Stage Of Dendritic Cells Research Articles

Interaction of immune cells and lymphatic vessels in corneal transplantation

AbstractImmune‐mediated graft rejections remain the most common cause for graft failure after organ and tissue transplantation. There is a great unmet medical need for pharmacologic strategies to promote graft survival without unduly compromising the health of the recipient.Corneal allograft rejection is histologically characterized by massive infiltration of CD4+ T‐cells, macrophages and the invasion of blood and lymphatic vessels.We could show that lymphatic vessels determine the high‐risk status of a corneal graft recipient. The healthy cornea is devoid of blood and lymphatic vessels. In contrast, in the high risk situation graft rejection is highly correlated with the degree of corneal vascularisation and presence of numerous pro‐inflammatory immune cells.CD11c+ CD11b+ myeloid dendritic cells (DCs) are present throughout the anterior stroma – even in the healthy cornea. Inflammatory stimuli determine the maturation stage of DCs and their immunogenic properties. It is known that an increasing number of MHC class II‐bearing, antigen specific DCs in cervical draining lymph nodes arise only a few hours after transplantation. This implies that DCs at different maturation stages might be the main initiators of this devastating tissue destruction.The origin of donor antigen presenting cells can be either from the donor tissue or the host. Thereby allo‐antigens from the grafted tissues travel via lymphatic vessels towards the draining lymph nodes of the recipient. Here they are recognized by host T cells either by the direct pathway, which involves the recognition of intact donor MHC molecules on donor‐derived APCs, or by the indirect pathway, which involves the recognition of donor major or minor MHC‐derived peptides, processed and presented by recipient APCs to host T cells. Nowadays it is widely accepted that resident MHC class II APC populations are present in the cornea. These cells are capable of expressing MHC class II antigen and accessory molecules (CD40, CD80) after transplantation. We and others could demonstrate that the immune reaction is triggered by the above described “direct allo‐sensitisation” where APCs travel from the donor tissue via corneal pathological lymphatic vessels into the local draining lymph nodes of the recipient and present the foreign antigen.Recent studies could show that lymphatic vessels not just display an exit route for leukocytes but are in close interaction with these cells and regulate the entry and trafficking of antigen presenting cells into and via lymphatic vessels.This presentation will summarize the current knowledge about the interaction of immune cells and lymphatic vessels in corneal transplantation.

Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells

Dendritic cells (DCs) translate local innate immune responses into long-lasting adaptive immunity by priming antigen-specific T cells. Accordingly, there is an ample interest in exploiting DCs for therapeutic purposes, e.g., in personalized immunotherapies. Despite recent advances in elucidating molecular pathways of antigen processing, in DCs the exact spatial organization of the underlying processes is largely unknown. Here, we unraveled the nanoscale organization of the transporter associated with antigen processing (TAP)-dependent peptide-loading machinery in human monocyte-derived DCs (moDC). We detected an unexpected accumulation of MHC I peptide-loading complexes (PLCs) and TAP-dependent peptide compartmentalization in protrusions of activated DCs. Using single-molecule localization microscopy we revealed that PLCs display homogeneously sized assemblies, independent of the DC activation status or cellular localization. Our data indicate that moDCs show augmentation of subcellular PLC density during DC maturation. We observed a twofold density increase in the cell body, while an even fourfold accumulation was detected in the tips of the protrusions at the mature DC stage in comparison to immature DCs. In these tip regions, PLC assemblies are found along highly compressed tubular ER networks. These findings provide novel insights into nanoscale organization of the antigen presentation machinery, and open new perspectives on the T cell stimulatory capacity of DCs.

