Tumor-educated tolerogenic dendritic cells induce CD3epsilon down-regulation and apoptosis of T cells through oxygen-dependent pathways

Defects in the CD3/TCR complex and impairment of T cell function are necessary for tumor evasion, but the underlying mechanisms are incompletely understood. We found that culture supernatants from several types of solid tumor cell lines drove human monocytes to become tolerogenic semimature dendritic cells (TDCs). Upon encountering T cells, the TDCs triggered rapid down-regulation of CD3epsilon and TCR-alpha/beta and subsequent apoptosis in autologous T cells. Consistent with these results, accumulation of immunosuppressive DCs coincided with CD3epsilon down-regulation and T cell deletion in cancer nests of human tumors. The impaired T cell function was mediated by factor(s) released by live TDCs after direct interaction with lymphocytes. Also, the TDC-induced effect on T cells was markedly reduced by blocking of NADPH oxidase but not by inhibition of arginase, inducible NO synthase (iNOS), IDO, or IFN-gamma. Moreover, we found that hyaluronan fragments constituted a common factor produced by a variety of human tumor cell lines to induce formation of TDCs. These observations indicate that tumor microenvironments, including hyaluronan fragments derived from cancer cells, educate DCs to adopt a semimature phenotype, which in turn aids tumor immune escape by causing defects in the CD3/TCR complex and deletion of T cells.     

Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles

A major limitation in studying molecular interactions between parasitic helminths and their hosts is the lack of suitable in vitro cultivation systems for helminth cells and larvae. Here we present a method for long-term in vitro cultivation of larval cells of the tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Primary cells isolated from cultivated metacestode vesicles in vitro showed a morphology typical of Echinococcus germinal cells, displayed an Echinococcus-specific gene expression profile and a cestode-like DNA content of approximately 300Mbp. When kept under reducing conditions in the presence of Echinococcus vesicle fluid, the primary cells could be maintained in vitro for several months and proliferated. Most interestingly, upon co-cultivation with host hepatocytes in a trans-well system, mitotically active Echinococcus cells formed cell aggregates that subsequently developed central cavities, surrounded by germinal cells. After 4 weeks, the cell aggregates gave rise to young metacestode vesicles lacking an outer laminated layer. This layer was formed after 6 weeks of cultivation indicating the complete in vitro regeneration of metacestode larvae. As an initial step toward the creation of a fully transgenic strain, we carried out transient transfection of Echinococcus primary cells using plasmids and obtained heterologous expression of a reporter gene. Furthermore, we successfully carried out targeted infection of Echinococcus cells with the facultatively intracellular bacterium Listeria monocytogenes, a DNA delivery system for genetic manipulation of mammalian cells. Taken together, the methods presented herein constitute important new tools for molecular investigations on host-parasite interactions in alveolar echinococcosis and on the roles of totipotent germinal cells in parasite regeneration and metastasis formation. Moreover, they enable the development of fully transgenic techniques in this group of helminth parasites for the first time.     

Echinococcus multilocularis: in vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells

Hybrid cells were produced from Echinococcus multilocularis metacestode germinal cells and murine tumor cells. Small colonies were formed which, while ceasing to grow after a few generations, remained viable for at least 10 weeks. These hybridoma cells secrete antigen(s) reacting in indirect immunofluorescence and ELISA specifically with sera from patients suffering from an E. multilocularis infection. The antigen(s) appear suitable for the differential diagnosis of E. multilocularis and E. granulosus. Thus, hybridoma cells may produce helminth antigens.     

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective ‘first generation’ cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these ‘second generation’ cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.     

Echinococcus multilocularis in the cotton rat: the in vitro protoscolicidal activity of peritoneal cells.

Protoscolices of Echinococcus multilocularis were incubated in vitro with peritoneal cells and with sera from normal and infected cotton rats. The cells from rats bearing massive subcutaneous and intraperitoneal hydatid cysts rapidly killed protoscolices; normal cells and cells from lightly infected animals did not. Only sera from rats bearing large, subcutaneous cysts displayed significant protoscolicidal activity. These data suggest that the phenomenon whereby large, established cysts of E. multilocularis effectively suppress the establishment, growth, and metastasis of distant foci of infection has an immunological component.     

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 × D2)F₁ and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host–parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.     

