Tesis sobre el tema "Immature and mature dendritic cells"

Thesis (M.S.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed May 8, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 45-47).

The ability of dendritic cells (DC) to be either tolerogenic or immunogenic offers many opportunities to manipulate DC function and genetically modify DC to promote allograft acceptance. However, the greatest challenge in DC gene therapy is the difficulty of achieving successful transfection. At present, DC-targeting gene delivery strategies are hindered by the lack of DC-specific target molecules. Therefore, the essential aim of this study is to isolate and identify peptides capable of targeting immature DC (iDC), in particular those that internalise well as they are likely to be more effective in gene delivery. Phage library of short cyclic peptides expressed on pIII protein of M13 filamentous phage was used first in selection against streptavidin. Phage bearing HPQ and HPM motifs were successfully isolated indicating the feasibility of the library in selecting streptavidin-binding peptides through specific biotin interaction. In order to develop a method for in vitro selection on cell surfaces, the library was used against a cell line expressing αVβ3 integrin. One phage clone with encoded sequence of CLSSPALLC bound to the cell surface and was taken up by the cells expressing the αVβ3 integrin. Using methods established in the previous panning, the library was panned against iDC, after two subtractive pannings against PBMC and CD14+ cells. The phage isolated were characterised by using several methods including flow cytometric analysis and confocal microscopy. Two phage selected, DC4.25 and DC4.44 with encoded peptide sequences of CFPPTFPAC and CTPLLSPFC, respectively, have demonstrated the ability to bind to, and be internalised by, iDC. Soluble biotinylated peptides, which contain the encoded peptide sequences, were synthesised along with sequence-scrambled control peptides. Flow cytometric analysis detected non-specific binding of the monomeric peptides to iDC when used at high concentration. In an attempt to increase the binding avidity of the peptides to iDC, Streptavidin conjugated with Alexa-488® flurophore was used to form complexes with the peptides. However, flow cytometric analysis revealed that the complexes also bound non-specifically to iDC, as binding was also seen in control peptides. Streptavidin conjugated with HRP was then used to form complexes with the peptides, in order to increase the sensitivity of the binding assay. The results corroborated the previous findings, which showed that the peptides bound iDC non-specifically. The failure of the synthetic peptides to bind to iDC might be due to a number of reasons, including loss of cyclised conformation of the peptides during synthesis and insufficient incorporation of sequences that is essential and accountable for the binding, in the synthetic peptides. However, this approach does offer the possibility of identifying cell binding peptides that may allow delivery of genes and other therapeutic or imaging agents.

The leukocyte immunoglobulin like receptors (LILRs) are a group of receptors with immunomodulatory effects. Group 1 LILRs comprise of LILRB1, among others, and bind to class 1 MHC molecules and transmits inhibitory signals. Studies have shown that LILRB1 ligation during the monocyte differentiation process into dendritic cells (DCs) results in the generation of a population of cells that are tolerogenic. Here we hypothesize that this tolerogenic nature of the resultant cells is due to the high expression of nuclear factor kappa – light chain enhancer of activated B cells (NF-κB) inhibitor – A20 binding inhibitor of NF-κB signalling 1 (ABIN1). In this study we analyzed the effect that ABIN1 exerts on the maturation of DCs and CD14+HLA-DRlow/- monocytes - a population of cells that have been recently been identified as myeloid derived suppressor cells (MDSCs) in humans. LILRB1 ligated DCs and CD14+HLA-DRlow/- monocytes, when treated with ABIN1 siRNA, displayed an increase in the expression of antigen presentation and co-stimulatory molecules such as CD80, CD86, HLA-DR and HLA-ABC and displayed a greater capacity to produce cytokines like IL-12 and IFN-α. Additionally, they displayed a greater capacity to stimulate the adaptive component of the immune system in terms of IFN-γ production, cell proliferation and adapter molecule and mitogen activated protein kinase (MAPK) activation in T cells. Based on the results we obtained, it can be concluded that ABIN1 plays a significant role in maintaining the immature and suppressive phenotype of immature myeloid cells (IMCs) by dampening NF-κB signalling, while also exerting a negative effect on antiviral signalling.