Assessing whether progesterone-matured dendritic cells are responsible for retention of fertilization products in missed abortion

We hypothesize that progesterone causes tolerogenic maturation of myeloid dendritic cells (DCs) in human decidua of threatened miscarriage or missed abortion characterized by a distinct phenotype and cytokine production, including reduction of the main NK cell proliferation and cytotoxic factor interleukin (IL)-15. During DC/NK cell interaction, progesterone-shaped DCs cannot efficiently multiply or equip NK cells with the cytotoxic mediators peforin and granulysin, which might harm trophoblasts and induce abortion. We propose that the presence, and maturation stage of decidual myeloid DCs be investigated using semi-quantitative immunohistological analyses and/or double-color immuno-fluorescent labeling of DC lineage and activation markers. The spatial arrangement of granulysin+ cells, NKp46+ NK cells, DCs, and trophoblasts might provide information about their mutual interactions in vivo. Multiple flow cytometry analyses of NK-receptors would provide insight into NK cell activation status. NK cell activation status could be also assessed by cytotoxicity assays against trophoblast cell lines, or isolated cognate extra-villous trophoblast cells. A correlation between decidual progesterone concentration or IL-15 expression, and the degree of DC maturation or the frequency of granulysin+ cells, might help to elucidate the mechanism of abortion retention in utero.

Dendritic Cell-based Immunotherapy for Rheumatoid Arthritis: from Bench to Bedside.

Dendritic cells (DCs) are professional antigen presenting cells, and play an important role in the induction of antigen-specific adaptive immunity. However, some DC populations are involved in immune regulation and immune tolerance. These DC populations are believed to take part in the control of immune exaggeration and immune disorder, and maintain immune homeostasis in the body. Tolerogenic DCs (tolDCs) can be generated in vitro by genetic or pharmacological modification or by controlling the maturation stages of cytokine-derived DCs. These tolDCs have been investigated for the treatment of rheumatoid arthritis (RA) in experimental animal models. In the last decade, several in vitro and in vivo approaches have been translated into clinical trials. As of 2015, three tolDC trials for RA are on the list of ClinicalTrial.gov (www.clinicaltrials.gov). Other trials for RA are in progress and will be listed soon. In this review, we discuss the evolution of tolDC-based immunotherapy for RA and its limitations and future prospects.

Development of uptake capacity and bactericidal activity in myeloid cells during GM-CSF-driven differentiation. (INC6P.326)

Abstract The antigen presenting function of dendritic cells (DC) is well defined, yet their ability to handle bacterial infection is poorly understood. We have previously shown that GM-CSF-derived DC can confine Listeria monocytogenes (Lm) to the phagosome, preventing its invasion of the cytosol, growth, and spread. However, it is not clear at what stage of development DC acquire the capacity to retain Lm in the phagosome. To measure this activity at different stages of development, GM-CSF driven bone marrow cells were sorted based on expression of Ly6C, CD115, and CD11c. This strategy enables isolation of cells representing five distinct myeloid cell types: CMP, GMP, monocytes, inflammatory dendritic precursors (IDP), and inflammatory DC (IDC). To measure bacterial invasion of the cytosol, we used an Lm strain that expresses GFP upon cytoplasmic entry, in combination with fluorescent bioparticles to measure bulk uptake. The CMP demonstrated minimal uptake capacity and little bacterial cytoplasmic entry. The GMP population showed greatly increased uptake capacity, but also high susceptibility to bacterial cytoplasmic entry. In the monocyte population, the uptake capacity reached its peak, and these cells gained the capacity to confine the bacteria to the phagosome. Finally, the uptake capacity decreased modestly in the IDP and IDC populations, but these cells also blocked cytoplasmic invasion of the bacteria. The mechanistic basis of phagosomal retention was further examined.

Pro-inflammatory, Th1, Th2, Th17 Cytokines and Dendritic Cells: A Cross-sectional Study in Chronic Periodontitis

