Function Of Human Dendritic Cells Research Articles

B Lymphocyte Stimulator Regulates Adaptive Immune Responses by Directly Promoting Dendritic Cell Maturation

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.

Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling

The Hedgehog (Hh) family of signaling molecules functions in the development of numerous tissues during embryogenesis and has also been involved in adult self-renewing tissues. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate whether thymic dendritic cells (DCs) are cell targets for Hh signaling. Both components of the Hh receptor, Patched and Smoothened, as well as other Hh-binding proteins with modulating functions, are expressed by human thymic DCs. The expression of Gli1, Gli2, and Gli3 transcription factors suggests that the Hh signaling pathway is active in thymic DCs, and approximately one-half of thymic DCs produces Sonic Hh (Shh). The culture of thymic DCs with Shh protects them from apoptosis [similarly to CD40 ligand (CD40L)], and these antiapoptotic effects are related to an up-regulation of Bcl-2 and Bcl-X(L) protein expression. The addition of the Hh pathway inhibitor, cyclopamine, decreases DC viability and impairs their allostimulatory function in vitro. In addition, the blockade of the Hh signaling pathway by cyclopamine treatment abrogates the up-regulation of HLA-DR, CD86, CD80, and CD83 expression induced by CD40L on thymic DCs. Finally, we also show that after activation with CD40L thymic DCs down-regulate the expression of Hh receptor components as well as Shh production. Taken together, these results suggest that the survival and function of thymic DCs are regulated by an autocrine Hh signaling.

Salp15 Binding to DC-SIGN Inhibits Cytokine Expression by Impairing both Nucleosome Remodeling and mRNA Stabilization

Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC) functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi–induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK)-dependent decrease of IL-6 and TNF-α mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor–induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

Norepinephrine modulates human dendritic cell activation by altering cytokine release

Norepinephrine (NE) can modulate dendritic cell (DC) activation in animal models, but the response of human DC to NE and other response modifiers is as yet not completely understood. Here we report the effect of NE on the cytokine response of a mixed population of human DC cells to extracellular stimuli. These cells were obtained by differentiating human cord blood CD34+ precursor cells. NE inhibited the lipopolysaccharide (LPS)-stimulated production of interleukin (IL)-23, IL-12 p40, tumor necrosis factor (TNF)-alpha and IL-6 whereas the expression of IL-10 was not significantly affected. Thus, human cord blood-derived DC respond to NE in a manner similar to mouse Langerhans cells (LC). Furthermore, forskolin also inhibited the LPS-induced levels of TNF-alpha, IL-12 p40, IL-23 p19 and IL-6, supporting the hypothesis that the effects of NE are mediated by cAMP. Data from experiments using inhibitors of adrenergic receptors suggest that NE acts through beta-adrenergic receptors. As IL-23 promotes the differentiation of CD4+ T cells required for T(H)1-mediated immunity, we suggest that NE decreases the differentiation of CD4+ T cells needed for T(H)1-mediated contact hypersensitivity and that NE is a candidate regulator of human DC functions in the skin.

Surface markers and functions of human dendritic cells exposed to mobile phone 1800 MHz electromagnetic fields

To investigate the effects of mobile phone 1800 MHz electromagnetic fields (EMF) on the surface markers and the functions of human dendritic cells (DC). Human DCs were exposed to intermittent 5 min on/10 min off EMF with specific absorption rates (SAR) 4 W/kg for 0 h, 1 h, 12 h or 24 h, respectively. FACS analysis was used to detect the positive percentage of DC surface markers including HLA-DR and co-stimulatory molecules such as CD80, CD86, CD40 and CD11c. CCK-8 kit was adopted to examine the function of allo-mixed lymphocyte reaction (allo-MLR) of DC, and enzyme linked immunosorbent assay (ELISA) to identify the levels of IL-12p70 and TNF-alpha secreted by DC. Compared with the sham radiation group, after exposure to the electromagnetic fields for 1 h, 12 h, or 24 h, HLA-DR, CD80,CD86 and CD40 were all declined except CD11c. The ability of DC allo-MLR in each exposure group was decreased significantly (P<0.05), especially in the 24 h exposure group. However, the secreted levels of IL-12p70 and TNF-alpha of DC in each exposure group remained no changed. The study showed that EMF exposure could down-regulate the surface molecules and stimulation ability of human DC.