Le succès de la réponse immunitaire repose en grande partie sur la capacité des leucocytes à se déplacer et à accomplir leur fonction au sein de structures anatomiques précises. Le fait qu’il puisse exister des mécanismes intrinsèques de coordination entre ces fonctions spécifiques et la migration de ces cellules n’a jamais été étudié auparavant. Nos travaux mettent en évidence, pour la première fois, l’existence d’un couplage entre la migration et la macropinocytose dans les cellules dendritiques qui explorent leur environnement en internalisant une grande quantité de matériel extra-cellulaire. C’est la Chaîne Invariante, protéine chaperon impliquée dans l’apprêtement des antigènes, qui est responsable de ce couplage en détournant le moteur Myosine II de l’arrière de la cellule, où elle promeut la migration, vers l’avant de la cellule. Ce recrutement transitoire de Myosin II autour des macropinosomes à l’avant favorise la macropinocytose et la délivrance de l’antigène dans les lysosomes, mais ralentit la cellule. L’implication de la Myosine II à la fois dans la migration et la capture d’antigène permet donc le couplage moléculaire entre ces deux processus et leur coordination spatio-temporelle. Cependant, les voies de signalisation impliquées dans le couplage avant/arrière dans les cellules dendritiques immatures restent encore méconnues. L’ensemble de mes travaux de thèse montrent que la libération de calcium du réticulum endoplasmique à travers les récepteurs IP3 (IP3Rs) est nécessaire pour maintenir le niveau de phosphorylation de la chaîne légère de Myosin (MLC) et la polarisation avant/arrière de Myosine II au cours de la migration des cellules dendritiques immatures. Nous montrons que les récepteurs IP3R1, 2 et 3 sont requis pour atteindre une vitesse maximale en 2- et 3-Dimension, et que le récepteur IP3R3, et dans une moindre mesure IP3R1, favorisent la persistance des cellules. En revanche, l’inhibition de l’expression du récepteur IP3R3 augmente la capacité des cellules dendritiques immatures à capturer l’antigène, ce qui est en accord avec notre résultat montrant que la capture de l'antigène est inversement reliée à la locomotion de cellules dendritiques. Nous proposons que le relargage du calcium par le réticulum endoplasmique favorise l’activité de la myosine II ce qui permet aux cellules dendritiques de ralentir de façon transitoire. Ce relargage calcique permet aux cellules dendritiques du optimiser l'internalisation des antigènes extracellulaires en maintenant leur polarité ce qui leur permet d’optimiser ainsi leur capacité d'échantillonnage de l’environnement
The immune response heavily relies on the migration capacity of leukocytes. These cells must stop in precise anatomical locations to fulfill a particular task. But whether and how specific functions are coordinated with migration by cell-intrinsic mechanisms is not known. We here show that in dendritic cells, which patrol their environment for the presence of antigens by internalizing extracellular material, macropinocytosis is coupled to cell migration. Coupling relies on the diversion of the Myosin II motor from its migratory function at the cell rear to macropinosomes at the cell front by the Invariant Chain, a cell-specific regulator of antigen presentation. Transient Myosin II recruitment at the cell front promotes antigen macropinocytosis and antigen delivery to endolysosomes but antagonizes cell migration. Thus, the requirement for Myosin II for both migration and antigen capture provides a molecular mechanism to couple these two processes and allow their coordination in time and space. However, the signaling pathways involved in back/front coupling in migrating immature DCs remain unknown. Here we show that calcium released from the endoplasmic reticulum through IP3 Receptors (IP3Rs) is required to maintain Myosin regulatory light Chain (MLC) phosphorylation and Myosin II back/front polarization during DC locomotion. We found that while IP3R1, 2 and 3 are required for immature DCs to reach maximal speed in 2-Dimensional and 3-Dimensional environments, IP3R3 and to a lesser extent IP3R1 positively regulate their persistency. On the contrary, silencing of IP3R3 increases antigen uptake by immature DCs, consistent with our finding showing that antigen capture is inversely coupled to DC locomotion (Appendix, manuscript #1). We propose that by promoting myosin II activity, calcium released from the ER help DCs to transiently slow-down to uptake extracellular antigens without losing their polarity and thereby optimizes their environment sampling capacity

