No description available.

No description available.

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

Allergic asthma is a chronic inflammatory airway disease that is dominated by Th2 immune responses, with accumulation of eosinophils, IgE and IgG1 production, and airway hyperresponsiveness. We reported previously that treatment of OVA-asthmatic mice with allergen-presenting IL-10-differentiated dendritic cells (DC) (DC10) leads to progressive and long-lasting full-spectrum asthma tolerance. However, little has been done in investigating a role for antigen-specific B cells in DC10-induced tolerance. In this study, we characterized the surface markers of DC10 and found that these cells expressed lower levels of CD40, CD80, MHC II, PD-L1 and PD-L2 relative to immunostimulatory LPS-differentiated DCs (DCLPS). Co-culturing DC10 or DC10-induced regulatory T cells (iTreg) with CD4+ Th2 effector T cells from asthmatic mice led to a marked suppression of DCLPS-induced T effector cell proliferation. Moreover, DC10 treatment of asthma phenotype mice down-regulated airway eosinophilic inflammation as determined 48 h after a recall allergen challenge, and reduced pulmonary parenchymal tissue OVA-specific IgG1-secreting (OVA-IgG1) plasma cell numbers. The number of lung OVA-specific IgG1 plasma cells decreased by 46.7% over a 2 week period in the absence of repeated allergen challenge, while the numbers of bone marrow OVA-specific IgG1 plasma cells stayed relatively stable over a 6 week period, as determined 48 h after a single allergen challenge of asthmatic mice. DC10 treatment had a significant impact on the serum of IgG1/IgE response. To address the question of how DC10 influence OVA-IgG1 plasma cells responses, we co-cultured enzymatically-dispersed lung total cells from asthmatic mice with or without DC10, and found that the DC10 significantly suppressed OVA-IgG1 plasma cell antibody production. To determine whether DC10 required input from T cells to accomplish this, we co-cultured CD4 T cell-depleted, B cell-enriched populations from the lungs of asthmatic mice with or without DC10, and found that DC10 strongly (65.4+/-3.5%) suppressed OVA-IgG1 plasma cells in CD4 T cell-depleted lung cell cultures. To assess whether DC10-induced Treg also suppress IgG1-secretion, we co-cultured lung CD4+ T cells from untreated or DC10-tolerized asthmatic mice with total lung cells from asthmatic donors, and found that the DC10-induced Tregs effectively (52.2+/-8.7%) suppressed OVA-IgG1 plasma cell responses. In summary, DC10 treatment strongly down-regulate OVA-specific IgG1 plasma cell responses of asthmatic mice, both in vivo and in vitro by at least two mechanisms: directly via DC10 as well as indirectly through DC10-induced Tregs.

No description available.

No description available.

