptake, maturation, and direction of T-cell T helper 1 (Th1)/Th2 skewing. Primary HSNECs were cultured from control (n = 8), chronic sinusitis without nasal polyps (CRSsNP) (n = 9), and chronic sinusitis with nasal polyps (CRSwNP) (n = 7) patients and exposed to Aspergillus. Conditioned media was placed upon monocyte-derived DCs from healthy controls. DC antigen uptake was assessed by dextran-fluorescein isothiocyanate (FITC) uptake. DC differentiation and maturation was assessed by immunostaining for CD209, CD80, and CD86 followed by flow cytometric analysis. DC direction of T-cell Th1/Th2 skewing was evaluated by immunostaining followed by intracellular flow cytometric analysis for interferon (IFN)-γ and interleukin (IL)-5. Control and CRSsNP HSNECs have the capacity to stimulate DC antigen uptake, differentiation, and maturation following Aspergillus exposure. CRSwNP HSNECs stimulate DC activation independent of Aspergillus exposure. Furthermore, Aspergillus-exposed CRSwNP HSNECs skew T-cells toward a Th2 phenotype. CRSwNP-derived HSNECs stimulate DC maturation and Th2 skewing independent of Aspergillus exposure. However, control and CRSsNP HSNECs induce DC maturation and Th2 skewing after Aspergillus exposure. These in vitro studies demonstrate that HSNECs are key regulators of DC functions in the sinus microenvironment.

The Cyclophilin-Binding Agent Sanglifehrin A Is a Dendritic Cell Chemokine and Migration Inhibitor

Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA.

2. Dendritic cells as regulators of immunity and tolerance

Dendritic cells (DCs) are antigen sampling sentinels of the peripheral tissue and specialists for antigen capture, processing, and presentation to T cells as well as T-cell priming. By virtue of their mission as antigen samplers, they populate the body surfaces that line the border of our organism with the environment such as the skin and the mucosa of the respiratory and the gastrointestinal tract. Although all DCs share some classic features, their individual organ-specific phenotype and function are ascertained by a complex, dynamic network of co-regulatory factors provided by their microenvironment. DC subtypes might initiate novel inflammatory immune responses as well as accelerate or break down ongoing inflammatory immune reactions in the skin, the gut, and the respiratory tract. On the basis of the recognition of self-signals or nonself-signals in the presence or absence of danger signals, the interplay of DC antigen uptake and presentation leads into immunosilencing or immunoactivating properties, which designate the outcome of tolerance or defensive immunity within the skin, the gut, or the respiratory tree.

Yersinia enterocolitica YopP inhibits MAP kinase-mediated antigen uptake in dendritic cells

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits this pathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.

Effects of Cyclophilin A on Myeloblastic Cell Line KG-1 Derived Dendritic Like Cells (DLC) Through p38 MAP Kinase Activation 1,2

Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as a secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. The role of p38 MAP kinase in DC functions was also investigated. KG1 cells (CD34+ human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell surface markers was analyzed by FACS analysis. The antigen-uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen-presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [3H] thymidine incorporation assay. To assess the T cell polarization stimulated by KG1 derived DCs, various Th1 and Th2 cytokines secreted by allostimulated CD4+ and CD8+ T cells were determined by Bioplex cytokine assay. Total and phosphorylated p38 MAPK activity in CypA treated DCs was detected by Bioplex p38 total and phosphoprotein assay. During the differentiation of KG1 cells to immature DCs, cell surface expression of CD11b was increased by 30.6% for CypA alone, 55% for CypA plus cytokines, and 44% for cytokines alone. Similarly, CypA alone increased the cell surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up-regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8+ T cells, and also increased the secretion of Th1 type cytokines TNF-alpha and IFN-gamma from the allogenic CD4+ T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pre-treatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8+ and CD4+ T cells (P < 0.05). CypA enhances DC differentiation and maturation by up-regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the p38 signaling pathway.