Dendritic cell activation by combined exposure to anti-CD40 plus interleukin (IL)-12 and IL-18 efficiently stimulates anti-tumor immunity

Abstract:  Despite as yet limited clinical effectiveness, dendritic cell (DC)-based immunotherapy remains a promising approach for the treatment of cancer, but requires further improvement in its immunostimulatory effectiveness. Potent anti-tumor immunity often depends on the induction of type 1 (T_H1) immune responses. Therefore, we combined different DC maturation stimuli that are known to induce T_H1 immunity [anti-CD40, interleukin (IL)-12, IL-18], with the aim to trigger a T_H1 driven anti-tumor CTL response. When compared with untreated DC or DC treated with anti-CD40 alone, DC matured with anti-CD40 plus IL-12 and IL-18 expressed significantly more IFN-γ and IL-12, induced enhanced CD8⁺ T-cell proliferation, prolonged synaptic interaction with T cells and increased CD8⁺ T-cell-mediated cytotoxicity. To analyse if these DC are able to induce efficient anti-tumor immunity, mice carrying a B16-OVA tumor were treated with tumor antigen (TA)-loaded DC that had been exposed to anti-CD40 or to anti-CD40 plus IL-12 and IL-18. Our data show that anti-CD40 plus IL-12 and IL-18 matured DC are superior to controls in retarding tumor growth. These data indicate that maturation of DC with anti-CD40 plus IL-12 and IL-18 potently stimulates the generation of an anti-tumor immune response and may lead to improved immunotherapeutic capacity of DC vaccination.     

Mice with heterozygous deficiency of manganese superoxide dismutase (SOD2) have a skin immune system with features of “inflamm-aging”

Dendritic cells (DC) are central in regulating skin immunity. Immunosenescence is associated with a chronic inflammatory state. Little is known about the contribution of DC to “inflamm-aging”. When determining langerhans cell (LC) numbers, we found a 60 % reduction of LC in aged epidermis. Reactive oxygen species(ROS) are linked with aging. The mitochondrial manganese superoxide dismutase (SOD2) is in the first line of antioxidant defense. We investigated the function of DC from SOD2 heterozygous mice (SOD2+/?) and found that at 4 months of age LC numbers are not altered, but activated LC have impaired expression of MHC-II and CD44. Immature SOD2+/? DC produced increased proinflammatory IL-6 and chemokines CXCL1 and CXCL2. Upon challenge SOD2+/? DC accumulated ROS. When activating SOD2+/? DC by LPS they less efficiently upregulated MHC-II, CD86 and CD44. Surprisingly, in vivo contact hypersensitivity (CHS) was enhanced in SOD2+/? mice although SOD2+/? DC were less potent in stimulating wt T cells. However, SOD2+/? T cells showed increased proliferation, even when stimulated with SOD2+/? DC, possibly explaining the increased CHS. Our findings suggest that SOD2 is a molecular candidate in the regulation of “inflamm-aging” conveying both immunosuppressive and proinflammatory signals through alteration of DC and T cell functions.     

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

Abstract:  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-γ 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.     

Iontophoresis with alternating current and direct current offset (AC/DC iontophoresis): a new approach for the treatment of hyperhidrosis

Summary Tap-water iontophoresis (TWI) using direct current (DC) is the most effective therapy in palmoplantar hyperhidrosis. Side-effects of this method are discomfort, with burning and tingling, and skin irritation, including erythema and vesicles. Incorrect use may induce iontophoretic burns at sites of minor skin injury. Elaborate safety measures are required to prevent electric shock.
The aim of this study was to minimize side-effects and to increase technical and safety standards of TWI, without loss of efficacy. In a controlled blind study, treatment of palmar hyperhidrosis by alternating current (AC) or by AC with DC-offset (AC/DC) was compared with the conventional DC method.
Palmar hyperhidrosis was completely controlled after an average of 11 treatments by either AC/DC iontophoresis or the conventional DC method. Virtually no effect was seen when AC without DC-offset was used for TWI. There were no signs of cutaneous irritation, or subjective sensations of discomfort when AC with or without DC-offset was employed. AC/DC iontophoresis should become the treatment of choice for palmoplantar hyperhidrosis.
The mechanism of action is unknown. It is hypothesized that an interrupted stimulus-secretion-coupling leads to a functional disturbance of sweat secretion.     

Spleen dendritic cells exhibit altered morphology and increased allostimulatory capacity after short-term culture.

Ia-bearing dendritic cells (DC) are a class of bone marrow-derived antigen-presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short-term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface Ia molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820-2826). In the present study we show, using immunoelectron microscopy with anti-Ia and anti-33D1 monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24-48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.     

Interleukin-21 inhibits dendritic cell-mediated T cell activation and induction of contact hypersensitivity in vivo

Interleukin (IL)-21 is a newly described cytokine that is produced by activated T cells and displays structural homology to IL-4 and IL-15. We here analyzed the role of IL-21 in dendritic cell (DC)-induced, T cell-mediated contact hypersensitivity (CHS) in vivo and on T cell activation and unspecific mixed lymphocyte reaction in vitro. By PCR, we demonstrate here constitutive expression of the specific IL-21 receptor and the common gamma-chain in DC, which together are able to mediate IL-21 signaling. Short-time incubation of in vitro generated DC with IL-21 significantly reduced their potential to induce an antigen-specific CD8+ T cell proliferation. Interestingly, 2h incubation of these DC with IL-21 before injection completely inhibited the potential of these DC to induce a CHS reaction to the hapten fluorescein 5-isothiocyanate in vivo. Mice injected with IL-21-treated DC even failed to mount a CHS response after repetitive injection of non-IL-21-treated DC 2 weeks later, suggesting that an antigen-specific unresponsiveness can be induced by IL-21-treated DC. Our data demonstrate that IL-21 is a new modulator of DC-T cell interaction with the potential to induce DC-mediated antigen-specific tolerance.     

Fc epsilon RI on dendritic cells: a receptor, which links IgE mediated allergic reaction and T cell mediated cellular response.

Dendritic cells (DC) are bone marrow derived cells with strong antigen presenting capacity and can induce primary immune responses by activating naive T cells. Cells of this lineage are called as professional antigen presenting cells (APC), because of their primary function as APC. Since the demonstration of IgE bound epidermal Langerhans cells (LC) in atopic dermatitis (AD) patients, a role of IgE bearing DC as a regulator of IgE mediated allergic reactions is emphasized. Indeed, IgE molecules on DC are functional. DC can take up, process and present IgE bound antigens to T cells more efficiently by means of their surface IgE receptor, FcepsilonRI. In addition to its role as an antigen focusing molecule, DC FcepsilonRI may have another function to induce co-stimulatory signals to T cells, by which Th2 type T cell activation is preferentially induced. Thus, this receptor could serve as an amplifying factor for T cell mediated allergic reactions.     

Formation and kinetics of MHC class I-ovalbumin peptide complexes on immature and mature murine dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells that are able to induce primary T cell responses. Therefore, several strategies employ peptide-pulsed DC in tumor immunotherapy. For efficient antigen presentation and induction of an immune response by DC the number and stability of MHC I-peptide complexes is crucial. We studied this issue by using the antibody 25-D1.16 that specifically detects OVA peptide SIINFEKL in conjunction with H-2 Kb molecules, and determined its kinetics on mature and immature bone marrow-derived murine DC. Optimal peptide loading was reached after 8-16 h at 50 microM peptide pulse, and was comparable in serum-free versus serum-containing medium. Stimulation of DC with LPS or Poly I:C, and to a lesser extent TNF-alpha, upregulated the total number of surface MHC I molecules and thus improved peptide loading. Pulse-chase experiments revealed a constant half-life of peptide/Kb complexes independent of preceding DC stimulation or their maturation stage. The duration of peptide/Kb complex expression on mature DC, however, could be extended from 24 h to 72 h when the cultures were pretreated with LPS or Poly I:C, but not TNF-alpha. These data might have important implications for the clinical application of peptide-pulsed DC in tumor immunotherapy.     

Recent advances in dendritic cell vaccines for cancer treatment.

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their unique abilities to efficiently take up, process, and present various forms of antigens to immunologically naive T cells. Recent advances in the understanding of molecular mechanisms regulating the functions of DC have allowed the investigators to "design" innovative DC-based vaccine formats for the treatment of cancer patients. In this article, we will review the most advanced DC vaccine strategies, focusing on three major areas: a) new ex vivo protocols for DC loading with tumor-associated antigens (TAA), b) genetic approaches to load skin-resident Langerhans cells (LC) with TAA, and c) chemokine-mediated LC entrapment and in situ loading of LC with TAA at the entrapment sites. We believe that these new classes of DC vaccine strategies will soon become applicable to the treatment of patients with malignant tumors.     