Generation of tolerogenic dendritic cells using Lactobacillus rhamnosus and Lactobacillus delbrueckii as tolerogenic probiotics

Systemic lupus erythematosus (SLE) concurs with excessive uncontrolled inflammatory immune responses that lead to the loss of immune tolerance. Dendritic cells (DCs) are important and determinant immune cells that regulate immune responses. Tolerogenic DCs with regulatory markers and cytokines could induce regulatory immune cells and responses. Tolerogenic probiotics are capable of producing regulatory DCs from monocytes in in vitro conditions. The purpose of this study was to evaluate the effect of Lactobacillus delbrueckii and Lactobacillus rhamnosus on the production of DCs in an in vitro condition. Peripheral blood mononuclear cells were isolated from the healthy and SLE donors. Monocytes were cultured with optimized concentrations of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin 4 (IL‐4) to produce immature DCs (IDCs). An IDC uptake assay was performed, and IDCs of healthy and SLE donors were divided into three subgroups following 48 hours of treatment with GM‐CSF and IL‐4, along with L. delbrueckii, L. rhamnosus, and mixed probiotics for the production of tolerogenic DCs. The surface expression of Human Leukocyte Antigen‐antigen D Related (HLA‐DR), CD86, CD80, CD83, CD1a, and CD14 was analyzed using flow cytometry, and the gene expression levels of indoleamine 2,3‐dioxygenase (IDO), IL‐10, and IL‐12 were measured using real‐time polymerase chain reaction. We observed significantly reduced expression of costimulatory molecules and other surface markers in the probiotic‐induced mature DCs (MDCs) in both healthy and SLE donor groups in comparison with lipopolysaccharide (LPS)‐induced MDCs. In addition, the expression of IDO and IL‐10 increased, whereas IL‐12 decreased significantly in probiotic‐induced MDCs compared with LPS‐induced MDCs. IDCs and especially mature tolerogenic DC of SLE patients highly expressed IDO. The results of the current study suggested that live probiotics could modify properties of DCs to modulatory cells, which might contribute to the induction of tolerance and renovation of immune hemostasis.     

Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.     

Ecology and epidemiology of Echinococcus multilocularis in Europe

Human alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis has a high mortality rate in untreated patients. The life-cycle of E. multilocularis in Europe predominantly involves foxes as definitive hosts. However, experimental studies demonstrated a comparable biotic potential of E. multilocularis in dogs and raccoon dogs but an insignificant potential in cats. AE occurs in central and eastern Europe at low incidence rates. Recent studies in foxes have shown that E. multilocularis has a wider geographic range (including Italy) than previously thought. In recent years, increases in fox populations have been observed in many European countries, especially in urban areas. As a result, the E. multilocularis cycle is now established in the urban environment. This presents an increased risk of infection for a large human population. Based on these facts and new epidemiological data, possible intervention strategies are presented.     

Transfer of regulatory properties from tolerogenic to proinflammatory dendritic cells via induced autoreactive regulatory T cells

Infectious tolerance is a term generally assigned to the process through which regulatory T cells (Tregs) transfer immunoregulatory properties to other T cells. In this study, we demonstrated that a similar process applies to human dendritic cells (DCs), albeit through a different mechanism. We induced and cloned proinsulin-specific Tregs using tolerogenic DCs and investigated mechanisms by which induced Ag-specific regulatory T cells (iaTregs) endorse the suppressive effects. iaTregs expressed FOXP3, programmed death-1, and membrane-bound TGF-β and upregulated IL-10 and CTLA-4 after stimulation with the cognate Ag. The iaTregs suppressed effector T cells only when both encountered the cognate Ags on the same APCs (linked suppression). This occurred independently of IL-10, TGF-β, programmed death-1, or CTLA-4. Instead, iaTregs used a granzyme B-mediated mechanism to kill B cells and monocytes, whereas proinflammatory DCs that resisted being killed were induced to upregulate the inhibitory receptors B7 (family) homolog 3 and ICOS ligand. These re-educated mature monocyte-derived dendritic cells (mDCs) suppressed effector T cells and induced IL-10-producing cells from the naive T cell pool. Our data indicated that human tolerogenic DCs confer infectious tolerance by inducing Ag-specific Tregs, which, in turn, re-educate proinflammatory mature DCs into DCs with regulatory properties.     

1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.     

