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Toleranz gegenüber Selbstantigenen in den peripheren Geweben kann durch CD4+ CD25+ Foxp3+ regulatorische T-Zellen (Tregs) vermittelt werden. Diese Zellen entstehen entweder in Folge der thymischen T-Zellselektion (natürlich vorkommende Tregs, nTreg) oder durch Konversion aus naiven T-Zellen in den peripheren lymphatischen Organen (induzierte Tregs, iTregs). Im Vorfeld der Arbeit war bereits bekannt, dass Dendritische Zellen (DZ) eine wichtige Rolle bei der Generierung von iTreg spielen. Allerdings bestand weitestgehend Unklarheit darüber, welche DZ in welchem Reifungszustand dazu in der Lage sind, iTregs gegen peripher-exprimierte Selbstantigene zu induzieren. Steady-state migratorische DZ (ssmDZ) gelten in dieser Hinsicht als potentielle Kandidaten, da bekannt ist, dass diese DZ bereits unter homöostatischen Bedingungen Selbstantigene aus peripheren Geweben in die drainierenden Lymphknoten transportieren und dort T-Zellen präsentieren können. Ein Ziel der vorliegenden Arbeit war daher, den Phänotyp und die tolerogene Kapazität der ssmDZ in den hautdrainierenden Lymphknoten näher zu untersuchen. Es konnte gezeigt werden, dass ssmDZ einen semireifen MHC IIint CD40hi CD80/CD86int CCR7+ Phänotyp aufweisen und in vitro mit Hilfe von endogenem TGF-β iTregs induzieren können. Darüber hinaus belegt diese Arbeit zusammen mit weiteren Daten aus unserer Arbeitsgruppe, dass ssmDZ in transgenen K5mOVA-Mäusen zellassoziertes epidermales OVA aus der Haut in die drainierenden Lymphknoten transportieren und dort an CD4+ OVA-spezifische TZR-transgene OT-II T-Zellen präsentieren können. Innerhalb der ssmDZ konnten die Langerin+ dermalen DZ als die DZ-Subpopulation eingegrenzt werden, die für die Konversion von naiven OT-II T-Zellen in CD4+ CD25+ Foxp3+ iTregs verantwortlich war. Ferner zeigte sich, dass CD103 nicht als Marker für ssmDZ in den hautdrainierenden Lymphknoten herangezogen werden kann. Ein weiteres Ziel dieser Arbeit war, herauszufinden, welchen Einfluss der Transkriptionsfaktor RelB auf die partielle Reifung und Migration der ssmDZ hat. RelB ist ein Mitglied der NF-κB-Familie und wird mit der Reifung von DZ in Verbindung gebracht. Erste Experimente zeigten eine nukleäre Translokation von RelB in ssmDZ sowie eine verringerte Frequenz dieser DZ in den hautdrainierenden Lymphknoten von relB+/- Mäusen und Mäusen mit einer Defizienz für den RelB-Bindungspartner p52. Allerdings konnte bei Mäusen mit einer DZ-spezifischen RelB-Inaktivierung (RelBDCko Mäuse) eine erhöhte Frequenz an ssmDZ in den hautdrainierenden Lymphknoten festgestellt, die nicht auf einer Zunahme an DZ in der Haut der Tiere zurückzuführen war. Diese Ergebnisse legen einerseits die Vermutung nahe, dass es sich bei den beobachteten Effekte in den relB+/- Mäusen um DZ-extrinsische Auswirkungen auf die ssmDZ handelt. Andererseits scheint RelB unter homöostatischen Bedingungen die Erhaltung und Migration der ssmDZ eher negativ zu beeinflussen. Weitere durchflusszytometrische Analysen wiesen zudem darauf hin, dass RelB in ssmDZ die Expression von Reifungsmarkern nur partiell reguliert. So konnte auf den ssmDZ in den hautdrainierenden Lymphknoten von RelBDCko Mäusen eine erhöhte Expression von CD40 beobachtet werden, während andere Reifungsmarker wie MHC II, CD80 und CD86 nicht signifikant in ihrer Expression betroffen waren. Im Rahmen dieser Arbeit wurde zudem untersucht, wie sich eine RelB-Defizienz in DZ auf die Homöostase und Induktion von Tregs auswirkt. Die hierzu analysierten RelBDCko Mäuse wiesen eine erhöhte Frequenz und absolute Zellzahl an Tregs in allen untersuchten lymphatischen Organen (hautdrainierende Lymphknoten, Milz und Thymus) auf. Darüber hinaus war in diesen Organen auch eine verstärkte Proliferation der Tregs gegenüber den Kontrolltieren