Background Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that play an important role in the peripheral tolerance mechanisms of the immune system. Their suppressive function on autoreactive T cells can prevent autoimmunity. In type 1 diabetes (T1D), Treg have been inconsistently reported to be impaired in their capability to suppress autoreactive T cells (Tan, et al., 2014; Zhang, et al., 2012). Treg can be thymus derived (tTreg) or generated from naïve CD4+ CD25- T cells in the periphery (pTreg), which exhibit similar suppressive qualities as tTreg. They have also been reported to be actively induced (iTreg) under tolerogenic conditions (Kleijwegt, et al., 2010; Yuan and Malek, 2012). Although several Treg subpopulations have been described, the archetypical Treg express the major markers CD4, CD25 and FOXP3, while CD127 is heavily downregulated. However, activated conventional T cells (Tconv) show a similar phenotype, at least transiently (Miyara, et al., 2009). Since Treg and Tconv have opposing functions and therapeutic indications, it is important to obtain markers that confidently identify bona fide Treg. Scientific aim The aim of my thesis is to define the heterogeneity of human T cells with a specific emphasis to identify bona fide Treg. I examined heterogeneity of this population in healthy controls and T1D patients, as my model disease, and examined how T cells that are exposed to antigen can be defined as Treg or Tconv. Material and Methods For marker phenotyping I used samples from new onset T1D patients (age 7-11 years), autoantibody positive (Aab+) patients and age-matched healthy controls, which were tested by flow cytometry with an array of Treg-associated markers. Separately, freshly isolated CD4+CD25+CD127lo Treg and CD+CD25- Tconv were used for transcriptomic analysis, which was done by RNAseq on isolated whole RNA. For functional analysis of antigen specific gene expression patterns I developed a multi-dye proliferation assay. Treg (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+/lo) were sorted from isolated peripheral blood mononuclear cells (PBMC). I recombined the sorted and proliferation dye stained subsets with CD4- cells to simulate whole PBMC assays and stimulated them with tetanus-, influenza- or auto-antigens (GAD65, proinsulin). Cells were incubated for 5 days and responding proliferating cells as well as non-responding cells were single cell sorted and analyzed by multiplex qPCR. In investigating therapeutic approaches to expand or generate Treg, I examined in vitro approaches for de novo induction of Tregs with tolerogenic dendritic cells (tDCs). The tDCs were differentiated from monocytes either in the presence of 1α,25-OH(2)Vitamin D3 and/or Dexamethasone and matured with lipopolysaccharide. In a multistep assay, naïve T cells were incubated with DCs for two rounds and functional suppression assays were performed. The resultant T cells were analyzed at the DNA, protein, and functional level. Results Substantial phenotypic heterogeneity of peripheral blood CD4+ T cells was observed and documented for three major populations: resting Tconv (CD25-CD127+/lo), activated Tconv (CD25+CD127+) and Treg (CD25+CD127lo) in healthy controls. Despite this, I observed no differences between the Treg subpopulations from new onset T1D patients, Aab+ patients and healthy controls. In addition, there were no differences in the Treg transcriptome of T1D patients and healthy controls by RNAseq. I was, however, able to identify a small set of differentially expressed genes was discovered in Tconv suggesting a role of neutrophils in the onset of T1D. Heterogeneity of antigen-responsive Tconv and Treg was