Uniform strobilar development of Echinococcus multilocularis in vitro from protoscolex to immature stages

The aim of this study was to obtain uniformity in strobilar development of Echinococcus multilocularis from protoscoleces in vitro. The isolate of E. multilocularis used was derived initially from a human case in France and subsequently maintained in the laboratory by intraperitoneal passage in Meriones unguiculatus. Protoscoleces used for culture were obtained using preparative procedures in which parasite tissue was disrupted gently with minimal exposure to pepsin and acidic conditions followed by immediate exposure to pancreatin in alkaline solution. Resultant cultures contained large numbers of evaginated, active, vermiform stages, which exhibited uniform strobilar development with formation of the first proglottid and segment and limited maturation of the first proglottid. All worms that exhibited proglottization subsequently segmented. Further proglottization did not occur and all worms degenerated within a few days following segmentation. The results are discussed in light of current knowledge of the relationships of somatic and germinal processes in Echinococcus. In view of these results, further studies should be encouraged to improve strobilar development of E. multilocularis in vitro.     

In vitro effects of isoprinosine and a dipeptide methyl ester on Echinococcus multilocularis protoscoleces

A protoscoleces/vesicles in vitro maintenance test with assessment of viability by eosin exclusion was used to evaluate the quantitative and qualitative activities of isoprinosine, its active component inosine and the dipeptide methylester L-Phe-Phe-OMe on isolated protoscoleces of Echinococcus multilocularis for 24 and 48 h. Isoprinosine and inosine showed dose- and time-dependent activity, the latter displaying a more rapid effect than the former. A high activity was shown with L-Phe-Phe-OMe, when compared to praziquantel. Ultrastructural alterations were much more striking with L-Phe-Phe-OMe, with an effect similar to that of praziquantel, whereas the chemotherapeutic activity of inosine and isoprinosine appeared to be directed against a metabolic target, with a lethal effect not immediately visible at the ultrastructural level. Thus, the previously reported in vivo activities of these drugs result largely from a direct effect on the parasite.     

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.     

Estriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity

Chronic inflammation contributes to numerous diseases, and regulation of inflammation is crucial for disease control and resolution. Sex hormones have potent immunoregulatory abilities. Specifically, estrogen influences immune cells and inflammation, which contributes to the sexual dimorphism of autoimmunity and protection against disease seen during pregnancy in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although long thought to act primarily on T cells, recent evidence demonstrated that myeloid cells, such as dendritic cells (DCs), are essential in mediating estrogen's protective effects. Estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE, and we evaluated whether E3 could act exclusively through DCs to protect against the inflammatory autoimmune disease EAE. Levels of activation markers (CD80 and CD86) and inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3, and B7-H4) were increased in E3 DCs. E3 DCs had decreased proinflammatory IL-12, IL-23, and IL-6 mRNA expression, increased immunoregulatory IL-10 and TGF-β mRNA expression, and a decreased ratio of IL-12/IL-10 protein production. Importantly, transfer of E3 DCs to mice prior to active induction of EAE protected them from developing EAE through immune deviation to a Th2 response. This protection was apparent, even in the face of in vitro and in vivo inflammatory challenge. In summary, our results showed that E3 generates tolerogenic DCs, which protect against the inflammatory autoimmune disease EAE. Targeted generation of tolerogenic DCs with immunomodulatory therapeutics, such as E3, has potential applications in the treatment of numerous autoimmune and chronic inflammatory diseases.     

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.     

耐受性树突状细胞预防和治疗卵清蛋白过敏小鼠的的研究

目的 比较腺病毒重组CTLA4Ig/α4β7诱导的耐受性树突状细胞对卵清蛋白(OVA)致敏小鼠的预防和治疗效果.方法 BALA/c小鼠32只,分为4组.基础致敏24h后回输耐受性树突状细胞为预防组;肠道激发4h后回输耐受性树突状细胞为治疗组;回输生理盐水为阴性对照组和OVA过敏小鼠为阳性对照组.观察各组小鼠过敏症状缓解情况;HE染色观察空肠形态;甲苯胺兰染色观察小肠固有层肥大细胞聚集及脱颗粒现象;ELISA测定各组血清中OVA特异性IgE水平.结果 与阳性对照组相比,治疗组小鼠OVA特异性IgE水平显著降低(0.25±0.05 vs 0.17±0.03) (P ＜0.05);体重增长明显(3.16 g±0.75 g vs 5.04 g±0.49 g)(P ＜0.05);腹泻症状减轻;HE染色可见治疗组小肠绒毛中上皮细胞局灶性坏死、脱落,固有层炎症细胞浸润现象明显改善;肥大细胞聚集和脱颗粒现象改善.而预防组较阳性对照组过敏症状、小肠形态学及OVA特异性IgE水平差异均无统计学意义.结论Ad CTLA4Ig/Adα4β7修饰的致耐受性树突状细胞对卵清蛋白过敏小鼠具有治疗作用而无预防作用.     

Surveillance and management of Echinococcus multilocularis in a wildlife park

The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts.