1. Epigenetic control of immune cell development, differentiation and function
Although it is widely accepted that epigenetic mechanisms contribute to fixation of immune cell fates, 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, commensal microbiota or chronic inflammation on immune cells’ epigenomes. These epigenetic modifications, particularly if acquired at young age, might have long-lasting and even life-long consequences for the functionality of the immune system. In three subprojects, we are investigating how epigenetic mechanisms contribute to immune cell development, differentiation and function.
a) Epigenetic fixation of Foxp3 expression – get a lesson from the thymus
Regulatory T cells (Tregs) play an important role for the maintenance of self-tolerance and are characterized by the expression of the transcription factor Foxp3, which acts as a lineage-specification factor determining the suppressive function of these immunoregulatory cells. 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 loose their suppressive capacity. Thus, stability of Foxp3 expression is a critical issue for the therapeutic application of Tregs for the suppression of unwanted immune responses, including allotransplant rejections. Therefore, a better understanding of those mechanisms controlling stable Foxp3 expression is needed before adoptive Treg transfer therapies can be 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 TSDR (Treg-specific demethylated region), is selectively demethylated only in Foxp3+ Tregs displaying stable Foxp3 expression. We could recently show that the Foxp3 locus 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. Currently, we are studying in detail which cellular players and molecular mediators contribute to the epigenetic fixation of the Treg fate within the thymus. This knowledge will be used to establish cell culture systems for the generation of stable, alloantigen-specific Foxp3+ Tregs for clinical applications.
This project is supported by the German Research Foundation (CRC738 “Optimization of conventional and innovative transplants”) (http://sfb738.de/project-c7.html)
b) Epigenetic signatures of murine and human T cell subsets
The differentiation of naïve conventional CD4+ T cells (Tconv) into highly specialized T helper cell subsets, such as Th1, Th2, Th17 and TFH cells, is accompanied by a number of epigenetic alterations, including global changes of the DNA methylation pattern, leading to the fixation of the unique effector cell phenotypes of the T helper cell subsets. By performing genome-wide methylome analyses of ex vivo isolated effector/memory T cell subsets, we could recently describe for the first time a Th17-specific epigenetic signature. Currently, we are analyzing corresponding signatures of human T helper cell subsets derived from healthy donors and allergic patients by performing whole-genome Bi‑seq.
In addition, we contributed to the identification of Treg-specific epigenetic signature since also the development of Foxp3+ Tregs, no matter whether they are thymus-derived or peripherally induced, goes hand-in-hand with the acquisition of a unique DNA methylation pattern. We applied the Treg- and Th17-specific epigenetic signatures to the RORgt+Foxp3+ T cell subset and could demonstrate that this cell population has a hybrid phenotype showing characteristics of both Tregs and Th17 cells with unique properties to suppress gastrointestinal inflammations. All experimental techniques as well as bioinformatics pipelines to perform epigenetic analyses are established in the laboratory. Interestingly, the aforementioned unique epigenetic signatures can also be used as biomarkers for the unequivocal and reliable identification and quantification of T helper cell subsets. We contributed to the establishment of an epigenetic assay that is based on the FOXP3 TSDR demethylation, and used this assay to quantify human FOXP3+ Tregs in atopic dermatitis and systemic lupus erythematodes patients in cooperation with colleagues from Hannover Medical School.
c) Microenvironmental factors imprint tolerogenic properties of stromal cells from gut-draining lymph nodes
A part of Foxp3+ regulatory T cells (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 mucosal 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 of the mucosal microenvironment (e.g. acute and chronic infections, chronic inflammation) have on the functional properties of LN stromal cells.
Immortalized LN stromal cells are used to identify the immunomodulatory factors secreted by them on a molecular level. 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 an individual fellowship within the Horizon 2020 Marie Skłodowska-Curie action. (http://ec.europa.eu/research/mariecurieactions/about-msca/actions/if/index_en.htm)
2) Development, differentiation and homeostasis of Foxp3+ Tregs and T helper cell subsets under steady-state and infectious conditions
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 CD4+ naive 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 most importantly by an intimate interplay with the naïve T cell population providing the essential T cell growth factor interleukin‑2. In close cooperation with the department Systems Immunology, we have recently developed the first mathematical model of peripheral Treg homeostasis, incorporating secondary lymphoid organs as separate entities and encompassing the aforementioned factors determining the size of the Treg population. Quantitative data were collected in the lab by monitoring naïve T cell homeostasis and Treg rebound after selective in vivo depletion of Foxp3+ Tregs. Using these data, the mathematicians built the model, which not only predicted the previously unanticipated possibility that Foxp3+ Tregs regulate migration of naïve T cells between spleen and peripheral lymph nodes, but also allowed the precise quantification of the number of pTregs generated in secondary lymphoid organs per day. This newly developed mathematical model will be basis for additional modeling approaches describing T cell-mediated immune responses during acute and chronic infections.
Using the gastrointestinal pathogen Yersinia pseudotuberculosis as an example for an acute infection, we could already demonstrate in close cooperation with the department Molecular Infection Biology that the high Treg-inducing capacity of the gut-draining mesenteric lymph nodes is severely impaired during an ongoing infection. Furthermore, we have collected first evidence that Yersinia can directly modulate differentiation and effector function of T cells in vivo by injecting Yersinia outer proteins (Yops) into the T cells with the help of their type III secretion system. In the future, we will investigate in more detail the cellular players and molecular pathways controlling development, differentiation, homeostasis and functional properties of both Foxp3+ Tregs and T helper cell subsets under infectious conditions (e.g. Yersinia pseudotuberculosis, Influenza A virus infection).
3) Control of immunological synapse formation in regulatory T cells
Regulatory T cells (Tregs) are maintaining self-tolerance and immune homeostasis by actively suppressing various immune cells including conventional T cells (Tconv). Notably, Tregs need to be activated via their T cell receptor (TCR) to exert their immunosuppressive functions. Although some evidence has been accumulated that a Treg-specific spatial organization of signaling components at the immunological synapse (IS) contributes to the unique signal control in Tregs, our knowledge about TCR-downstream signaling mechanisms controlling these 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. At the moment, we are investigating how the dynamic and spatial organization of these specific signaling modules controls IS formation in Tregs.
This project is supported by the German Research Foundation (CRC854 “Molecular organization of cellular communication within the immune system”) (SFB854)
4) Molecular targeting of Foxp3+ Tregs
Accumulating evidence suggests that tumor-bearing individuals and patients suffering from chronic infections harbor increased numbers of Foxp3+ Tregs, which prevent the development of efficient anti-tumor and pathogen-specific immune responses, respectively. Thus, the overall aim of this project is to develop novel therapeutic strategies that 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. This project is based on a selection of promising natural compounds derived from myxobacteria that were recently identified in a screening approach to inhibit Foxp3 expression in ex vivo isolated Tregs and to prevent de novo induction of Foxp3 in TGF‑b-induced Tregs. We will use primary T cells from mice and men to study the effect of the candidate compounds on the Tregs’ suppressive capacity to unravel their mode-of-action and to rule out any undesired effects on differentiation and function of effector T cells. Furthermore, we will investigate the efficacy of the in vivo therapeutic administration of the candidate compounds in pre-clinical disease and vaccination models.
This project is supported by the Wilhelm-Sander Foundation (http://www.sanst.de).
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