Dendritic Cells as a Novel Target for Erythropoietin: Studies in a Human System.

The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system have been described for some time; however, the mechanism of action by which EPO affects lymphocyte number and function has yet to be elucidated. Our search for possible mechanisms by which EPO affects these parameters led us to the novel discovery that EPO receptors (EPO-R) are expressed in dendritic cells (DCs), the most potent antigen presenting cells and most efficient T cell primers. Furthermore, we show that EPO has direct effects on the phenotype and function of human DCs. When added in vitro, EPO increased the percentage of peripheral blood originated DCs that express the co-stimulatory molecules CD80, CD86 and CD40. We also show that EPO up-regulates the level of expression of these molecules, as well as that of HLA-DR in monocyte-derived DCs. When added to immature DCs, EPO alone can induce their maturation. Furthermore, we demonstrate that EPO enhances DC function, as revealed by increased antigen uptake, secretion of interleukin (IL)-12 and stimulatory function in allogeneic mononuclear cell proliferation. We propose that DCs may represent a missing link which might explain previously observed immunomodulatory actions of EPO. Hence, our findings are of crucial importance and may open new clinical avenues for EPO in the optimization of ex-vivo DC-based vaccines, or when administered to patients in order to enhance immune system responses.

Extracellular Branched-Chain Amino Acids, Especially Valine, Regulate Maturation and Function of Monocyte-Derived Dendritic Cells

The functions of dendritic cells (DCs) are impaired in patients with liver cirrhosis. It is well-known that cirrhotic patients show decreased levels of plasma branched-chain amino acids (BCAA). Although amino acids are associated with maintaining the cell structure and function in many organs, limited data are available regarding the role of amino acids including BCAA in the immune system. We aimed to investigate the roles of BCAA in the function of human monocyte-derived DCs (MoDC). CD14-positive monocytes (CD14 (+)) were isolated from PBMC from healthy volunteers and hepatitis C virus (HCV) cirrhotic patients. In medium deprived of BCAA or valine, monocytes were able to differentiate into immature, but not into mature, DCs and showed weak expression of CD83. The deprivation of leucine or isoleucine did not affect this process. The MoDC allostimulatory capacity was significantly decreased in medium deprived of BCAA or valine (p = 0.017, p = 0.012, Bonferroni's analysis, respectively). Annexin V(FITC)/propidium iodide staining showed that the DC yield and viability were not significantly different under any medium. Immunoblotting demonstrated that depletion of valine or leucine decreased phospho-S6 kinase expression. Valine increased dose-dependently the allostimulatory capacity and IL-12 production of MoDC from both healthy volunteers and HCV cirrhotic patients. An elevated extracellular concentration of valine could improve the DC function in cirrhotic patients. These data provide a rationale for nutrition therapy that could be beneficial to patients with cirrhosis.

PPARγ regulates the function of human dendritic cells primarily by altering lipid metabolism

AbstractActivation of the lipid-regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) modifies the immunophenotype of monocyte-derived dendritic cells (DCs). However it has not been analyzed in a systematic manner how lipid metabolism and immune regulation are connected at the transcriptional level via this receptor. Here we present the genome-wide expression analyses of PPARγ-instructed human DCs. Receptor activation was achieved by exogenous, synthetic as well as endogenous, natural means. More than 1000 transcripts are regulated during DC development by activation of PPARγ; half of the changes are positive effects. These changes appear to enhance and modulate the robust gene expression alterations associated with monocyte to DC transition. Strikingly, only genes related to lipid metabolism are overrepresented among early induced genes. As a net consequence, lipid accumulation appears to be diminished in these cells. In contrast, genes related to immune response are regulated after 24 hours, implying the existence of indirect mechanisms of modulation. Receptor dependence was established by using DCs of patients harboring a dominant-negative mutation of PPARγ. Our data show that PPARγ acts as a mostly positive transcriptional regulator in human developing DCs, acting primarily through controlling genes involved in lipid metabolism and via this, indirectly modifying the immune phenotype.