Das human pathogene Varicella-Zoster Virus (VZV) gehört zur Familie der Herpesviren und ist weltweit verbreitet. Die Primärinfektion verursacht Varicellen, welche durch einen bläschenartigen Hautausschlag charakterisiert ist. Im Anschluss daran etabliert VZV eine lebenslange Latenz und verursacht nach Reaktivierung Herpes Zoster. Seit 2004 ist der Lebendimpfstoff aus attenuierten Virionen des VZV-Stammes V-Oka in Deutschland empfohlen. Im Gegensatz zur Infektion mit zirkulierenden virulenten VZV Stämmen tritt nach Verimpfung des Vakzin-Stammes V-Oka kein Exanthem auf. Die Haut ist der Hauptreplikationsort von VZV und immunologische Unterschiede zwischen virulentem VZV und dem Vakzin-Stamm treten hier am deutlichsten auf. In der vorliegenden Arbeit konnte eine neue Immunevasionsstrategie virulenter VZV Stämme aufgedeckt werden, welche erklären könnte, wie virulente VZV Stämme frühe antivirale Immunantworten umgehen. In Hautläsionen von Herpes Zoster Patienten konnte eine massive Infiltration von myeloiden inflammatorischen Dendritischen Zellen beobachtet werden. In vitro Studien mit Monozyten abgeleiteten Dendritischen Zellen (DC), welche inflammatorische DC repräsentieren, zeigten, eine signifikant erhöhte Expression von CD1c Molekülen nach Infektion mit dem Vakzin-Stamm, sowie virulentem VZV. Funktionelle Untersuchungen mit intraepithelialen CD1c-restringierten gamma delta T Zellen zeigten, dass DC nach Infektion mit dem Vakzin-Stamm phänotypisch und funktionell reiften und somit die T Zellen zur IFN-gamma Sekretion stimulierten. Im Gegensatz dazu wurde die funktionelle Reifung von DC, die mit virulentem VZV infiziert waren, geblockt. Folglich wurde kein bioaktives IL-12 sezerniert, welches als entscheidendes Cytokin zum Aufbau einer antiviralen T-Helfer 1 Immunantwort beiträgt. Darüber hinaus konnte gezeigt werden, dass virulentes VZV die Signalkaskade des Toll-like Rezeptors 2 (TLR2) in DC inhibiert und somit die IL-12 Produktion verhindert.
Varicella-zoster virus (VZV) which belongs to the family of herpesviruses is restricted to humans and distributed worldwide. Primary infection of VZV causes chickenpox characterized by a disseminated rash. Thereafter, VZV establishes a lifelong latency and can be reactivated to cause herpes zoster. Since 2004 the attenuated strain V-Oka of VZV was licensed for Germany to immunize children against VZV infection. In contrast to infection by circulating virulent VZV strains, vaccination with V-Oka remains asymptomatic. The skin is the major replication site of VZV and immunological differences between virulent VZV and the vaccine should become most apparent within this immune organ. In summary, this study discovered a new immune evasion strategy of virulent VZV strains which might explain how virulent VZV strains overcome innate antiviral responses. A strong infiltration of myeloid-derived inflammatory DCs has been detected in skin lesions of herpes zoster patients. In vitro studies with monocyte-derived dendritic cells (DCs), reflecting inflammatory DCs, showed that they were efficiently infected by both, the vaccine and a virulent VZV strain. Intriguingly, a significant upregulation of CD1c molecules on VZV-infected DCs was observed. Functional investigations using intraepithelial CD1c-restricted gamma delta T cells revealed that DCs infected with the vaccine virus were fully instructed to mature, thereby promoting IFN-gamma secretion of gamma-delta T cells. In striking contrast, DCs infected with virulent VZV strains were efficiently blocked to mature functionally. In detail, they did not secrete bioactive IL-12 which is an instrumental cytokine for generation of antiviral T helper 1 responses. Moreover, virulent VZV blocked Toll-like receptor 2 (TLR2) signaling in DCs thereby preventing production of bioactive IL-12 which in turn inhibited IFN-gamma secretion by gamma-delta T cells.