Fetal calf serum-free generation of functionally active murine dendritic cells suitable for in vivo therapeutic approaches

Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins. The yield of fetal calf serum-free cultured DC was further enhanced by addition of the proinflammatory cytokines TNF-alpha and IL-1beta with the combination resulting in up to 10% more DC. Phenotypically, CD11c + DC cultured fetal calf serum-free homogenously coexpressed the DC-specific molecule DEC-205 as well as the costimulatory molecules CD40, CD80, and CD86. In contrast, only a subpopulation of the CD11c + DC cultured in fetal calf serum-containing medium coexpressed these molecules. Functionally, fetal calf serum-free DC showed strong stimulatory capacity for na?ve allogeneic CD4 + and CD8 + T cells. Importantly, fetal calf serum-free DC showed spontaneous in vivo migratory activity. Moreover, 5 x 105 subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneous injection of a single dose of 5 x 105 OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in na?ve TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models. Keywords: DC vaccines/dendritic cell development/fetal calf serum-free culture conditions for DC/in vivo therapeutic DC approaches.     

New technologies toward dendritic cell-based cancer immunotherapies

Immunologically naive T cells are activated most efficiently or even exclusively by special subsets of antigen presenting cells, termed dendritic cells (DC). Members of the DC family have been identified in virtually all epithelial tissues that are constantly exposed to environmental antigens or infectious microbes. For example, skin is equipped with at least two members of this family, epidermal Langerhans cells (LC) and dermal DC. DC have been shown to play pathogenic roles in several different inflammatory/immunological disorders and protective roles against infectious pathogenes and cancer development. In this review article, we will overview the recent progress in the development of DC-based immunotherapies for the prevention and treatment of cancers.     

Immunohistochemical localization of activated and mature CD83+ dendritic cells in granulomas of sporotrichosis.

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) and CD83 is expressed at a high level on immune-competent, activated and mature DC. In order to obtain insight into the immune response in sporotrichosis, we searched immunohistochemically for CD83+ DC in the granulomatous skin lesions of patients. A small but significant subpopulation of CD83+ DC (approximately 0.2 to 0.8% of inflammatory cells) was found. The most common distribution pattern of CD83+ DC was as clusters with mononuclear lymphoid cells within the granulomas. In addition, CD83+ DC were occasionally scattered around the granulomas. High-intensity staining of CD83 antigens was detected not only on the surface, but also in the cytoplasm of DC. No CD83+ DC were observed in the epidermis. A considerable amount of CD1a+ cells were detected in the epidermis, papillary dermis and around the granulomas, but CD1a+ cells were rarely found within the granulomas. There were large numbers of CD68+ monocytes and macrophages in the granulomas. These results indicate that activated and mature DC may play a role in the immune response to sporotrichosis.     

Langerhans cell migration is modulated by N-sulfated glucosamine moieties in heparin

Dendritic cell (DC) migration into and out of tissues is important for the generation of primary immune responses to antigens encountered in tissues. In order to study the mechanisms involved in DC migration we used a skin explant system and quantitated the number of Langerhans cells (LC), which are immature precursors of DC in skin-draining lymph nodes, remaining in the epidermis in response to incubation with various biomolecules. This paper shows that LC trafficking in epidermis is a metabolically active process that is modulated by heparin, specifically by N-sulfated glucosamine moieties in heparin. This is the first demonstration of structural specificity in the biochemical requirements for DC migration in a tissue and therefore is important to understanding DC migration in general.     

Induction of tumor peptide-specific cytotoxic T cells under serum-free conditions by mature human dendritic cells

Abstract Tumor vaccination strategies using antigen-pulsed dendritic cells (DC) are currently under development. We established an in vitro system using cultured DC from HLA-typed volunteers for the induction of tumor peptide-specific CD8⁺ T cells. The strength and specificity of the resulting CTL responses were investigated. For stimulation of syngeneic CD8⁺ T cells two well-defined DC populations were generated: CD1a⁺ immature DC cultured in the presence of GM-CSF and IL-4 and mature CD83⁺ DC generated by additional stimulation with a cytokine cocktail. Stimulations were performed under serum-free conditions and in the absence of exogenous cytokines. Analysis of T cell responses showed that mature DC, but not immature DC, were able to induce the expansion of syngeneic tumor peptide-specific CD8⁺ T cells. Priming of CD8⁺ T cells with peptide-pulsed mature DC rapidly increased the frequency of antigen-specific T cells (ELISPOT technique). T cells induced by mature DC showed strong antigen-specific cytotoxicity in ⁵¹Cr-release assays whereas no antigen-specific cytotoxicity was detectable in CTL generated by immature DC. These data show that terminally differentiated mature DC are necessary for the induction of tumor antigen-specific CTL responses. Received: 13 January 2000 / Revised: 23 March 2000 / Accepted: 23 March 2000     

Leishmaniasis,contact hypersensitivity and graft‐versus‐host disease: understanding the role of dendritic cell subsets in balancing skin immunity and tolerance