festzustellen. Weitere Untersuchungen zeigten, dass die Proliferation der Tregs in RelBDCko Mäusen in den hautdrainierenden Lymphknoten sogar stärker ausfiel als in der Milz. RelB scheint somit die tolerogene Kapazität der DZ zur Regulation der Treg-Expansion im Thymus und in der Peripherie zu beeinflussen. Unter Verwendung von neutralisierenden αIL-2-Antikörpern konnte zudem belegt werden, dass die periphere Proliferation der Tregs in den RelBDCko Mäusen von IL-2 abhängig ist. Damit einhergehend zeigten erste Vorversuche eine erhöhte IL-2-Produktion in den peripheren lymphatischen Organen von RelBDCko Mäusen. Zusammenfassend legen die Daten dieser Arbeit den Schluss nahe, dass ssmDZ in den hautdrainierenden Lymphknoten in der Lage sind, Toleranz durch Induktion von iTregs gegen epidermale Selbstantigene zu induzieren. Untersuchungen an neuartigen Mäusen mit einer konditionalen RelB-Inaktivierung spezifisch in DZ deuten darauf hin, dass die Migration und Reifung von ssmDZ partiell durch RelB reguliert wird. Da Tregs eine Schlüsselrolle bei der Erhaltung der peripheren Toleranz einnehmen, ist die Beobachtung, dass eine RelB-Defizienz in allen DZ zu einer verstärkten Treg-Proliferation und somit zu einer veränderten Treg-Homöostase führt, ein intererssanter Ausgangspunkt für weitere Untersuchungen.
Effective T cell immunity was believed to occur by mature DC, whereas tolerogenicity was attributed strictly to immature DC phenotypes. However, intermediate DC maturation stages were identified conditioned by inflammatory mediators like TNF. Furthermore, the T cell tolerance mechanisms are dependent on distinct modes and intensities of co-stimulation. Therefore, in this study it was addressed how distinct DC maturation signatures instruct CD4+ T cell tolerance mechanisms. DC acquire antigens from apoptotic cells for self-peptide-MHC presentation and functionally adapt presumed tolerogenic DC phenotypes. Here, immature murine bone-marrow derived DC representing both inflammatory and conventional DC subsets adapted a maturationresistant DC signature upon apoptotic cell recognition but no additional tolerogenic features. Immature DC instruct CD4+ FoxP3+ regulatory T cells in a TGF-β prone micro-environment or generate anergic CD4+ T cells hampered in the TCR-induced proliferation and IL-2 secretion. Secondary stimulation of such anergic CD4+ T cells by immature DC increased primarily IL-10 production and conferred regulatory function. These IL-10+ regulatory T cells expressed high levels of CTLA-4, which is potently induced by immature DC in particular. Data in this work showed that anergic T cells can be re-programmed to become IL-10+ regulatory T cells upon ligation of CTLA-4 and CD28 signalling cascades by B7 costimulatory ligands on immature DC. In contrast, semi-mature DC phenotypes conditioned by the inflammatory mediator TNF prevented autoimmune disorders by induction of IL-10+ Th2 responses as demonstrated previously. Here, it was shown that TNF as an endogenous maturation stimulus and pathogenic Trypanosoma brucei variant-specific surface glycoproteins (VSG) induced highly similar DC gene expression signatures which instructed default effector Th2 responses. Repetitive administration of the differentially conditioned semi-mature DC effectively skewed T cell immunity to IL-10+ Th2 cells, mediating immune deviation and suppression. Collectively, the data presented in this work provide novel insights how immature and partially mature DC phenotypes generate T cell tolerance mechanisms in vitro, which has important implications for the design of effective DC-targeted vaccines. Unravelling the DC maturation signatures is central to the long-standing quest to break tolerance mimicked by malignant tumours or re-establish immune homeostasis in allergic or autoimmune disorders.
