CD4+ helper T cells come in multiple flavors and are highly specialized to orchestrate immune responses directed against invading pathogens. CD4+ effector T cell subsets, such as Th1, Th2 and Th17 cells, are required to promote inflammatory immune responses, while regulatory T cells (Tregs) antagonize these responses to prevent severe immunopathology. The main goal of the Department Experimental Immunology is to identify cellular players and molecular factors controlling development, differentiation and functional properties of inflammatory and regulatory CD4+ helper T cell subsets with the final aim to discover novel biomarkers and molecular targets to develop therapeutic approaches for the selective modulation of helper T cell-mediated immune responses. Particular emphasis is laid on the elucidation of epigenetic mechanisms contributing to the fixation of T cell fates, and on how these processes are modulated by infections as well as environmental cues such as microbiota and nutrition. Key expertise lies in multi-color flow cytometry, transcriptomic and epigenetic profiling (RNA-seq, scRNA-seq, Bi-seq, ATAC-seq), tailored epigenetic modulation of immune cells, as well as murine infection models.
Impact of infections on thymic Treg development and peripheral homeostasis
Regulatory T cells (Tregs), characterized by the expression of the lineage specification factor Foxp3, are essential for maintaining self-tolerance and modulating inflammatory immune responses. They either develop within the thymus (thymus-derived Tregs, tTregs) or are converted from naive CD4+ T cells in the periphery (peripherally-induced Tregs, pTregs). The size of the Treg population is tightly controlled by numerous factors, namely thymic output, homeostatic proliferation, peripheral conversion, trans-organ migration, apoptosis and an intimate interplay with the naïve T cell population providing the essential T cell growth factor interleukin 2. Using a combined experimental and theoretical approach, we have recently developed the first mathematical model of peripheral Treg homeostasis under steady-state conditions in close cooperation with the Department Systems Immunology (Prof. Michael Meyer-Hermann), incorporating secondary lymphoid organs as separate entities and encompassing the aforementioned factors determining the size of the Treg population. This newly developed mathematical model is currently used as the basis for additional modeling approaches describing T cell-mediated immune responses during acute Influenza A virus (IAV) infection. We aim to dissect the influence of viral infections on both thymic development and peripheral homeostasis of Foxp3+ Tregs in adult individuals. We will utilize results from the mathematical modeling to determine experimentally inaccessible avenues to precisely quantify and predict durable consequences of viral infections on Tregs.
This project is supported by a DAAD fellowship.
The neonatal period is particularly important to establish a functional adaptive immune system. During this crucial time window after birth a first wave of Tregs egresses from the thymus that is required to durably establish self-tolerance and prevent the development of autoimmune diseases in adulthood. Using Treg-specific fate-mapping approaches we are studying the impact of neonatal infections on the functional properties of neonatally developing Tregs. We aim to unravel how infections early after birth can permanently shape the adaptive immune system by modulating a key component of cellular immune regulation.
Control of immunological synapse formation in Tregs
Tregs have to be stimulated in order to exert their immunosuppressive function. Generally, T cell activation takes place at the immunological synapse (IS), the contact zone between T cells and antigen-presenting cells. Our knowledge regarding T cell receptor (TCR)-downstream signaling mechanisms controlling Treg-specific functions is only fragmentary. Recently, we performed comparative proteome, phosphoproteome and toponome analyses using ex vivo isolated Tregs and Tconv and could identify a number of Treg-specific TCR-proximal signaling modules encompassing Treg-specific phosphorylation patterns. Currently, we are investigating how the dynamic and spatial organization of these specific signaling modules enables the formation of a Treg-specific IS and allows the Tregs to exert their unique function. This knowledge will pave the way for specific molecular targeting of Tregs to modulate the immunosuppressive function of this clinically relevant cell type.
This project is supported by the German Research Foundation (CRC854 “Molecular organisation of cellular communication within the immune system”).
Helper T cell differentiation: modulation and more
Accumulating evidence suggests that Foxp3+ Tregs prevent the development of efficient immune responses. Thus, we aim to develop novel therapeutic strategies to selectively modulate the suppressive activity of Foxp3+ Tregs in order to unleash preexisting anti-tumor or pathogen-specific immunity or to enhance the efficacy of antigen-specific vaccinations. We utilise a selection of promising natural compounds derived from myxobacteria that were recently identified in a screening approach to inhibit Foxp3 expression and induction. We are using primary T cells from mice and men to study the effect of these candidate compounds on the Tregs’ suppressive capacity to unravel their mode-of-action. Furthermore, we are investigating the efficacy of the therapeutic in vivo administration of these candidate compounds in pre-clinical disease and vaccination models.
This project received support from the Wilhelm Sander Foundation.