Please cite this paper as: Leishmaniasis, contact hypersensitivity and graft‐versus‐host disease: understanding the role of dendritic cell subsets in balancing skin immunity and tolerance. Experimental Dermatology 2010; 19 : 760–771. Abstract: Dendritic cells (DC) are key elements of the immune system. In peripheral tissues, they function as sentinels taking up and processing antigens. After migration to the draining lymph nodes, the DC either present antigenic peptides by themselves or transfer them to lymph node–resident DC. The skin is the primary interface between the body and the environment and host’s various DC subsets, including dermal DC (dDC) and Langerhans cells (LC). Because of their anatomical position in the epidermis, LC are believed to be responsible for induction of adaptive cutaneous immune responses. The functions of LC and dDC in the skin immune system in vivo are manifold, and it is still discussed controversially whether the differentiation of T‐cell subtypes (e.g. effector T cells and regulatory T cells) may be initiated by distinct DC subtypes. As skin DC are able to promote or downmodulate immune responses, we chose different skin diseases (cutaneous leishmaniasis, contact hypersensitivity, UV radiation‐induced suppression, and graft‐versus‐host disease) to describe the biological interactions between different DC subtypes and T cells that lead to the development of efficient or unwanted immune responses. A detailed knowledge about the immune modulatory capacity of different cutaneous DC subsets might be helpful to specifically target these cells through the skin during therapeutic interventions.     

Apoptosis in dendritic cell biology

Apoptosis or programmed cell death regulates many aspects in immunological homeostasis and, thus, controls the initiation, magnitude, duration, and termination of immune responses. Recent studies on dendritic cells (DC), including Langerhans cells (LC), have reinforced this concept by documenting that these antigen presenting cells express surface receptors and ligands that are known to mediate apoptotic cell death and that they are highly susceptible to apoptotic signals. In this review article, four major topics concerning apoptosis in the biology of DC will be overviewed: (a) molecular mechanisms of apoptosis; (b) DC apoptosis induced by various stimuli; (c) regulation of DC apoptosis; and (d) cross-priming and cross-tolerance induced by DC ingesting apoptotic bodies.     

In situ localization of CD83-positive dendritic cells in psoriatic lesions

BACKGROUND: Dendritic cells (DC) are considered to be the most potent antigen-presenting cells, and CD83 is expressed at a high level on immune-competent, activated and mature DC. Although changes in the number or localization of mature and activated CD83+ DC could be expected in psoriasis, there is little information on such changes. AIM: Morphological identification of CD83+ DC in psoriatic skin lesions. MATERIALS AND METHODS: Immunohistochemical staining was performed in 5 specimens of psoriasis vulgaris and 6 specimens of pustular psoriasis. Formalin-fixed, paraffin-embedded sections were used for examination in this study. The skin sections were pretreated with 0.1% trypsin for 60 min at 37 degrees C prior to immunostaining for CD83. RESULTS: A small but significant subpopulation of CD83+ DC was found in the upper dermis. In addition, CD83+ DC were occasionally scattered in the epidermis. The most common distribution pattern of CD83+ DC was as clusters with mononuclear lymphoid cells in the upper dermis. CD83+ DC were in close contact with lymphocytes. High-intensity staining of CD83 antigens was detected not only on the surface, but also in the cytoplasm of DC. CONCLUSION: These results indicate that activated and mature CD83+ DC may play a role in the immune response in psoriasis and provide in vivo support for the concept that CD83+ DC provide signals for direct intralesional T cell activation.     

单纯疱疹病毒2型对小鼠树突状细胞体外影响的研究

目的探讨单纯疱疹病毒2型(HSV2)对小鼠树突状细胞(DC)存活率、形态学及分泌白介素12(IL12)的影响。方法用HSV2感染小鼠DC系,倒置显微镜观测其形态学的变化,台盼蓝染色计数细胞存活率。HSV2感染和/或LPS刺激后,ELISA法检测DC分泌IL12的变化。结果HSV2感染3天后,小鼠DC树突回缩,变圆,细胞间间隙增大,存活率迅速降至79.73%,至7天后存活率仅为31.53%,均低于对照组(P均<0.05)。HSV2感染后能抑制LPS刺激DCIL12的分泌(P<0.05)。结论HSV2感染后,DC树突回缩,存活率降低,分泌IL12功能受损,均不利于对HSV2的免疫应答。     

Dendritic cells and their role in skin-induced immune responses

This artide gives a brief review on dendritic cells (DC) with regard to their origin, life cyde and functions. The regulation of immune responses by DC functioning as antigen-presenting cells is discussed. Special attention is given to epidermal DC, e.g. Langerhans cells. The perspectives of DC-based therapy are also mentioned.