Leishmania amazonensisinfection impairs differentiation and function of human dendritic cells

Dendritic cells (DCs) are of utmost importance in initiating an immune response and may also function as targets for pathogens. The presence of pathogens inside DCs is likely to impair their functions and thus, influence immune responses. In the present report, we evaluated the impact of the presence of Leishmania amazonensis during differentiation and maturation of human monocyte-derived DCs. The presence of live L. amazonensis parasites during DC differentiation led to a significant decrease in CD80 (92%) and CD1a (56%) expression and an increase in CD86 (56%) cell surface expression. Phenotypic changes were accompanied by a lower secretion of IL-6, observed after 6 days of DC differentiation in the presence of L. amazonensis. DCs differentiated in the presence of L. amazonensis were used as APC in an autologous coculture, and lower amounts of IFN-gamma were obtained compared with control DCs differentiated in the absence of parasites. The effect of heat-killed parasites, but not of Leishmania antigen, during DC differentiation and maturation was similar to that observed with viable parasites. During maturation, the presence of live L. amazonensis parasites, but not of soluble Leishmania antigen, led to a decrease in IL-6 and IL-10 production. In this way, we observed that the parasite is able to abrogate full DC differentiation, causing a delay in the immune response and likely, favoring its establishment in human hosts.

Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.

Amyloid β peptide promotes differentiation of pro-inflammatory human myeloid dendritic cells

A key event of Alzheimer's disease (AD) pathogenesis is the production of amyloid β peptides (Aβ), which are hypothesized to lead to neurodegeneration by still unclear mechanisms, including a chronic inflammatory response characterized by innate immune cell activation and pro-inflammatory molecule release. Since dendritic cells (DCs) are central players of innate immune response and brain dendritic-like cells may have a crucial role in AD pathogenesis, this study investigates the effects of Aβ on human DC functions. Myeloid DCs differentiated in the presence of Aβ42 showed an increase in survival and soluble antigen uptake, a reduction in HLA molecule expression and in IL-10 and IL-12 production. Accordingly, Aβ42-treated DCs were impaired in inducing T cell proliferation and IL-2 production. On the other hand, Aβ42 treatment provided DCs with the ability to release higher levels of IL-1β, IL-6 and IL-18, than control DCs. These results demonstrate that Aβ42 can modulate the immune system by inducing pro-inflammatory DC differentiation, thus gaining new insights into AD pathogenesis and immune-based therapeutic intervention.

Sorafenib but not sunitinib affects the induction of immune responses

3504 Background: The tyrosine kinase inhibitors (TKIs) sorafenib and sunitinib are approved for the treatment of patients with metastatic renal cell cancer. To analyze the possible use of these compounds in combination with immunotherapeutic approaches we investigated the effects of both TKIs on function of human dendritic cells (DCs) and induction of primary immune responses in vitro and in vivo. Methods: Human monocytes-derived DCs were treated with DMSO, sorafenib or sunitinib. Functional and phenotypic analyses as well as the possible impact on signal transduction pathways were performed. Furthermore, induction of immune responses in vivo was analyzed in animals treated with both compounds. Results: Sorafenib but not sunitinib inhibits function of DCs. Exposure of DCs to sorafenib reduces expression of CD1a, MHC and costimulatory molecules in response to stimuli via TLR ligands. Sorafenib reduces cytokine production by DCs as well as their ability to migrate and stimulate T cell (TC) responses. We found that these inhibitory effects of sorafenib are mediated via inhibition of PI3K, MAP kinases and NFκB signaling. The TKIs have no influence on phenotype and proliferation of TCs. To analyze the effects of TKIs on the generation of immune responses in vivo, induction of TC responses was assessed by peptide vaccination with the model antigen OVA-001 in C57BL/6 mice. When mice were pretreated with both TKIs which were also given during vaccination, it was observed that sorafenib, but not sunitinib significantly reduces the generation of vaccine-specific CD8+ TCs. Numbers of CD4+ CD25+ regulatory TCs are reduced in sunitinib-treated mice, but not in sorafenib-treated animals. All effects of the TKIs are reversible, and the immune responses go back to normal levels if mice are immunized after discontinuation of treatment. Conclusions: In summary, sunitinib represents an interesting compound to be used in combination with immunotherapeutic approaches to treat cancer patients. No significant financial relationships to disclose.