Epigenetic mechanisms contribute to the fixation of immune cell fates. However, molecular details are largely unknown. A better understanding of the events leading to engraved gene expression profiles will enable us to generate tailored immune cell subsets with epigenetically fixed functional properties for therapeutic purposes. In addition, only fragmentary knowledge has been accumulated about the impact of infections and environmental cues such as diet, microbiota or chronic inflammation on the epigenomes of immune cells. These epigenetic modifications, particularly if acquired at young age, might have durable and even life-long consequences for the functionality of the immune system. In four subprojects, we are investigating how epigenetic mechanisms contribute to immune cell development, differentiation and function.
Generation of stable Tregs: Get a lesson from the thymus
Tregs play an important role for the maintenance of self-tolerance. It has been demonstrated that Tregs require continuous expression of Foxp3 to ensure long-term stability of suppressive activity. Cells showing only instable Foxp3 expression rapidly lose their suppressive capacity. Thus, stability of Foxp3 expression is a critical issue for the therapeutic immune-suppressive application of Tregs. Therefore, a better understanding of the mechanisms controlling stable Foxp3 expression is essential, before adoptive Treg transfer therapies can be successfully brought into the clinics.
We have previously demonstrated that stable Foxp3 expression is under epigenetic control, and that a CpG-rich evolutionarily conserved element within the Foxp3 locus, the Treg specific demethylated region (TSDR), is selectively demethylated only in Foxp3+ Tregs displaying stable Foxp3 expression. We could recently show that the TSDR becomes progressively demethylated during maturation of thymic Tregs via an active mechanism that involves enzymes of the ten-eleven-translocation (TET) family causing hydroxylation of methylated cytosines.
Recent studies revealed that vitamin C enhances the enzymatic activity of TET enzymes. We could demonstrate that alloantigen-specific Foxp3+ Tregs (allo iTregs) in vitro generated in presence of vitamin C display an enhanced TSDR demethylation accompanied by increased stability of Foxp3 expression and superior suppressive capacity in vivo. Currently, we are focusing on which cellular players and molecular mediators contribute to the epigenetic fixation of the Treg fate. We are particularly focusing on the influence of the thymic microenvironment on the induction of ‘demethylating’ features mediated by thymic antigen-presenting cells. The gained knowledge will be used for the establishment of novel protocols for the generation of stable, Foxp3+ allo iTregs for clinical applications.
This project received support from the German Research Foundation (CRC738 “Optimization of conventional and innovative transplants”).
Epigenetic profiling of helper T cells
The differentiation of naïve conventional CD4+ T cells (Tconv) into highly specialized helper T cell subsets is accompanied by vast epigenetic modifications, including global changes of the DNA methylation pattern. Although it is known that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, global analyses of the changes in the DNA methylation pattern during T cell differentiation are still fragmentary. We have recently described the first Th17-specific epigenetic signature in the murine system, and currently we are identifying and functionally characterizing uniquely differentially methylated regions in human helper T cell subsets. Our central goal is to understand the molecular pathways controlling the activity and reprogramming of differentiated T cell subsets and how functional properties are maintained within the epigenome. Furthermore, the identified unique epigenetic signatures can be used as novel biomarkers for the unequivocal, reliable identification and quantification of helper T cell subsets in clinical settings.
Maintenance of T cell homeostasis requires the establishment of specific subsets of Tregs harbouring distinct functional and tissue-specific tropisms. Particularly for intestinal immune responses unique Treg subsets have been identified, with RORgt+ Treg majorly contributing to intestinal tolerance. Using Treg- and Th17-specific epigenetic signatures, we demonstrated that the RORgt+Foxp3+ T cell subset has a hybrid phenotype showing characteristics of both Tregs and Th17 cells with unique properties to suppress gastrointestinal inflammations.
To gain a deeper understanding of the development of effector T cell responses we also implemented mathematical modeling approaches, based on extensive studies on the influence of key T cell differentiation cytokines that define lineage commitment. Based on the in silico results of in depth profiling of cytokine-dependent effector T cell lineage commitment, we aim to better understand how neuroinflammatory diseases shape T cell differentiation and in turn regulate the innate arm of immunity, namely microglia, driving pathogenicity.
This project is supported by the Helmholtz Association (Personalized Medicine Initiative “iMed” and Future Theme “Immunology&Inflammation”) and by the European Union’s Horizon 2020 research and innovation program (ENLIGHT-TEN Innovative Training Network under the Marie Skłodowska-Curie grant agreement No.: 675395), and received funding from the German Research Foundation (CRU250 “Genetic and cellular mechanisms of autoimmune diseases”).
Identification and functional characterisation of epigenetic signature genes in innate lymphoid cell lineages
In CD4+ helper T cells, specific demethylated regions have been identified that are critical for the identity, function and lineage stability of the different subsets. Considering the striking similarities between T cell lineages and innate lymphoid cell (ILC) populations regarding the dependency on specific transcription factors and expression of signature cytokines, we are dissecting how ILC populations can be functionally characterised by unique modifications in their genomic DNA methylation pattern. Using a genome-wide sequencing approach, we dissect methylomes of murine ILC subtypes as well as classical NK cells. The identified differentially methylated regions are agglomerated to unique epigenetic signatures that will allow characterising ILCs under several specific inflammatory conditions in vivo. In addition, we are also translating our findings into the human system by inspecting the methylation status of homologous regions in ILCs derived from human tissues and align these to detailed expression analysis. Together, the identification and functional characterisation of differentially methylated regions in ILC subsets will be an important step for the discovery of key pathways and molecular mechanisms in ILC differentiation and function.