Herpes simplex virus Typ 1 (HSV-1) ist ein humanpathogenes Virus der Familie Herpesviridae. Für eine erfolgreiche Virusreplikation besitzt HSV-1 mehrere Gene, die in den meisten infizierten Zelltypen Apoptose verhindern. Im Gegensatz dazu führt die HSV-1 Infektion eines zentralen Zelltyps des Immunsystems, den unreifen dendritischen Zellen (iDCs), zu Apoptose. Dies könnte ein Aspekt der HSV-1 Immunevasion sein. Bisher waren die Ursachen der Apoptose von HSV-1 infizierten iDCs unzureichend aufgeklärt. Es wurde jedoch gezeigt, dass das antiapoptotische zelluläre Protein c-FLIP in HSV-1 infizierten iDCs reduziert ist. In dieser Arbeit wurde die c-FLIP Menge in iDCs erstmalig mit Hilfe von RNA Interferenz erfolgreich reduziert. Dies bestätigte die Bedeutung von c-FLIP für die Lebensfähigkeit von iDCs. Folglich könnte auch die Reduktion der c-FLIP Menge nach HSV-1 Infektion iDCs für Apoptose empfindlich machen. Die HSV-1 induzierte c-FLIP Reduktion erfolgte in späten Stadien der Infektion, abhängig von der ordnungsgemäßen Expression viraler „early“ und „leaky late“ Gene. Sie fand nicht auf RNA Ebene statt und war unabhängig vom Proteasom und der Bindung an den „death inducing signaling complex“. Stattdessen wurde c-FLIP wahrscheinlich von einer viralen oder zellulären Protease abgebaut. In dieser Arbeit wurde erstmals gezeigt, dass zusätzlich zu Veränderungen im zellulären Apoptosesignalnetzwerk der Mangel an einem antiapoptotischen viralen Faktor zur Apoptose von HSV-1 infizierten iDCs beiträgt. Eine Microarray Analyse der HSV-1 Genexpression ergab, dass HSV-1 Latenz-assoziierte Transkripte (LATs) in apoptotischen iDCs signifikant geringer exprimiert waren als in nicht-apoptotischen epithelialen Zellen. LATs besitzen in Neuronen und epithelialen Zellen eine antiapoptotische Aktivität. Diese könnte den Mangel an c-FLIP kompensieren. Übereinstimmend mit dieser Hypothese induzierte eine HSV-1 LAT-Deletionsmutante mehr Apoptose in iDCs im Vergleich zum Wildtyp-Virus.
Herpes simplex virus type 1 (HSV-1) is a human pathogen which belongs to the family Herpesviridae. HSV-1 encodes several genes, which serve to efficiently prevent apoptosis in most infected cell types, thereby ensuring successful virus replication. In contrast, HSV-1 infection of one central cell type of the immune system, immature dendritic cells (iDCs), results in apoptosis. This could be one aspect of HSV-1 immunevasion. So far, the mechanisms underlying apoptosis of HSV-1 infected iDCs were poorly defined. However, it has been shown that the antiapoptotic cellular protein c-FLIP is reduced in HSV-1 infected iDCs. In this work, the amount of c-FLIP was for the first time successfully reduced in iDCs by RNA interference. This confirmed the importance of c-FLIP for viability of iDCs. Therefore, it is likely that c-FLIP reduction after HSV-1 infection also sensitizes iDCs to apoptosis. HSV-1 induced c-FLIP reduction occurred at late stages of infection and was dependent on proper expression of early and leaky late virus genes. Furthermore, it was not operative at the RNA level and was independent from the proteasome and binding to the death inducing signaling complex. Rather, c-FLIP was presumably degraded by a viral or cellular protease. In this work it was shown for the first time, that in addition to changes in the cellular apoptosis signaling network, the lack of one antiapoptotic viral factor contributes to apoptosis of HSV-1 infected iDCs. HSV-1 latency-associated transcripts (LATs) were significantly lower expressed in apoptotic iDCs compared to non-apoptotic epithelial cells, determined by microarray analysis of HSV-1 gene expression. It is known that in neurons and epithelial cells, LATs possess a potent antiapoptotic activity. This could compensate the lack of c-FLIP. Consistent with this hypothesis, a LAT deletion mutant of HSV-1 induced more apoptosis in iDCs compared to the respective wild type virus.

