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ECRT Projekte

Das ECRT will insbesondere den wissenschaftlichen Nachwuchs beim Aufbau eigener Forschungsvorhaben unterstützen. Dafür bietet das ECRT Einstein Kickboxen mit einer Startfinanzierung für erste Experimente zur Validierung der Forschungsideen sowie ECRT Research oder Consumable Grants zur Weiterentwicklung der Forschungsvorhaben.

Die Zusammenfassungen der Projekte stehen nur auf Englsich zur Verfügung.

Sie befinden sich hier:

Laufende ECRT geförderte Projekte

Einstein Kickbox - Förderperiode 2021

Homeostatic proliferation of naïve CD4+ T cells, induces epigenetic changes, which contributes to immune aging

Aging is associated with a functional decline of the immune system, which is linked to the inferior immune response of elderly people to infections and vaccinations. Naïve T cells (TN) harbor the greatest T cell receptor diversity and hence, are important for reactivity against new infections and vaccines. In humans however, the generation of new TN clones ceases as early as puberty. To maintain a high number of CD4+ TN, the cells must undergo activation-independent proliferation, known as homeostatic proliferation. CD4+ TN which lack the surface marker CD31 (CD31-), are enriched for cells which have undergone several rounds of homeostatic proliferation compared to CD31 expressing TN (CD31+). Indeed, the number of CD31+ CD4+ TN decreases with age as well as the telomere length in TN, demonstrating that cells from older individuals experienced a higher number of homeostatic proliferation events. In our preliminary data we could show that already in individuals <= 30 years, CD31+ and CD31- CD4+ TN show significant epigenetic differences on the level of DNA methylation.
With the support of the Einstein funding we will further characterize potential differences in transcriptional regulation in CD31+ and CD31- TN, which we hypothesize to increase with accumulation of homeostatic proliferation events.

Team: Dania Hamo, Andreas Magg, Mai Phan

Funding: Einstein Kickbox - Young Scientists

Impact of myeloid derived suppressor cells on bone metabolism in periprosthetic joint infection

Periprosthetic joint infection (PJI) is a severe complication occurring in approximately 1-5% of all patients after total joint replacement. Despite successful revision surgery, risk of aseptic loosening is significantly elevated due to the impaired capability of bone regeneration. Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive cells that are activated during inflammation. In PJI, MDSCs were reported to represent a large proportion of all leukocytes. Recently, their ability to differentiate into osteoclasts has been observed. Thus, we speculate MDSCs to be a viable target to prevent osteolysis and promote bone regeneration in patients suffering from PJI.

Programmed cell death 1 (PD-1) receptor, an immune regulatory receptor currently targeted in various cancer treatments, has been suggested to be linked to osteoclast differentiation. Targeting PD-1 may be beneficial for bone regeneration and an effective and safe novel therapeutic approach in patients with PJI to improve implant survival.

In our project, we aim to investigate the role of PD-1 in septic activation of MDSCs in patients with PJI. Our results may improve the understanding of the pathological mechanisms in PJI and provide a novel therapeutic approach to improve implant survival.

Team: Yi Ren, Denise Jahn, Arne Kienzle, Weijie Du

Funding: Einstein Kickbox - Young Scientists

Investigation of flow-dependent BMP9 binding and development of an intramolecular FRET sensor for the study of vascular ALK1/ALK5 signalling

Endothelial cells (ECs) are exposed to fluid-sheer stress (FSS) originating from blood flow, which is translated and crosstalks with a variety of signal pathways, i.e. the BMP pathway. FSS-dependent modulation of the BMP signalling pathway is highly cell type, concentration and force dependent. The key quiescence factors BMP9/10 show strong potency in the vasculature, due to the high abundance of their high-affinity receptor ALK1 on ECs. Mutations in ALK1 or in its co-receptor Endoglin alter the vascular BMP signalling response and are associated with vascular pathologies e.g. Hereditary Haemorrhagic Telangiectasia (HHT).

The visualization of BMP receptors bears difficulties due to the lack of specific antibodies. We therefore developed a Halo- and SNAP-labelled BMP receptor library, which in combination with fluorescently labelled BMP9-SiR-d12 ligand allowed for visualisation of ligand-bound receptor complexes.

Using a microfluidic flow set-up, we now aim to visualize BMP9/10 binding to ECs under fluid flow. In a later stage of the project this knowledge will be transferred to in vivo experiments. Ventricular injections of labelled BMP9/10‑SiR-d12 into developing zebrafish embryos carrying an endothelial specific reporter will allow for visualization of vascular bed-specific BMP9/10-SiR-d12 binding towards ECs.