There are a limited number of studies correlating the different stages of dendritic cells (DC) maturation with cytokines in individuals presented chronic periodontitis (CP). The aim of the study was to evaluate the correlation among the expression of IL-2, IL-10, IL-4, IL-6, IFN-, TNF-α, and IL-17A with the presence of DC and mild-moderate or advanced CP. Gingival samples were obtained from 24 individuals with CP and six samples of normal mucosa (NM) overlapping third molar for controls of the levels of cytokines. Periodontal examination was performed. Immunohistochemical staining was carried out, revealing CD1a+ immature, Fator XIIIa+ immature, and CD83+ mature DCs. The inflammatory infiltrate was counted, and the cytokines were measured by flow cytometry. Densities of DCs and inflammatory infiltrate, cytokines, subtypes of CP, and clinical periodontal parameters were correlated and compared. IL-6 expression was correlated positively with the increased numbers of CD1a+ immature DCs. Levels of IL-2, TNF-α, IFN-, IL-10, and IL-17A were increased when compared with NM. The percentage of sites with clinical attachment level (CAL)>3 were positively correlated with densities of inflammatory infiltrate and negatively correlated with densities of immature DCs. IL-6 can contribute to the increase of the immature DCs in the CP. Higher levels of IL-2, TNF-α, IFN-, IL-10, and IL-17A cytokines were observed in CP. Higher densities of inflammatory infiltrate as well as lower densities of immature DCs can result in a more severe degree of human CP.

Molecular Characterization of Antigen-Peptide Pulsed Dendritic Cells: Immature Dendritic Cells Develop a Distinct Molecular Profile when Pulsed with Antigen Peptide

As dendritic cells (DCs) are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC), immature DC (iDC), and mature DC (mDC) from human peripheral blood monocytes (PBMC) obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20), a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC). When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA), pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN), anti-apoptosis (NFKBIA), DNA repair (MRPS11, RAD21, TXNRD1), and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61) in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE) induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.

Hepatitis B e Antigen Seroconversion Is Related with the Function of Dendritic Cells in Chronic Hepatitis B Virus Infection.

Aim. To investigate the relationship between hepatitis B e antigen seroconversion and the function of dendritic cells (DC) in patients with hepatitis B virus. Methods. The peripheral blood mononuclear cells (PBMC) from 21 chronic HBV patients in immune tolerance state, 23 patients in inactive HBsAg carrier state, and 10 healthy HBV-naive blood donors were incubated and induced into DC in presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), respectively. The expressions of surface markers on DC were detected by flow cytometry, and the stimulatory capacity of DC in allogenic mixed leukocyte reaction (MLR) was tested by CCK-8, and the level of cytokines released by DC was analyzed by enzyme-linked immunosorbent assay (ELISA). Results. DC from patients in immune tolerance showed a remarkably lower surface expression of CD80, CD86, and HLA-DR and exhibited an impaired stimulatory capacity in MLR and reduced secretion of IL-12, as compared to the patients in inactive HBsAg carrier state. There was no significant difference between the indicators from the patients in inactive HBsAg carrier state and healthy subjects. There was a significant difference of HBV DNA level between immune tolerance and inactive HBsAg carrier group (P < 0.01) and a negative correlation between HBV DNA level and the expressions of dendritic cells in both groups, respectively (P = 0.01). Conclusion. DC from patients in inactive HBsAg carrier state shows stronger function in comparison with patients in immune tolerance, the expressions of dendritic cells correlate with HBV DNA level, and the function stage of DC may play an important role in HBeAg seroconversion.

Altered MARCH1 ubiquination-regulated dendritic cell immune functions during the early stage of zymosan-induced multiple organ dysfunction syndrome (MODS) in mice