Tolerogenic dendritic cells are monocyte-derived dendritic cells (DC) cultured such that they adopt an immunoregulatory phenotype. In vitro, these cells are able to induce and maintain T cell tolerance through deviation of naive T cells to an anti-inflammatory phenotype and induction of anergy in memory T cells. Equivalent cells suppress established arthritis in murine models and tolerogenic DC are presently the subject of a phase I safety and efficacy trial at Newcastle University as part of the AutoDeCRA study. However, in spite of these promising data, we are yet to rigorously explore the basis of the phenotype of tolerogenic DC and lack markers to unequivocally distinguish them from other types of DC. This body of work is concerned with the development of a workflow to enable these questions to be addressed using mass spectrometry-based quantitative proteomics. Specifically, methods have been optimized and validated to enable a) enrichment and proteolytic digestion of membrane proteins, favouring their detection over more abundant cytoplasmic and nuclear proteins in LC/MS; b) differential stable isotope labelling of peptide N- and C-termini, enabling ‘isobaric peptide termini labelling’-based relative quantitation at the MS2 level; c) pipette-tip based anion exchange fractionation of IPTL-labelled peptides prior to LC/MS analysis, broadening depth of proteome coverage. Efforts to apply aspects of the workflow to perform quantitative comparisons of the whole cell proteomes and qualitative profiling of the membrane proteomes of mature and tolerogenic DC are also documented. It is envisaged that future application of this optimized workflow as a whole will enable the identification and relative quantitation of significant numbers of mature and tolerogenic DC plasma membrane proteins. Differentially expressed proteins of interest identified through this approach may then be further investigated for putative roles in tolerance induction.

Orientador: Lair Zambon
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Os resultados terapêuticos globais do carcinoma de pulmão não pequenas células em estádio avançado são bem limitados. A imunoterapia com células dendríticas foi desenvolvida como uma nova estratégia para o tratamento de câncer de pulmão. O objetivo deste estudo foi avaliar a viabilidade, segurança e respostas imunológicas em pacientes com carcinoma de pulmão não pequenas células tratados com vacina autóloga de células dendríticas maduras pulsadas com antígenos. Cinco pacientes HLA-A2 com carcinoma de pulmão não pequenas células inoperável (estádio III ou IV) foram selecionados para receber duas doses de 5 x 107 de células dendríticas administradas por vias subcutânea e intravenosa, duas vezes em intervalos de duas semanas. A segurança, tolerabilidade e respostas imunológica e tumoral à vacina foram avaliadas pela evolução clínica e laboratorial, ensaio de linfoproliferação e critérios de RECIST, respectivamente. A dose utilizada para a imunoterapia demonstrou ser segura e bem tolerada. O ensaio de linfoproliferação mostrou uma melhora na resposta imune específica após a imunização, com uma resposta significativa após a segunda dose (p = 0,001). Esta resposta não foi persistente e houve uma tendência à redução após duas semanas da segunda dose da vacina. Dois pacientes apresentaram uma sobrevida quase duas vezes maior que a média esperada e foram os únicos que expressaram os antígenos tumorais HER-2 e CEA Apesar do pequeno tamanho da amostra, os resultados sobre o tempo de sobrevida, resposta imune, segurança e tolerabilidade, combinado com os resultados de outros estudos, são animadores para a condução de um estudo clínico com doses múltiplas em pacientes com câncer de pulmão que foram submetidos a tratamento cirúrgico, seguindo as diretrizes do Cancer Vaccine Clinical Trial Working Group
Abstract: Overall therapeutic outcomes of advanced non-small-cell lung cancer (NSCLC) are poor. The dendritic cell (DC) immunotherapy has been developed as a new strategy for the treatment of lung cancer. The purpose of this study was to evaluate the feasibility, safety and immunologic responses in use in mature, antigen-pulsed autologous DC vaccine in NSCLC patients. Five HLA-A2 patients with inoperable stage III or IV NSCLC were selected to receive two doses of 5x107 DC cells administered subcutaneous and intravenously two times at two week intervals. The safety, tolerability and immunologic and tumor responses to the vaccine were evaluated by the clinical and laboratorial evolution, lymphoproliferation assay and RECIST's