This project is supported by the German Research Foundation (SPP1937 “Priority Program Innate Lymphoid Cells”).
The interest in personalized adoptive T-cell therapy has increased in the recent past, since it was found to be an effective and safe treatment option for immunocompromised patients suffering from infectious complications of persistent viruses like cytomegalovirus (CMV) or virus-induced malignancies after stem cell and solid organ transplantation. Adoptive T-cell therapies enhance the long-term protection of patient’s immunity after engraftment and immune reconstitution without increasing the risk of graft-versus-host disease. Despite this success, reliable biomarkers predicting the therapeutic outcome are lacking, the adoptively transferred T cells are still only poorly defined and the efficacy of the antiviral T-cell products can be further improved.
We – together with colleagues from Hannover Medical School – aim to advance adoptive T-cell therapy beyond current state-of-the-art as a personalized and GMP-compliant T-cell immunotherapy using epigenome-profiling and -editing. Thereto, we will epigenetically profile CMV-specific human CD8+ T cells by whole-genome ATAC-seq and Bi-seq to analyze the chromatin accessibility and DNA methylation pattern, respectively. These data will enable us to identify novel biomarkers to predict the cytotoxic activity and functional properties of the antiviral T-cell products that can be used for an optimized antiviral T-cell donor selection. In addition, the whole-genome epigenetic signatures will be the basis for a selection of candidate regions for tailored, CRISPR-Cas9-mediated epigenome-editing using a highly versatile and flexible toolbox of novel vectors and recombinant proteins to improve the efficacy of personalized CMV-specific T-cell immunotherapy for immunocompromised patients.
This project is supported by the Helmholtz Association (Future Theme “Immunology&Inflammation”) and the LifeScience Foundation.
A fraction of Foxp3+ Tregs, named peripherally-induced Tregs (pTregs), are very efficiently de novo generated within gut-draining lymph nodes (LNs), and are known to play a key role for the maintenance of intestinal tolerance. On our search for factors influencing the high Treg-inducing capacity of gut-draining LNs, we could recently identify the non-hematopoietic stromal cells as key players since gut-draining LNs transplanted under the skin still induced high frequencies of Foxp3+ Tregs. Interestingly, these properties depended on environmental factors as mesenteric LNs (mLN) of germ-free mice (lacking any microbiota) and celiac LNs (celLN) from vitamin A-deficient mice did not retain their high Treg-inducing capacities. Currently, we are studying if the tolerogenic properties of the LN stromal cells are epigenetically imprinted, which bacterial species, molecular mediators and signaling pathways are involved and which influence perturbations early during development and adult life (e.g. acute and chronic infections, chronic inflammation) have on the functional properties of LN stromal cells. Furthermore, we are dissecting the underlying molecular regulatory networks that allow LN stromal cells to retain and exert their tolerogenic functions.
To circumvent the scarcity of LN stromal cells, we have generated LN stromal cell lines in close cooperation with the Research Group Model Systems for Infection and Immunity (Prof. Dagmar Wirth). We have utilized these cell lines to confirm immunomodulatory factors identified from ex vivo experiments in greater depth. This detailed understanding of the processes by which LN stromal cells influence the generation of Foxp3+ Tregs will be highly instrumental for the development of strategies to selectively manipulate pTreg generation.
This project is supported by the German Research Foundation (SPP1656 “Priority Program Intestinal Microbiota – a Microbial Ecosystem at the Edge between Immune Homeostasis and Inflammation”), by the Helmholtz Association (Future Theme "AMPro") and the China Scholarship Council, and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 656319.
Pathogens modulate T cell differentiation
Using the gastrointestinal pathogen Yersinia pseudotuberculosis as an example for an acute intestinal infection, we could already demonstrate in close cooperation with the Department Molecular Infection Biology (Prof. Petra Dersch) that the high Treg-inducing capacity of the gut-draining mLNs is severely impaired during an ongoing infection. Furthermore, we could show that Yersinia can directly modulate differentiation and effector function of T cells in vivo by injecting Yersinia outer proteins (Yops) into T cells via the type III secretion system.
Currently, we are expanding our insights into pathogen-mediated modulation of T cell differentiation and focus on soluble bacteria-derived mediators from Staphylococcus aureus, a bacterium colonizing one third of the human population, mostly without causing diseases. Particularly, in patients undergoing surgery or afflicted with immune suppression, Staphylococcus aureus causes severe pathologies ranging from wound infection to endocarditis or sepsis. Staphylococcus aureus secretes a variety of toxins and enzymes, some of which play an important role during pathogenesis and can modulate innate and adaptive immune responses. We are studying the host response to several Staphylococcus aureus toxins both in vitro and in vivo and focus on their impact on T cell differentiation.
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