Using a zymosan-induced mouse model of multiple organ dysfunction syndrome (MODS), we previously found profound increases in spleen immune cells’ expressions of ubiquitin and MHC-II molecules and increased CD11c+ dendritic cells (DCs) within 24h of zymosan injection. We postulated that the early stage of MODS altered DCs function via an ubiquitination-associated mechanism. We intraperitoneally injected zymosan into 100 male C57BL/6 mice (0.8mg/g) and randomly divided them into 5 groups based on the days after injection (20mice/group): 1d, 3d, 5d, 7d, and 10d. Mice were examined for spleen CD11c+ DC functions at the indicated days. Untreated mice were used for normal spleen tissue and T cell samples. By qPCR, IL-12 and TNF-α mRNA expressions in spleen CD11c+ DCs were significantly increased in MODS 1d mice; on subsequent days post-injection, these mRNA levels gradually returned to control levels. The same patterns were found for MODS mice DCs induction of untreated mouse T cells proliferation and IL-2 and IFN-γ mRNA expressions. When T cell functions were examined using MODS 1d DCs with and without MG132 treatment, an inhibitor of ubiquitinated protein degradation, T cell functional activities were enhanced by DCs treated with MG132. MODS 1d DCs also had significantly reduced MARCH1 mRNA expression, a key ubiquitin ligase that regulates DCs MHC-II expression. Silencing DCs MARCH1 expression with siRNA resulted in enhancing their induction of T cell functional activities. Using co-immunoprecipitation, Western blot, and flow cytometry assays, we deduced that MARCH1 ubiquitinated DC surface MHC-II molecules to regulate DC's immune functions in MODS mice. Our results suggest that aberrant degradation of spleen DCs MARCH1-mediated ubiquitinated proteins is involved during the earliest stage of MODS development.

Phosphatidylinositol-3-kinase activity during in vitro dendritic cell generation determines suppressive or stimulatory capacity

Modulating PI3K at different stages of dendritic cells (DC) generation could be a novel means to balance the generation of immunosuppressive versus immunostimulatory DC. We show that PI3K inhibition during mouse DC generation in vitro results in cells that are potently immunosuppressive and characteristic of CD8alpha- CD11c+ CD11b+ DC. These DC exhibited low surface class I and class II MHC, CD40, and CD86 and did not produce TNF-alpha. In allogeneic MLR, these DC were suppressive. Although in these mixed cultures, there was no increase in the frequency of CD4+ CD25+ Foxp3+ cells, the Foxp3 content on a per cell basis was significantly increased. Sustained TLR9 signaling in the presence of PI3K inhibition during DC generation overrode the cells' suppressive phenotype.

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and delivery of encoded antigens. Our findings imply that bioavailability of recombinant RNA vaccines in vivo highly depends on the density and the maturation stage of DCs at the administration site and are of importance for the design of RNA-based clinical immunotherapy protocols.

Dendritic cells are equally distributed in intrauterine and tubal ectopic pregnancies

To examine different stages of dendritic cells (DCs) in intrauterine (IUPs) and viable tubal (VTPs) pregnancies to further elucidate mechanisms of fetomaternal tolerance and extravillous trophoblast invasion. Experimental study on patient-controlled material. University hospital. Seven women with normal IUPs and ten with VTPs in the first trimester. Suction curettage in IUP, laparoscopy in VTP. Immunohistochemistry for cytokeratin-7 (trophoblast), CD83 (mature DCs), DEC205 (activated but not fully mature DCs), DC-SIGN (immature macrophage-like DCs), and CD14 (macrophages) alone and in double staining. The numbers of CD83+ and DEC205+ cells were similarly low in IUP and VTP (0.83 and 0.44 cells/mm2; 2.28 and 2.96 cells/mm2). The number of DC-SIGN+ cells was higher, though without significant differences among the entities examined (57.5 and 47.4 cells/mm2). About two-thirds of DC-SIGN+ cells were also CD14+ in IUP and VTP. The almost equal distribution of CD83+, DEC205+, and DC-SIGN+ cells in IUP and VTP suggests analogue control mechanisms in intrauterine and extrauterine DC differentiation and a comparable role of these DCs for the development of fetomaternal tolerance.

Immune Responses in AML Patients Following Vaccination with GRNVAC1, Autologous RNA Transfected Dendritic Cells Expressing Telomerase Catalytic Subunit hTERT.