criteria, respectively. The dose of the vaccine has shown to be safe and well tolerated. The lymphoproliferation assay showed an improvement in the specific immune response after the immunization, with a significant response after the second dose (p = 0.001). This response was not long lasting and a tendency to reduction two weeks after the second dose of the vaccine was observed. Two patients had a survival almost twice greater than the expected average and were the only ones that expressed HER-2 and CEA together. Despite the small sample size, the results on the survival time, immune response, and safety and tolerability, combined with the results of other studies, are encouraging to the conduction of a large clinical trial with multiples doses in patients with early lung cancer who underwent surgical treatment, following the guidelines of the Cancer Vaccine Clinical Trial Working Group
Doutorado
Clinica Medica
Doutor em Ciências

碩士
國立陽明大學
醫學生物技術暨檢驗學系
103
H5N1 is one of avian influenza viruses with high mortality rate. From 2003, H5N1 has accounted for more than 440 people death. Previous studies revealed that H5N1-induced cytokine dysregulation may be a main reason of high mortality. Besides, our past study also demonstrated that dendritic cells capture H5N1 to infect other cells. H5N1 infected cells produced considerable amounts of cytokines and even caused severe cell death. Nevertheless, there is no clear answer of how H5N1 causes cytokine dysregulation. Autophagy is a highly conserved behavior in the eukaryotes. Previous studies indicated some viruses such as HCV could manipulate autophagy-related proteins to help viral replication and regulate immune response during infection. Recent studies showed H5N1 could cause autophagic cell death in lung epithelial cells. There are numerous pattern recognition receptors on dendritic cells associated with autophagy process, but few studies discussed the role of C-type lectins in autophagy. Also in our previous study, we have found that H5N1 showed better infectivity in immature dendritic cells, induced cytokine production and caused more severe CPE in DC-SIGN expressing cell. Our present study focuses on the role of autophagy in H5N1 induced cell death, cytokine secretion and viral replication in immature dendritic cell and lung epithelial cells. We found that considerable decrease in H5N1 induced cell death and cytokine secretion in H5N1-infected immature dendritic cells while inhibiting autophagy and the same phenomenon occurred in H5N1-infected lung epithelial cells. However, we demonstrated that autophagy blockade reduced viral replication only in immature dendritic cells but not in lung epithelial cells. Furthermore, we used DC-SIGN expressing cell line and anti-DC-SIGN mAb or downstream molecule inhibitor to investigate whether DC-SIGN is involved in H5N1-mediated autophagy. Our results proved that DC-SIGN had no direct influence on H5N1-induced autophagy.

碩士
國立成功大學
微生物暨免疫學研究所
88
Tumor vaccines derived from genetically modified tumor cells or from tumor antigen-pulsed dendritic cells (DCs) show promise in cancer immunotherapy. DCs can be generated from bone marrow-derived cells and splenocytes in the presence of GM-CSF and IL-4. DCs would mature into functional tumor antigen-presenting cells under the exposure to tumor cells or tumor extracts. In this study, the strategies based on DCs combined with genetically engineered tumor cells expressing GM-CSF and IL-4 were exploited to test the feasibility of generation of mature DCs in vitro for tumor immunotherapy. Murine GM-CSF and/or IL-4 cDNA were each cloned into recombinant retroviral vectors (pRufCD), which combine the expression of a suicide gene, E. coli cytosine deaminase (CD), for drug selection. These retroviral vectors were transfected into MBT-2 (murine bladder tumor cell line) and LL2 (murine lewis lung carcinoma cell line). The GM-CSF and IL-4 genes were integrated into the chromosomal DNA of MBT-2 and LL2 cells as confirmed by PCR. The expressions of these two cytokines were also verified by RT-PCR. Furthermore, the GM-CSF protein was detectable in the supernatants of GM-CSF-transfected tumor cells. When splenocytes from naive mice were cultured with MBT-2 cells transduced with GM-CSF gene and those transduced with IL-4 gene, matured DCs were generated, which were characterized for expression of MHC class II antigen, CD11c and CD86. The matured DCs can induce T-cell maturation as well as generate more matured DCs when cocultured with naive splenocytes. We will exploit this DC-based strategy for tumor immunotherapy.