We further want to assess dependence of ligands, co-receptors or FSS on BMP9/10 signalling in rare-signalling receptor complexes and therefore intend to develop a cell-impermeable dual-dye FRET sensor.

Team: Wiktor Burdzinski, Olya Oppenheim, Jerome Jatzlau

Funding: Einstein Kickbox - Young Scientists

Fat Signals – How Adipose Tissue-Derived Factors Affect Epithelial Regeneration

Regeneration of the intestinal epithelium and its modulation by signalling molecules of other cell types plays a critical role in many diseases. An often neglected and to date scarcely investigated cell type in this context are adipocytes. Besides storing lipids, adipose tissue produces various humoral factors, so called adipokines, that are known to critically affect immune function. We want to investigate the effects of adipokines on epithelial regeneration using primary epithelial organoids. With this in vitro system, stem cells of the intestinal epithelium are grown into 3-dimensional self-organizing mini-organs that model not only basic tissue architecture but also regeneration after injury. Modifications of the culture medium and basement membrane matrix further allow for controlling cell identity and signalling activity. Following induction of a regenerative phenotype through specific culture conditions, the cells are treated with different adipokines to analyse their effects on proliferation, functionality, gene expression, protein localization, and barrier integrity. Besides the basic 3D culture model, we are using additional sophisticated in vitro systems, such as transwell filter systems or custom-made flow chambers, to investigate functional aspects and behaviour under flow. Analysis methods include real-time quantitative PCR, immunofluorescence microscopy, metabolic analysis via Seahorse, and transepithelial electrical resistance measurements.

Team: Lorenz Gerbeth, Max Brunkhorst, Jörn Felix Ziegler, Marcus Lindner

Funding: Einstein Kickbox - Young Scientists

Normothermic extracorporeal gen modification of the rat liver

Liver transplantation still presents the only curative treatment for end-stage liver disease, but viable donor organs are very limited. Normothermic ex vivo liver perfusion (NEVLP) has been developed as an alternative to static cold storage. NEVLP aims at maintaining the liver metabolism by perfusing the graft with oxygenated medium at 37°C and reducing ischemia time. Gene modification during NEVLP may be a possibility to tackle organ failure through immunogenic injury but may also allow for future therapies. Aim of the proposed project would be to develop an effective method to influence the gene expression of liver grafts via transfection with coding RNA sequences during small animal NEVLP. A plasmid coding for luciferase mRNA as well as a shRNA sequence against the CIITA gene expression is used for the downregulation of the expression of major histocompatibility antigens in rats, allowing for detection by recipient immune system after transplantation, thus reducing the need for immunosuppressive therapy after transplantation.

Team: Joseph Gaßner, Nathanael Raschzok, Manfred Gossen, Igor Sauer, Julian Michelotto

Funding: Einstein Kickbox - Advanced Scientists

3D immunomodulation model in vitro – Closing the gap in translatability of MSC products

Mesenchymal stem cell (MSC) therapy was proposed as a treatment for alloreactive conditions such as transplant rejection or GvHD due to their immunomodulatory effects observed in controlled 2D culture conditions. However, in a clinical setting MSCs are delivered into a harsh and poorly understood in vivo environment. Thus, when evaluating the therapeutic potential of a cell product, the predictive value of effects observed in standard 2D culture is low:

As the microenvironment (be it a tissue or an engineered delivery carrier) is known to heavily influence MSC biology, we propose that isolated 2D culture should no longer be the state-of-the-art testing platform to predict therapeutic potential in vivo. Instead we aim to evaluate MSC-mediated immunomodulatory effects in an integrated 3D co-culture model, where a tissue organoid is infiltrated by alloreactive immune cells and treated with MSCs at the same time.

For our first model, we will look at patient-derived whole bone marrow aspirate. Novel engineering challenges lie in designing a biomaterial that allows for immune cell infiltration, as well as in developing a biofabrication technique that will bring together all 3 components in a meaningful 3D architecture. Readouts will include immune profiling of different in-house MSC products, flow cytometry and cytokine analysis of immune cells and histological analysis of co-culture constructs.

Team: Norman Drzeniek, Stefania Martini

Funding: Einstein Kickbox - Young Scientists

Biomechanical comparison of titanium versus magnesium miniplates for fracture fixation in a sheep mandible model

As a standard of care, titanium miniplates are used for osteosynthesis in midfacial and mandible fractures. Titanium implants can be either permanently left in situ or removed once the bone healing is achieved. In the first case, negative long-term side effects such as particle debris leading to a foreign-body-reaction, possible stress-shielding effects, interference with skeletal growth as well as diagnostic imaging artifacts may occur. On the other hand, plate removal exposes the patient to all risks linked to a second surgery.