identified by gene expression profiling. I was able to define Treg specific as well as activation specific profiles, and found different expression profiles if T cells are foreign antigen or autoantigen activated and if the responding cells are Treg or Tconv. Genes that define the specific profiles include FOXP3, CD127, several cytokines, transcription factors and activation markers. The manipulation of naïve CD4+CD25- T cells by tDCs led to an unstable CD25+CD127loFOXP3+ phenotype of the generated cells. However, none of the subsequently performed functional assays could confirm that the resultant cells were iTreg or exhausted activated Tconv. In particular, methylation status of the Treg-specific demethylated region (TSDR) was inconsistent with stable Treg, suggesting that so-called tolerogenic protocols may not lead to a long-lived Treg phenotype. Conclusion CD4+CD25+ T cells are heterogeneous. I defined marker combinations that will help distinguish Treg from ex vivo and in vitro activated Tconv cells. With these tools, I was able to show that healthy controls and patients with type 1 diabetes cannot be distinguished by Treg phenotype. Comprehensive single cell analysis of antigen activated T cells provided the most promising avenue for identifying antigen-specific Treg and opens new possibilities to analyze immune therapeutic approaches, particularly when Treg expansion is the therapeutic objective. The findings will be used for monitoring children participating in antigen-based prevention studies in children at risk for T1D. / Hintergrund Regulatorische T Zellen (Treg) sind eine Subpopulation der CD4+ T Zellen, welche eine wichtige Rolle in den peripheren Toleranzmechanismen des Immunsystems spielen. Ihre suppressive Funktion auf autoreaktive T Zellen kann Autoimmunität verhindern. Verschiedene Studien berichteten widersprüchlich, dass Treg in Typ 1 Diabetes (T1D) in ihrer Fähigkeit beeinträchtigt sind autoreaktive T Zellen zu supprimieren (Tan et al., 2014; Zhang et al., 2012). Treg können im Thymus differenzieren (tTreg) oder aus peripheren naïven CD4+CD25- T Zellen generiert werden (pTreg), welche ähnliche suppressive Eigenschaften wie tTreg besitzen. Es wurde außerdem berichtet, dass Treg aktiv unter tolerisierenden Konditionen induziert werden können (iTreg) (Kleijwegt et al., 2010; Yuan and Malek, 2012). Obwohl verschiedene Treg Subpopulationen beschrieben wurden, exprimieren die archetypischen humanen Treg die Hauptmarker CD4, CD25 und FOXP3 exprimieren, während CD127 herunterreguliert ist. Jedoch zeigen auch aktivierte konventionelle T Zellen (Tconv) diesen Phänotyp (Miyara et al., 2009). Da Treg und Tconv gegensätzliche Funktionen und therapeutische Indikationen aufweisen, ist es wichtig Marker zu erhalten, die sicher bona fide Treg identifizieren. Fragestellung Das Ziel meiner Arbeit ist es, die Heterogenität von humanen T Zellen zu definieren mit einen spezifischen Fokus bona fide Treg zu identifizieren. Dafür untersuchte ich die Heterogenität dieser Zellpopulation in gesunden Individuen und T1D Patienten, als Krankheitsmodell, und wie T Zellen als Treg oder Tconv definiert werden können wenn sie einem Antigen ausgesetzt sind. Material und Methoden Für das Phänotypisieren habe ich Proben von Patienten mit beginnendem T1D (Alter 7-11 Jahre), Autoantikörper positiven Patienten (Aab+) und gesunden Individuen mittels Durchflusszytometrie auf eine Reihe von Treg-assoziierten Markern getestet. Des Weiteren wurden frisch isolierte CD4+CD25+CD127lo Treg und CD+CD25- Tconv für die Transkriptomanalyse (RNAseq) genutzt, welche mit der Gesamt-RNA durchgeführt wurden. Für die funktionelle Analyse von Antigen-spezifischen