Characterization of tolerogenic rat bone marrow-derived dendritic cells and regulatory T cells
(2010)
Tolerogenic dendritic cells (DC) and regulatory T (Treg) cells are able to prevent destructive immune responses. There is reason to hope that it may soon be possible to use DC and Treg cells to suppress immune responses antigen-specific, not only after transplantation, but also in the case of autoimmunity and allergy. At the moment, the generation of such cell types is very time-consuming and not suitable for clinical routine. In addition, it is not yet fully understood how these cells elicit a desired protective immune response in vivo and how the risks of an excessive immune suppression can be managed. The rat is one of the most important animal models in biomedical research. It is therefore surprising that tolerogenic DC and Treg cells in particular have not been more thoroughly investigated in this model. Thus, the aim of the present study was to systematically characterize these immune cells and investigate their impact on the immune system. Tolerogenic DC were generated from bone marrow precursors cultured with GM-CSF and IL-4 (= IL-4 DC). The proportion of naturally occurring Treg cells with a CD4posCD25posFoxp3pos phenotype comprises approximately 5-8% of the peripheral CD4pos T cells. The characterization of IL-4 DC revealed an up to 26-fold reduced expression of surface molecules such as MHC class II molecules, CD80, CD86, ICAM-1 and CD25 in comparison to mature splenic DC (S-DC). This low expression did not change when the cells where stimulated with different maturation-inducing signals such as replating, LPS, TNF- α and CD40L. Thus, these cells possess a robust phenotype resistant to maturation-inducing stimuli. IL-4 DC take up antigen via endocytosis and are not able to activate naïve T cells or to restimulate antigen-specific T cells. Furthermore, they are able to inhibit and prolongate mature S-DC induced T cell proliferation as well as mature S-DC induced restimulation of antigen-specific T cells, respectively. Thereby, the T cell proliferation was reduced up to 95%. This strong inhibitory effect was mediated within 24 hours in association with a reduced cytokine production (IL-2 about 49% and IFN-γ about 92%). The inhibitory properties of IL-4 DC don´t seem to be caused exclusively by the reduced expression of co-stimulatory molecules. In this study, the detection of the inhibitory molecules PD-L1 and PD-L2 on IL-4 DC suggests they have an impact on mediating inhibitory signals to the T cells. In addition, a suppressive effect of soluble factors was shown. The supernatant of one million IL-4 DC, collected after a 24 hour culture, suppressed mature S-DC induced proliferation of naïve T cells by about 90%. TGF-β, which was detected in the supernatant (up to 300 pg/ml), appears to be the causing soluble factor for this immune inhibition. By contrast, the supernatants of mature S-DC, which did not inhibit the activation of T cells, showed a TGF-β concentration of only about 100 pg/ml. The cytotoxic nitric oxide does not contribute to the IL-4 DC-mediated inhibition of T cell proliferation. The NO synthase inhibitor NMMA reduced the amount of NO by about 50%, but the decreased NO levels did not influence T cell proliferation. Indeed, IL-4 DC are not able to induce T cell proliferation, but this doesn´t mean that there is no change on the molecular level. For instance, T cells co-cultured with IL-4 DC during a first culture are not able to proliferate in the presence of mature S-DC during a second culture. This anergic-like state, however, could be abolished by adding exogenous IL-2. In addition, T cells co-cultured with IL-4 DC are able to inhibit the activation of naïve T cells. Naïve and activated T cells were not able to inhibit the mature S-DC induced T cell proliferation. This observation suggests the induction of Treg cells and was investigated in more detail. Indeed, flow cytometric analysis showed a 1.6-fold expansion of CD4posCD25posFoxp3pos T cells from naturally occurring Treg cells in the presence of IL-4 DC. Thereby, the expansion of CD4posCD25posFoxp3pos T cells occurs independently of the maturation state of DC. Both immature IL-4 DC as well as mature S-DC were able to expand the percentage of naturally occurring Treg cells. However, Treg cells pre-incubated with mature S-DC demonstrated a diminished inhibitory effect compared to Treg cells pre-incubated with IL-4 DC. Treg cells pre-incubated with IL-4 DC were able to inhibit the activation of naïve T cells. In this study it was shown that the regulatory potential of DC cannot be deduced solely by their phenotype or maturation state. Other factors, such as functional properties, need to taken into consideration, too. The induction of Treg cells with suppressive properties induced by in vitro generated tolerogenic IL-4 DC might provide an important mechanism for the maintenance of peripheral tolerance. However, for clinical application further investigation is necessary, not only to understand the interactions between tolerogenic DC and Treg cells, but also to investigate the impact of the transfer of a larger quantity of regulatory cells on the immune system of the recipient.