Immune Complexes Inhibit Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcgammaRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcgammaR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcgammaRI and to a lesser extend with FcgammaRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.

A Key Role for Poly(ADP-Ribose) Polymerase-1 Activity during Human Dendritic Cell Maturation

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-kappaB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-kappaB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.

Phenotypic and Functional Effects of Heat Shock Protein 90 Inhibition on Dendritic Cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-gamma secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.

Immunomodulatory effects of aqueous birch pollen extracts and phytoprostanes on primary immune responses in vivo

We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids, such as phytoprostanes, that modulate human dendritic cell (DC) function in a way that results in an enhanced T(H)2 polarization in vitro. Here we analyzed the immunomodulatory capacities of Betula alba (white birch) aqueous pollen extracts (Bet-APEs) and pollen-associated phytoprostanes in the murine system in vitro and in vivo. DC function was analyzed in vitro by using BALB/c bone marrow-derived DCs. T-cell responses were analyzed with DO11.10 peptide 323-339 from chicken ovalbumin (OVA)-specific CD4 T cells as responder cells. For in vivo studies, OVA-specific CD4 T cells were adoptively transferred into BALB/c mice. Twenty-four hours later, mice were challenged by means of intranasal application of OVA in the absence or presence of Bet-APEs or phytoprostanes. Polarization of T-cell responses in vivo was analyzed in draining lymph node cells. In vitro Bet-APEs and E(1)-phytoprostanes dose-dependently inhibited LPS-induced IL-12p70 of DCs. In addition, Bet-APEs induced a T(H)2 polarization in vitro. Similarly, intranasal instillation of Bet-APEs in vivo, together with the antigen, lead to increased IL-4, IL-5, and IL-13 secretion and decreased IFN-gamma secretion from antigen-specific T cells in the draining lymph nodes. In contrast, intranasal E1- and F1-phytoprostanes downregulated both T(H)1 and T(H)2 cytokine production in vivo. Pollen release water-soluble factors that display T(H)2-polarizing capacities in vivo independently of E(1)- and F(1)-phytoprostanes. Identification of the underlying mechanisms might open new approaches for pharmacologic intervention.

Complement protein C1q induces maturation of human dendritic cells

Maturation of dendritic cells (DCs) is known to be induced by several stimuli, including microbial products, inflammatory cytokines and immobilized IgG, as demonstrated recently. Since immune complexes formed in vivo also contain C1q, moreover apoptotic cells and several pathogens fix C1q in the absence of antibodies, we undertook to investigate whether this complement protein has an impact on various functions of human DCs. Maturation of monocyte-derived immature DCs (imMDCs) cultured on immobilized C1q was followed by monitoring expression of CD80, CD83, CD86, MHCII and CCR7. The functional activity of the cells was assessed by measuring cytokine secretion and their ability to activate allogeneic T lymphocytes. Cytokine production by T cells co-cultured with C1q-matured DCs was also investigated. C1q, but not the structurally related mannose-binding lectin was found to bind to imMDC in a dose-dependent manner and induced NF-κB translocation to the nucleus. Immobilized C1q induced maturation of MDCs and enhanced secretion of IL-12 and TNF-α, moreover, elevated their T-cell stimulating capacity. As IFN-γ levels were increased in supernatants of MDC–T cell co-cultures, our data suggest that C1q-induced DC maturation generates a Th1-type response. Interestingly, IL-10 levels were elevated by C1q-treated MDCs but not in the supernatant of their co-cultures with allogeneic T cells. Taken together, these results indicate that C1q-opsonized antigens may play a role in the induction and regulation of immune response. Moreover our data are relevant in view of the role of C1q in removal of apoptotic cells and the association between C1q-deficiency and autoimmunity.