Abstract 633 Background:Telomerase activity of leukemic cells from AML patients is frequently increased, especially among cases with the most aggressive clinical features. The major protein catalytic subunit of telomerase (hTERT) is known to include immunogenic epitopes which can stimulate generation of hTERT-specific cytotoxic T cells. Methods:This study was designed to evaluate the feasibility, safety, and immunologic effects of telomerase catalytic subunit (hTERT)-expressing autologous dendritic cells (DC) in 20 or more evaluable adult AML patients in CR1 or CR2. GRNVAC1 (VAC1) was produced from patient-specific leukapheresis harvests. Monocyte-enriched PBMCs were cultured with cytokines at a central facility to the immature DC stage, then electroporated with an RNA construct encoding hTERT and a lysosomal targeting sequence (LAMP-1). Further culture with cytokines induced a mature DC phenotype. The RNA construct enables expression of multiple HLA appropriate hTERT epitopes. CD83+CD14low phenotype, hTERT protein expression, and sterility were required for release of the cryopreserved doses. CR1 patients were eligible with intermediate or high risk cytogenetics; all CR2 patients were eligible. Patients would have PBMCs harvested after induction, but could complete up to 4 chemotherapy consolidation cycles prior to start of study vaccination, at which point they were re-evaluated for remission status. Patients in ongoing CR or early relapse were vaccinated. Early relapse was defined as marrow blasts < 20%, ANC > 400/ μL with no new infection, and plts > 30,000/ μL. VAC1 in doses of 1 × 107 cells was administered by intra-dermal injections weekly X 6, followed by 4 weeks off, then boost vaccinations every other week X 6. Injections could then continue on a q28 day extended boost schedule. Immune response was evaluated by delayed type hypersensitivity (DTH) and cell based and peptide pool ELISPOT assays. Levels of WT-1 in blood samples were monitored (qPCR, normalized to abl) as an exploratory MRD marker. Bone marrows were performed at specified intervals to evaluate remission status. Results:VAC1 cells having mature DC immunophenoptype and that were functional in mixed lymphocyte cultures and in vitro migration assays could be produced from leukapheresis product. 18 patients have now commenced vaccination (median age 55 yrs; range 34 to 75) and leukapheresis harvests from 2 additional patients are currently in manufacture. Sixteen patients who have started vaccination were in CR1 and 2 in CR2. Two patients were in early relapse, one of whom progressed following 3 vaccinations, while the other continues on study following 3 vaccinations. Five of the patients who started vaccination had leukemias with cytogenetic abnormalities.VAC1 was well tolerated, with the exception of one patient who presented with ITP 2.4 months following start of VAC1 treatment. This patient had initial immunophenotype consistent with minimally differentiated subtype, and continued in AML remission for 7.3 months from treatment start. Three additional patients who started vaccination in CR relapsed at 1.2 to 2.3 months, while 12 continue in CR at 1.4 to 17.5 months following vaccination start. The patient who has continued in CR the longest had flt-3 ITD+ leukemia with normal cytogenetics. This patient had elevated WT-1 levels at the time of leukapheresis, persisting unchanged to treatment week 7, but then gradually declining to within the normal range by week 21. Three patients who relapsed within 2.3 months had elevated and increasing WT-1 levels at and following start of vaccination.Seven of 13 patients on whom data is currently available had ELISPOT immune response, as defined by ≥ 2 fold increases over baseline. Five of 13 had evidence of DTH reactions. To date, there is no clear correlation between measured immune response and clinical outcome in this small number of patients. Conclusions:We have produced and administered GRNVAC1 to 18 AML patients in complete remission (16) or early relapse (2). Treatment has been generally well tolerated, with the exception of 1 patient who developed ITP. Twelve of 16 CR patients continue in remission. There was evidence of hTERT specific immune response by ELISPOT in over half the evaluated patients. The study is ongoing, now focused on patients with AML with poor risk cytogenetic or molecular features or in CR2. Disclosures:DiPersio:Genzyme: Honoraria. Blum:Celgene: Research Funding. Elias:Geron Corporation: Employment, Equity Ownership. Reddy:Geron Corporation: Employment, Equity Ownership. Smith:Geron Corporation: Employment, Equity Ownership.

PU.1 Is Indispensable for Development of Mature Dendritic Cells and Their Function That Plays a Critical Role in Maintenance of Normal T Cell Pool.

