HIV gp120 receptors on human dendritic cells

Dendritic cells (DCs) are important targets for human immunodeficiency virus (HIV) because of their roles during transmission and also maintenance of immune competence. Furthermore, DCs are a key cell in the development of HIV vaccines. In both these settings the mechanism of binding of the HIV envelope protein gp120 to DCs is of importance. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte-derived DCs (MD-DCs) rather than CD4. In this study a novel biotinylated gp120 assay was used to determine whether CLR or CD4 were predominant receptors on MDDCs and ex vivo blood DCs. CLR bound more than 80% of gp120 on MDDCs, with residual binding attributable to CD4, reconfirming that CLRs were the major receptors for gp120 on MDDCs. However, in contrast to recent reports, gp120 binding to at least 3 CLRs was observed: DC-SIGN, mannose receptor, and unidentified trypsin resistant CLR(s). In marked contrast, freshly isolated and cultured CD11c(+ve) and CD11c(-ve) blood DCs only bound gp120 via CD4. In view of these marked differences between MDDCs and blood DCs, HIV capture by DCs and transfer mechanisms to T cells as well as potential antigenic processing pathways will need to be determined for each DC phenotype. (Blood. 2001;98:2482-2488) (C) 2001 by The American Society of Hematology.

A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera

CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/ activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.

Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease

Dendritic cells (DC) have a key role in controlling the immune response, by determining the outcome of antigen presentation to T cells. Through costimulatory molecules and other factors, DC are involved in the maintenance of peripheral tolerance through modulation of the immune response. This modulation occurs both constitutively, and in inflammation, in order to prevent autoimmunity and to control established immune responses. Dendritic cell control of immune responses may be mediated through cytokine or cell-contact dependent mechanisms. The molecular and cellular basis of these controls is being understood at an increasingly more complex level. This understanding is reaching a level at which DC-based therapies for the induction of immune regulation in autoimmunity can be tested in vivo. This review outlines the current state of knowledge of DC in immune tolerance, and proposes how DC might control both T cell responses, and themselves, to prevent autoimmunity and maintain peripheral tolerance.

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

The fundamental role of dendritic cells (DC in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6,0 AutoPBPC) provides an operator-independent reliable source of motionuclear cells (MNC for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-1 procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 151. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. (C) 2002 Published by Elsevier Science B.V.

Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123hi and CD11c+ subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of new in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction. (C) 2002 by The American Society of Hematology.

Human peripheral blood V alpha 24(+) V beta 11(+) NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells

Background and Objectives We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. Materials and Methods Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. Results No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24(+) Vbeta11(+) NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. Conclusions Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24(+) Vbeta11(+) NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24(+) Vbeta11(+) NKT cells, or for autoimmune diseases.

Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells

Background: The response of hepatocellular carcinoma (HCC) to therapy is often disappointing and new modalities of treatment are clearly needed. Active immunotherapy based on the injection of autologous dendritic cells (DC) co-cultured ex vivo with tumor antigens has been used in pilot studies in various malignancies such as melanoma and lymphoma with encouraging results. Methods: In the present paper, the preparation and exposure of patient DC to autologous HCC antigens and re-injection in an attempt to elicit antitumor immune responses are described. Results: Therapy was given to two patients, one with hepatitis C and one with hepatitis B, who had large, multiple HCC and for whom no other therapy was available. No significant side-effects were observed. The clinical course was unchanged in one patient, who died a few months later. The other patient, whose initial prognosis was considered poor, is still alive and well more than 3 years later with evidence of slowing of tumor growth based on organ imaging. Conclusions: It is concluded that HCC may be a malignancy worthy of DC trials and sufficient details in the present paper are given for the protocol to be copied or modified. (C) 2002 Blackwell Publishing Asia Pty Ltd.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-IBBL into murine DCs, and monitored the ability of these recombinant DO to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DO increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DO. We also report an additive effect of 4-IBBL and receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand coexpression in E7-transduced DC inummogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. ReIB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which ReIB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4(+) T cells induced by the DCs transfer antigen-specific Infectious tolerance to primed recipients in an interleukin10-dependent fashion. Thus CD40, regulated by ReIB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.

Recent advances on the role of CD40 and dendritic cells in immunity and tolerance

CD40 is a key signaling pathway for the function of B cells, monocytes, and dendritic cells in the immune system, and plays an important role in inflammatory pathways of nonhemopoietic cells. The NFkappaB family of transcription factors is a critical mediator in inflammation. NFkappaB is involved both in the regulation of CD40 expression and in cell signaling after CD40 ligation. This positive feedback loop linking NFkappaB and CD40 plays an important role in the control of the adaptive immune response, with fundamental implications for immunity and tolerance in vivo.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Optimization of production of anti-cancer vaccines based on dendritic cells

Cancer remains as one of the top killing diseases in first world countries. It’s not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a “new” breath on cancer treatment, taking use of the patients’ immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs’ antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.

Cross-presentation by WASp deficient B cells and dendritic cells

The immune system comprises of different cell types whose role is to protect us against pathogens. This thesis investigates a very important mechanism for our organism protection in a specific disorder: cross-presentation in Wiskott-Aldrich Syndrome (WAS). WAS is caused by loss-of-function mutations in the cytoskeletal regulator WASp and WAS patients suffer from eczema, thrombocytopenia, and immunodeficiency. X-linked neutropenia (XLN) is caused by gain-of-function mutations in WASp and XLN patients suffer from severe congenital neutropenia and immunodeficiency. This thesis was focused on the role of B and T lymphocytes and dendritic cells (DCs). This work will be divided into two main topics: 1) In the first part I studied the capacity of B cells to take up, degrade and present antigen. Moreover I studied the capacity of B cells to induce T cell proliferation. 2) In the second part, I studied T cell proliferation induced by dendritic cells. To increase our understanding about this mechanism, additional experiments were performed, including acidification capacity of CD8+ and CD8- DCs, reactive oxygen species (ROS) production since it is directly connected to acidification. These assays were measured by flow cytometry. Localization of Rac1 and Rac2 GTPases was assessed by confocal microscopy. Proliferation, acidification and ROS production assays were performed also with cells from X-linked neutropenia (XLN) mice. From this study we concluded that B cells cannot induce CD8+ T cell proliferation however they take up and present antigen. Moreover I have shown that increased cross-presentation by WASp KO DCs with ovalbumin is associated with decreased capacity to acidify endosomal compartment; and WASp KO CD8- DCs have increased Rac2 localization to the phagosome. XLN dendritic cells have similar acidification and ROS production capacity than wildtype cells. In conclusion, our data suggests that WASp regulates antigen processing and presentation in DCs.