Genexpressionsmustern habe ich ein Multifarbenproliferationstest entwickelt. Treg (CD4+CD25+CD127lo) und Tconv (CD4+CD25-CD127+/lo) wurden aus isolierten mononukleären Zellen des peripheren Blutes (PBMC) sortiert. Ich habe die sortierten und gefärbten Zellen mit CD4- Zellen zusammengefügt, um einen Gesamt-PBMC-Test zu simulieren und habe die Zellen mit Tetanus-, Influenza- oder Auto-antigen (GAD65, Proinsulin) stimuliert. Die Zellen wurden für 5 Tage inkubiert und die Antigen-reagierenden und -proliferierenden Zellen sowie die nicht-reagierenden Zellen Einzelzell sortiert und mittels Multiplex qPCR analysiert. Um therapeutische Ansätze zum Expandieren oder Generieren von Treg zu untersuchen, habe ich in vitro Ansätze für die de novo Induktion von Treg durch die Nutzung von tolerisierenden dendritischen Zellen (tDCs) untersucht. Die tDCs wurden von Monozyten in Anwesenheit von 1α,25-OH(2)Vitamin D3 und/oder Dexamethason differenziert und mit Lipoploysaccharid maturiert. Naïve T Zellen wurden in einem Mehrschrittverfahren mit DCs inkubiert. Die resultierenden T Zellen wurden auf DNA, Protein und funktioneller Ebene analysiert. Ergebnisse Substantielle phänotypische Heterogenität von peripheren Blut CD4+ T Zellen wurde in drei Hauptpopulationen in gesunden Individuen beobachtet und dokumentiert: ruhende Tconv (CD25-CD127+/lo), aktivierte Tconv (CD25+CD127+) und Treg (CD25+CD127lo). Weiterführend ergab der phänotypische Vergleich von Patienten mit beginnender T1D, Aab+ Patienten und gesunden Individuen keine Unterschiede in den Treg Subpopulationen. Außerdem zeigten sich keine Unterschiede in den durch RNAseq gemessenen Treg Transkriptomen von T1D Patienten und gesunden Individuen. Jedoch wurde ein kleine Gruppe von differentiell exprimierten Genen in Tconv entdeckt, welche eine mögliche Rolle von Neutrophilen in T1D andeuten. Heterogenität von Antigen-spezifischen Tconv und Treg Antworten wurde durch Genexpressionsanalysen identifiziert. Ich konnte Treg- sowie Aktivierungs-spezifische Muster definieren und verschiedene Expressionsprofile finden, wenn T Zellen durch Fremd- oder Autoantigen aktiviert wurden und ob sie die reagierenden Zellen Treg oder Tconv sind. Folgende Gene waren hauptsächlich in die Profilbildung involviert: FOXP3, CD127, mehrere Zytokine, Transkriptionsfaktoren und Aktivierungsmarker. Die Manipulation von naïven CD4+CD25- T Zellen durch tDCs führte zu einem instabilen CD25+CD127loFOXP3+ Phänotyp der generierten Zellen. Jedoch konnte keiner der weiterführenden funktionellen Analysen unterscheiden, ob die resultierenden Zellen iTreg oder aktivierte erschöpfte T Zellen waren. Insbesondere war der Methylierungsstatus der Treg-spezifisch demethylierten Region (TSDR) nicht konsistent mit einen stabilen Treg Phänotyp, was darauf hinweist, dass sogenannte tolerisiernde Protokolle nicht zu einem langlebigen Treg Phänotyp führen. Schlussfolgerungen CD4+CD25+ T Zellen sind heterogen. Ich habe Markerkombinationen definiert die helfen werden Treg von ex vivo und in vitro aktivierten Tconv Zellen zu unterscheiden. Mit diesen Mitteln war ich in der Lage zu zeigen, dass gesunde Individuen und Patienten mit Typ 1 Diabetes nicht anhand ihres Treg Phänotyps unterschieden werden können. Umfassende Einzelzell-Analysen von Antigen aktivierten T Zellen lieferten den vielversprechendsten Ansatz für die Identifizierung von Antigen-spezifischen Treg und eröffnen neue Möglichkeiten um immuntherapeutische Ansätze zu analysieren, insbesondere wenn Treg Expansion das therapeutische Ziel ist. Diese Erkenntnisse werden zukünftig für das Monitoring von Kindern, mit einem hohen T1D Risiko, genutzt die an Antigen-basierten Präventionsstudien teilnehmen.