Influence of heat stress on human monocyte-derived dendritic cell functions with immunotherapeutic potential for antitumor vaccines

Mild heat stress can modulate the activities of immune cells, including dendritic cells (DC) and theoretically, would constitute an innovative approach capable of enhancing the antitumor functions of DC. Therefore, we tested the effects of mild heat stress on the physiology and viability of human monocyte-derived DC, the major type of DC used in tumor immunotherapy trials. We first designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout the generation of DC. Using this protocol, we observed that heat stress did not perturb the morphology and the phenotype of immature or mature DC or the capacities of immature DC to uptake antigens efficiently. It is noteworthy that in response to heat stress, mature DC produced higher levels of IL-12p70 and TNF-alpha, which are two cytokines involved in the stimulation of inflammatory reaction, whereas IL-10 production remained low. After heat-stress exposure, mature DC have the full ability to stimulate naive T cells with Th1 response polarization (high IFN-gamma and low IL-4 production) in an allogeneic MLR. It is interesting that heat stress enhanced the migratory capacities of DC in response to MIP-3beta/CCL19. Finally, heat stress partly protected DC from apoptosis induced by cytokine withdrawal. Overall, these findings validate the feasibility of improving immune response by heating human monocyte-derived DC and provide a strong rationale for using mild heat stress in combination with DC vaccination to increase antitumor response.

Diterpenes inhibit IL-12 production by DC and enhance Th2 cells polarization

Sugiol and 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (Secoferruginol) are diterpenes isolated from the heartwood of Cryptomeria japonica and are pharmacologically active substances. Dendritic cells (DC) have a key influence on the differentiation of naïve T cells into Th1 or Th2 effector cells. We demonstrate that Sugiol and Secoferruginol activate human DC as documented by phenotypic and functional maturation and altered cytokine production. Human monocytes were exposed to Sugiol or Secoferruginol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD83 on Sugiol-primed DC were enhanced. Sugiol dose-dependently inhibited IL-12p70 production by LPS-primed DC and to a lesser extent, the production of the proinflammatory cytokines. Naïve T cells co-cultured with Sugiol-primed DC, turned into typical Th2 which produced large quantities of IL-4 and released small amounts of IFN-γ and reduced Th1 cell polarizing capacity. Sugiol-primed DC induced the development of Th2 cells via the enhanced expression of OX40L and augmented the Th2 cell polarizing capacity of DC via the inhibition of IL-12p70. Similar results were obtained with Secoferruginol. These results suggest that some diterpenes modulate human DC function in a fashion that favors Th2 cell polarization and might have implication in autoimmune diseases.

Impact of transfection with total RNA of K562 cells upon antigen presenting, maturation, and function of human dendritic cells from peripheral blood mononuclear cells

Vaccination of dendritic cells (DCs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. The purpose of this study was to investigate whether human monocyte-derived DCs were able to present P210(Bcr-Ab1) protein and induce antigen-specific cytotoxic T lymphocyte (CTL) responses in vitro after transfected with total RNA of K562 cells (K562-RNA). DCs derived from human peripheral blood mononuclear cells were transfected with K562-RNA with electroporation or DOTAP lipofection. The successful transfection was determined by reverse transcription-polymerase chain reaction and Western blot. The phenotypes of the DCs were analyzed by flow cytometry (FCM), and cytotoxicity of CTL was assessed by propidium iodide staining followed by FCM analysis. The CD1a expression and purity of DCs were measured by FCM. The Bcr-Abl fusion gene was detected in the DCs with 24 hours after the transfection. The transfected cell expressed increased levels of CD80, CD83, CD86, and HLA-DR. Moreover, the transfected DCs strongly stimulated the T lymphocytes to gain cytotoxic activity against K562 cells. Culture medium containing 1 percent human plasma was the most effective for DC growth. Human DCs transfected with K562-RNA effectively induce specific immune responses. This method can be used to induce tumor-specific immune response and may have potential application in immunotherapy of tumors.