Solid organ transplantation is an established therapeutic approach in modern medicine to extend and to improve the life of patients in the final stages of organ failure. Transplantation between genetically non-identical individuals leads to the activation of the transplant recipient's immune system. This alloimmune response is a consequence of the recognition of foreign MHC molecules by alloreactive host T cells. To prevent their activation and the subsequently induced activation of further cell subsets (e.g. B cells, cytotoxic T cells, macrophages)immunosuppressive drugs are absolutely necessary in the clinic. However,permanent immunosuppression leads to severe side effects such as nephrotoxicity, diabetes and hyperlipidaemia, and a reduced immunity to infections and malignant diseases. At the moment, there is no real alternative to immunosuppression. The purpose of this study was to analyse the importance of rat dendritic cells with immune inhibitory properties to prevent the immune activation after experimental transplantation. The rat is one of the most important animal models for experimental organ transplantation in a clinic-relevant procedure. In order to modulate the immune response after transplantation in an antigenspecific manner, the strategy should include the alloantigens. These antigens have to be presented by immature dendritic cells in the absence of costimulatory signals in order to turn alloreactive T cells into anergic or regulatory T cells instead of effector T cells. For a certain rat model of allograft rejection,the immunodominant peptide P1 was identified as an important alloantigen which accelerates graft rejection. Such a model offers an attractive and practical approach to analyse the potential of host tolerogeneic dendritic cells pulsed with P1 to suppress the allograft-induced immune response in an antigen-specific manner without the need of chronic immunosuppression. A homogenous population of rat immature dendritic cells was generated from bone marrow precursors cultured with GM-CSF and IL-4 (= IL-4 DCs) or GM65 CSF and IL-10 (= IL-10 DCs). These cells with an identical immature phenotype showed no or a very low surface expression of costimulatory molecules like CD80 and CD86 and a 10-fold reduced expression of MHC class II molecules in comparison to mature splenic DCs. No obvious difference was observed between the phenotype of the IL-4 DCs and the IL-10 DCs. Neither IL-4 DCs nor IL-10 DCs were able to activate naïve T cells or to restimulate antigen-specific T cells. This strong inhibitory effect, mediated within 24 hours, was dependent on the number of immature dendritic cells added to the proliferation assay. Antigen-specific T cells pre-incubated with IL-4 DCs and IL-10 DCs, respectively, were not able to proliferate in the presence of P1-pulsed mature DCs. This anergic state was reversible with the addition of exogenous IL-2. T cells incubated with IL-4 DCs (= IL-4 DC-Ts) were able to inhibit the T cell proliferation in a cell number dependent manner. In contrast, antigen-specific T cells pre-incubated with P1-pulsed IL-10 DCs (= IL-10 DC-Ts)showed no effect on the proliferation assay. This was the unique difference between IL-4 DCs and IL-10 DCs found in the present study. Immature DCs influenced also the immune response after transplantation. Different numbers of P1-loaded immature IL-4 DCs and IL-10 DCs were transferred intravenously into Lewis rats one day before transplantation. The best results were obtained with 30 million P1-pulsed immature DCs which prolonged the survival time to a median of 11.2 ± 1.6 days. In addition, the antigen specificity of this effect was demonstrated with a third-party graft from Brown Norway donors. These findings suggest that an antigen-specific modulation of the immune response is possible using immature dendritic cells loaded with the allogeneic antigens. Even more, the protocols described in the present study show that the immune system can be, at least temporarily, controlled after transplantation without the use of immunosuppressive drugs