Background Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that play an important role in the peripheral tolerance mechanisms of the immune system. Their suppressive function on autoreactive T cells can prevent autoimmunity. In type 1 diabetes (T1D), Treg have been inconsistently reported to be impaired in their capability to suppress autoreactive T cells (Tan, et al., 2014; Zhang, et al., 2012). Treg can be thymus derived (tTreg) or generated from naïve CD4+ CD25- T cells in the periphery (pTreg), which exhibit similar suppressive qualities as tTreg. They have also been reported to be actively induced (iTreg) under tolerogenic conditions (Kleijwegt, et al., 2010; Yuan and Malek, 2012). Although several Treg subpopulations have been described, the archetypical Treg express the major markers CD4, CD25 and FOXP3, while CD127 is heavily downregulated. However, activated conventional T cells (Tconv) show a similar phenotype, at least transiently (Miyara, et al., 2009). Since Treg and Tconv have opposing functions and therapeutic indications, it is important to obtain markers that confidently identify bona fide Treg. Scientific aim The aim of my thesis is to define the heterogeneity of human T cells with a specific emphasis to identify bona fide Treg. I examined heterogeneity of this population in healthy controls and T1D patients, as my model disease, and examined how T cells that are exposed to antigen can be defined as Treg or Tconv. Material and Methods For marker phenotyping I used samples from new onset T1D patients (age 7-11 years), autoantibody positive (Aab+) patients and age-matched healthy controls, which were tested by flow cytometry with an array of Treg-associated markers. Separately, freshly isolated CD4+CD25+CD127lo Treg and CD+CD25- Tconv were used for transcriptomic analysis, which was done by RNAseq on isolated whole RNA. For functional analysis of antigen specific gene expression patterns I developed a multi-dye proliferation assay. Treg (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+/lo) were sorted from isolated peripheral blood mononuclear cells (PBMC). I recombined the sorted and proliferation dye stained subsets with CD4- cells to simulate whole PBMC assays and stimulated them with tetanus-, influenza- or auto-antigens (GAD65, proinsulin). Cells were incubated for 5 days and responding proliferating cells as well as non-responding cells were single cell sorted and analyzed by multiplex qPCR. In investigating therapeutic approaches to expand or generate Treg, I examined in vitro approaches for de novo induction of Tregs with tolerogenic dendritic cells (tDCs). The tDCs were differentiated from monocytes either in the presence of 1α,25-OH(2)Vitamin D3 and/or Dexamethasone and matured with lipopolysaccharide. In a multistep assay, naïve T cells were incubated with DCs for two rounds and functional suppression assays were performed. The resultant T cells were analyzed at the DNA, protein, and functional level. Results Substantial phenotypic heterogeneity of peripheral blood CD4+ T cells was observed and documented for three major populations: resting Tconv (CD25-CD127+/lo), activated Tconv (CD25+CD127+) and Treg (CD25+CD127lo) in healthy controls. Despite this, I observed no differences between the Treg subpopulations from new onset T1D patients, Aab+ patients and healthy controls. In addition, there were no differences in the Treg transcriptome of T1D patients and healthy controls by RNAseq. I was, however, able to identify a small set of differentially expressed genes was discovered in Tconv suggesting a role of neutrophils in the onset of T1D. Heterogeneity of antigen-responsive Tconv and Treg was identified by gene expression profiling. I was able to define Treg specific as well as activation specific profiles, and found different expression profiles if T cells are foreign antigen or autoantigen activated and if the responding cells are Treg or Tconv. Genes that define the specific profiles include FOXP3, CD127, several cytokines, transcription factors and activation markers. The manipulation of naïve CD4+CD25- T cells by tDCs