Abstract 1471Poster Board I-494PU.1, a hematopoietic transcription factor, is indispensable for development of myelo-lymphoid cells from hematopoietic stem cells (HSCs). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. By disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool and their functions. We crossed PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC. In these mice, PU.1 gene was disrupted in all conventional DCs but not in other hematopoietic cells, including lymphoid cells, myeloid cells and their progenitors. Development of DC precursors such as Lin−c-KitloFLT3+MCSFR+, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <40%, <25%, <10% and <5% as compared with the wild-type control, respectively. Moreover, the number of mature T cells reduced to ∼60% in the spleen as compared to the control. PU.1-deficient DCs displayed impaired functions to induce antigen-driven T cell proliferation, and to produce inflammatory cytokines (TNFa, IL-6, IL-12) in response to Toll like receptor (TLR) stimulation. These results clearly show that PU.1 is required for development of the peripheral DC pool and for maintenance of their immunological functions, which might be required for maintenance of the peripheral T cell pool. Disclosures:No relevant conflicts of interest to declare.

PU.1 Plays a Critical Role in Maintenance of Mature Dendritic Cell Pool

PU.1, a hematopoietic transcription factor, is absolutely required for development of myelo-lymphoid cells from hematopoietic stem cells (HSC). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. In this study, by disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool. By crossing PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC, we disrupted PU.1 at the CD11c+ DC stage. In these mice, development of DC precursors such as Lin−c-KitloFLT3+MCSFR+ DC progenitors, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <30%, <10%, <10% and <5% of DC numbers in control mice, respectively. In contrast, mice possessed normal numbers of granulocytes/monocytes, B cells, and naïve, effector or regulatory T cells. These mice have not developed any significant hematological or immune disorders at least until 6 months after birth. These results clearly show that PU.1 is required not only for DC development but also for maintenance of the peripheral DC pool. We are currently trying to elucidate the underlying mechanism for PU.1 to maintain mature DC numbers in peripheral organs.

Dendritic Cell Differentiation State and Their Interaction with NKT Cells Determine Th1/Th2 Differentiation in the Murine Model ofLeishmania majorInfection

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4(+) T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Ralpha signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-alpha or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.

Trypanosoma cruziInduces Regulatory Dendritic Cells In Vitro

A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor beta (TGF-beta) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-beta, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-beta and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.

Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and purified. DCs were isolated and cultured from human peripheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 106 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37°C for 0.5, 1, 2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluorescence. The result detected by FCM indicated that after incubation for 0.5 h at 37°C, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37°C, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09 ± 31.78/105 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36 ± 15.73). There was no significant difference between the 2 groups (P < 0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78 ± 1.40)/105 cells and EGFP-loaded (23.13 ± 4.57)/105 cells DC groups in the amount of DCs secreting IL-12 (P < 0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP. With increasing incubation time, pinocytosis begins to dominate. HSP70-EGFP may promote DCs to secret cell factor IL-12.

Late dendritic cells are still able to evoke a potent alloreactive CTL response

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime naïve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF- α, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN- γ production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20 h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN- γ production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.

An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+ monocytes

In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DCs for electroporation. We further show a stable electroporation efficacy resulting in 60% of GFP positive cells. Expression of co-stimulatory molecules and maturation markers such as CD80, CD86, CD83 as well of the chemokine receptor 7 (CCR7) was found in 90% of the mature DCs. Importantly, production of IL-12p70 was 10 times higher in cells electroporated at the monocyte stage compared to cells electroporated at the immature DC stage. Stimulation of autologous naïve lymphocytes by DCs electroporated at monocytes stage elicited proliferation of CD8+ T-cell with 7-fold increase in IFN-γ release. Blocking of the MHC-Class I molecules significantly inhibited the IFN-γ release, indicating that antigen presentation was MHC-Class I mediated. In summary, electroporation of CD14+ monocytes with RNA results in a high yield of antigen presenting DCs with high immuno-stimulatory capacity and antigen presentation on MHC-Class I molecules. This improved method may represent an attractive approach for RNA-based DC immunotherapy.