Die allogene Organtransplantation, d.h. die Übertragung zwischen genetisch nicht-identischen Individuen der gleichen Spezies, ist bei irreversiblen Organerkrankungen nach wie vor die Therapie der Wahl. Die Transplantatabstoßung ist eine zum Funktionsverlust von Organtransplantaten führende T-Zellvermittelte Immunantwort. Ihre Ursache liegt in der Inkompatibilität von Organtransplantat und Transplantat-Empfänger hinsichtlich der Moleküle des Haupthistokompatibilitätskomplexes,die auch als Alloantigene bezeichnet werden. Zwar lässt sich die Transplantatabstoßung mit immunsuppressiven Medikamenten hemmen, doch vermindern diese die Immunabwehr und begünstigen die Entstehung von Infektionen und Tumorerkrankungen. Für die klinische Transplantation gibt es momentan keine Alternativen zur Immunsuppression. Um das Transplantat ohne Immunsuppression dauerhaft zu schützen, müssen die regulatorischen Komponenten des Immunsystems gezielt gestärkt werden. Das Ziel dieser Arbeit war es deshalb, die inhibierende Wirksamkeit unreifer dendritischer Zellen auf die nach Transplantation einsetzende Alloimmunantwort zu überprüfen. Charakteristisch für die Alloimmunantwort ist die Vielzahl der beteiligten Alloantigene. Doch ist es in den letzten Jahren gelungen, Peptidantigene mit einer nachweisbaren Funktion bei der Transplantatabstoßung (vermittelt über den indirekten Weg der Alloantigenerkennung) zu identifizieren. Für die in dieser Arbeit verwendete experimentelle Spender-Empfänger-Kombination ist die Bedeutung des Alloantigens P1, hierbei handelt es sich um ein aus 19 Aminosäuren bestehendes Peptid, für die Alloimmunantwort bekannt. Autologe unreife dendritische Zellen lassen sich aus Knochenmarkvorläuferzellen mit GM-CSF und IL-4 (diese Zellen werden als IL-4 DCs bezeichnet) bzw. mit GM-CSF und IL-10 (IL-10 DCs) kultivieren. Sowohl für IL-4 DCs als auch IL-10 DCs wurde keine bzw. eine sehr geringe Expression der kostimula67 torischen Moleküle CD80 und CD86 auf ihrer Zelloberfläche nachgewiesen. Die Oberflächenexpression von MHC-Klasse II Molekülen war im Vergleich zu reifen, aus der Milz isolierten dendritischen Zellen, um den Faktor 10 reduziert. In einem nächsten Schritt wurde die Wirkung von IL-4 DCs und IL-10 DCs auf T-Lymphozyten getestet. Sie können weder naive T-Lymphozyten aktivieren noch antigenspezifische T-Lymphozyten restimulieren. Der von diesen Zellen vermittelte suppressive Effekt wurde innerhalb von 24 Stunden wirksam und war eindeutig abhängig von der Zellzahl. Antigenspezifische T-Lymphozyten waren nach ihrer Inkubation mit IL-4 DCs oder IL-10 DCs nicht mehr mit P1- beladenen reifen DCs zu restimulieren. Dieser anergische Zustand ließ sich aber nach Zugabe von IL-2 aufheben. Anergische T-Lymphozyten, die mit IL-4 DCs kokultiviert wurden (= IL-4 DC-Ts), zeigten ihrerseits einen inhibierenden Effekt auf antigenspezifische T-Lymphozyten. Im Gegensatz dazu waren IL-10 DC-Ts hierzu nicht in der Lage. Dies ist der einzige Unterschied zwischen IL-4 DCs und IL-10 DCs, der in dieser Arbeit gefunden wurde. Auch in vivo zeigten IL-4 DCs und IL-10 DCs sowohl eine inhibierende Wirkung auf die lokale T-Zellpopulation als auch einen protektiven Effekt auf die Transplantatfunktion. Diese ließ sich in Abhängigkeit von der Zellzahl um 4 Tage ohne jegliche Unterstützung mit Immunsuppressiva verlängern. Dabei wurden maximal 30 Millionen unreife DCs pro Lewis Ratte eingesetzt, was ca. 10 Millionen Zellen pro 100 g Körpergewicht entspricht. Ihr immunprotektiver Effekt war dabei eindeutig antigenspezifisch. Insgesamt lassen die Ergebnisse den Schluss zu, dass autologe unreife dendritische Zellen, beladen mit Alloantigenen, eine hochattraktive Strategie zur antigenspezifischen Modulation der Alloimmunantwort nach Transplantation darstellen. In weiteren Studien soll die Effizienz dieser Zellen gesteigert werden