led to an unstable CD25+CD127loFOXP3+ phenotype of the generated cells. However, none of the subsequently performed functional assays could confirm that the resultant cells were iTreg or exhausted activated Tconv. In particular, methylation status of the Treg-specific demethylated region (TSDR) was inconsistent with stable Treg, suggesting that so-called tolerogenic protocols may not lead to a long-lived Treg phenotype. Conclusion CD4+CD25+ T cells are heterogeneous. I defined marker combinations that will help distinguish Treg from ex vivo and in vitro activated Tconv cells. With these tools, I was able to show that healthy controls and patients with type 1 diabetes cannot be distinguished by Treg phenotype. Comprehensive single cell analysis of antigen activated T cells provided the most promising avenue for identifying antigen-specific Treg and opens new possibilities to analyze immune therapeutic approaches, particularly when Treg expansion is the therapeutic objective. The findings will be used for monitoring children participating in antigen-based prevention studies in children at risk for T1D. / Hintergrund Regulatorische T Zellen (Treg) sind eine Subpopulation der CD4+ T Zellen, welche eine wichtige Rolle in den peripheren Toleranzmechanismen des Immunsystems spielen. Ihre suppressive Funktion auf autoreaktive T Zellen kann Autoimmunität verhindern. Verschiedene Studien berichteten widersprüchlich, dass Treg in Typ 1 Diabetes (T1D) in ihrer Fähigkeit beeinträchtigt sind autoreaktive T Zellen zu supprimieren (Tan et al., 2014; Zhang et al., 2012). Treg können im Thymus differenzieren (tTreg) oder aus peripheren naïven CD4+CD25- T Zellen generiert werden (pTreg), welche ähnliche suppressive Eigenschaften wie tTreg besitzen. Es wurde außerdem berichtet, dass Treg aktiv unter tolerisierenden Konditionen induziert werden können (iTreg) (Kleijwegt et al., 2010; Yuan and Malek, 2012). Obwohl verschiedene Treg Subpopulationen beschrieben wurden, exprimieren die archetypischen humanen Treg die Hauptmarker CD4, CD25 und FOXP3 exprimieren, während CD127 herunterreguliert ist. Jedoch zeigen auch aktivierte konventionelle T Zellen (Tconv) diesen Phänotyp (Miyara et al., 2009). Da Treg und Tconv gegensätzliche Funktionen und therapeutische Indikationen aufweisen, ist es wichtig Marker zu erhalten, die sicher bona fide Treg identifizieren. Fragestellung Das Ziel meiner Arbeit ist es, die Heterogenität von humanen T Zellen zu definieren mit einen spezifischen Fokus bona fide Treg zu identifizieren. Dafür untersuchte ich die Heterogenität dieser Zellpopulation in gesunden Individuen und T1D Patienten, als Krankheitsmodell, und wie T Zellen als Treg oder Tconv definiert werden können wenn sie einem Antigen ausgesetzt sind. Material und Methoden Für das Phänotypisieren habe ich Proben von Patienten mit beginnendem T1D (Alter 7-11 Jahre), Autoantikörper positiven Patienten (Aab+) und gesunden Individuen mittels Durchflusszytometrie auf eine Reihe von Treg-assoziierten Markern getestet. Des Weiteren wurden frisch isolierte CD4+CD25+CD127lo Treg und CD+CD25- Tconv für die Transkriptomanalyse (RNAseq) genutzt, welche mit der Gesamt-RNA durchgeführt wurden. Für die funktionelle Analyse von Antigen-spezifischen Genexpressionsmustern habe ich ein Multifarbenproliferationstest entwickelt. Treg (CD4+CD25+CD127lo) und Tconv (CD4+CD25-CD127+/lo) wurden aus isolierten mononukleären Zellen des peripheren Blutes (PBMC) sortiert. Ich habe die sortierten und gefärbten Zellen mit CD4- Zellen zusammengefügt, um einen Gesamt-PBMC-Test zu simulieren und habe die Zellen mit Tetanus-, Influenza- oder Auto-antigen (GAD65, Proinsulin) stimuliert. Die Zellen wurden für 5 Tage inkubiert und die Antigen-reagierenden und -proliferierenden Zellen sowie die nicht-reagierenden Zellen Einzelzell sortiert und mittels Multiplex qPCR analysiert. Um therapeutische Ansätze zum Expandieren oder Generieren von Treg zu untersuchen, habe ich in vitro Ansätze für die de novo Induktion von Treg durch die Nutzung von tolerisierenden dendritischen Zellen (tDCs) untersucht. Die tDCs wurden von Monozyten in Anwesenheit von 1α,25-OH(2)Vitamin D3 und/oder Dexamethason differenziert und mit Lipoploysaccharid maturiert. Naïve T Zellen wurden in einem Mehrschrittverfahren mit DCs inkubiert. Die resultierenden T Zellen wurden auf DNA, Protein und funktioneller Ebene analysiert. Ergebnisse Substantielle phänotypische Heterogenität von peripheren Blut CD4+ T Zellen wurde in drei Hauptpopulationen in gesunden Individuen beobachtet und dokumentiert: ruhende Tconv (CD25-CD127+/lo), aktivierte Tconv (CD25+CD127+) und Treg (CD25+CD127lo). Weiterführend ergab der phänotypische Vergleich von Patienten mit beginnender T1D, Aab+ Patienten und gesunden Individuen keine Unterschiede in den Treg Subpopulationen. Außerdem zeigten sich keine Unterschiede in den durch RNAseq gemessenen Treg Transkriptomen von T1D Patienten und gesunden Individuen. Jedoch wurde ein kleine Gruppe von differentiell exprimierten Genen in Tconv entdeckt, welche eine mögliche Rolle von Neutrophilen in T1D andeuten. Heterogenität von Antigen-spezifischen Tconv und Treg Antworten wurde durch Genexpressionsanalysen identifiziert. Ich konnte Treg- sowie Aktivierungs-spezifische Muster definieren und verschiedene Expressionsprofile finden, wenn T Zellen durch Fremd- oder Autoantigen aktiviert wurden und ob sie die reagierenden Zellen Treg oder Tconv sind. Folgende Gene waren hauptsächlich in die Profilbildung involviert: FOXP3, CD127, mehrere Zytokine, Transkriptionsfaktoren und Aktivierungsmarker. Die Manipulation von naïven CD4+CD25- T Zellen durch tDCs führte zu einem instabilen CD25+CD127loFOXP3+ Phänotyp der generierten Zellen. Jedoch konnte keiner der weiterführenden funktionellen Analysen unterscheiden, ob die resultierenden Zellen iTreg oder aktivierte erschöpfte T Zellen waren. Insbesondere war der Methylierungsstatus der Treg-spezifisch demethylierten Region (TSDR) nicht konsistent mit einen stabilen Treg Phänotyp, was darauf hinweist, dass sogenannte tolerisiernde Protokolle nicht zu einem langlebigen Treg Phänotyp führen. Schlussfolgerungen CD4+CD25+ T Zellen sind heterogen. Ich habe Markerkombinationen definiert die helfen werden Treg von ex vivo und in vitro aktivierten Tconv Zellen zu unterscheiden. Mit diesen Mitteln war ich in der Lage zu zeigen, dass gesunde Individuen und Patienten mit Typ 1 Diabetes nicht anhand ihres Treg Phänotyps unterschieden werden können. Umfassende Einzelzell-Analysen von Antigen aktivierten T Zellen lieferten den vielversprechendsten Ansatz für die Identifizierung von Antigen-spezifischen Treg und eröffnen neue Möglichkeiten um immuntherapeutische Ansätze zu analysieren, insbesondere wenn Treg Expansion das therapeutische Ziel ist. Diese Erkenntnisse werden zukünftig für das Monitoring von Kindern, mit einem hohen T1D Risiko, genutzt die an Antigen-basierten Präventionsstudien teilnehmen.

Utilization of tolerogenic dendritic cells (tolDCs) as a cell-based therapy represents a promising strategy in treatment of autoimmune diseases including type 1 diabetes (T1D). Numerous protocols have been established to generate tolDCs ex vivo and their therapeutic effect has been demonstrated in animal models of autoimmune diseases. In this thesis we compared three different variants of such protocols which are based on the combined treatment of bone marrow- derived DCs with vitamin D and dexamethasone applied at different time points of their maturation towards tolDCs. We assessed the efficiency of these protocols in regards of their effect on the expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II and the chemokine receptor CCR7 on the surface of tolDCs. Then, we evaluated the migration pattern of antigen unloaded tolDCs in vivo as well as their effect on the induction of immune responses and cell proliferation of lymph node cells. This was achieved by labelling of tolDCs with membrane dye PKH26 and by following their migration path by flow cytometry after intraperitoneal (i.p) or subcutaneous (s.c.) injection into either left or right side of the body. On day 1, 3, 5, 7, and 9, the presence of PKH26+ tolDCs was examined in spleen, pancreatic, mesenteric, inguinal and axillary...