Therefore, biodegradable implants gained increasing interest to overcome the limitations of traditional biomaterials for fracture fixation. As a promising product biodegradable magnesium implants are characterized by Young’s modulus similar to cortical bone, which we hypothesize can also guarantee enough stability at the fracture site. A known complication associated with magnesium degradation is the release of hydrogen ions, which negatively impacts the bone healing process. A possible solution is given by slow-degrading magnesium alloy WE43 and new coating techniques, as Plasma Electrolytic Oxidation (PEO).

In this study, we aim to test the novel WE43-based magnesium implants with PEO-surface modifications in a sheep mandible fracture model ex vivo in order to compare their mechanical performance to conventional miniplates.

We hypothesize that magnesium miniplates are suitable for load sharing indications if the mechanical properties are respected.

Team: Claudius Steffen, Heilwig Fischer, Vincenzo Orassi, Dag Wulsten, Oskar Schmidt-Bleek

Funding: Einstein Kickbox - Young Scientists

BMP9/Alk1 signaling in vascular repair and remodeling: from guardian of endothelial quiescence to activator of angiogenesis

Successful wound repair and tissue regeneration rely on controlled re-vascularization of the trauma region by angiogenic sprouting. Maturation of newly formed blood vessels notably depends on BMP9/10/Alk1 signaling, for which deficiency can result in the rare disease Hereditary Hemorrhagic Telangiectasias. BMP9/10 signal via Alk1 in endothelial cells (ECs) and prompts SMAD1/5/9 signaling, thereby promoting vascular quiescence and curbing post-injury angiogenesis which, if left unchecked, can impair scarring.

However, recent work reported on pro-angiogenic effects of BMP9, highlighting BMP9 functions as context-dependent and cell-type specific; but the mechanisms modulating the bipartite role of BMP9/alk1 signaling as pro- or anti-angiogenic are still unclear.

To study the processes regulating BMP9 dual roles, we will use fibrin-embedded endothelial spheroids as 3D models of angiogenesis, using diverse endothelial cell types and ligand concentrations. Super resolution confocal imaging and immunofluorescence will help assess sprouts morphology. Moreover, we will use 3D traction force microscopy to evaluate the crosstalk between endothelial BMP9/Alk1 signaling and mechanobiology, and 3D biomaterials to investigate the influence of BMP9/Alk1 signaling on stromal cell-induced ECM remodeling during angiogenesis.

A better mechanistic understanding of BMP9/Alk1-signaling roles during angiogenesis and vascular homeostasis could help develop therapeutics to better control the angiogenic response after injury, and thus improve scarring and tissue repair.

Team: Mounir Benamar, Erik Brauer

Funding: Einstein Kickbox - Young Scientists

Can skin fibroblasts from patients with HFpEF be used as a read-out of cardiac fibroblasts?

Heart failure (HF) is responsible for substantial morbidity and mortality and is increasing in prevalence. Up to one half of HF patients have preserved ejection fraction (HFpEF). Although there has been remarkable progress in the treatment of HF with reduced ejection fraction (HFrEF), translation of those therapies to HFpEF has been disappointing, indicating the need to further understand the emergence of HFpEF. HFpEF and HFrEF differ in their underlying pathogenesis. In contrast to HFrEF, which is driven by an initial cardiac/cardiomyocyte damage, a low-grade systemic inflammation induced by comorbidities underlies HFpEF-specific cardiac remodeling and dysfunction.

Serum S100A8/A9 alarmin levels are increased in HFpEF patients as well as in patients with systemic sclerosis, which are characterized by skin fibrosis and cardiac diastolic dysfunction. These findings support the rationale that a systemic inflammatory response can provoke fibroblast activation in the heart and in the skin. They further raise the hypothesis whether skin fibroblasts from HFpEF patients could be used as readouts for the activated state of fibroblasts in the heart and could hereby potentially serve as a prognostic tool and a tool to test anti-fibrotic therapies. Here, we investigate whether skin fibroblasts from HFpEF patients can be used to mirror cardiac fibroblast activation (upon a pro-inflammatory stimulus), when they are grown under stiffness conditions mimicking the cardiac environment.

Team: Isabell Matz, Erik Brauer

Funding: Einstein Kickbox - Young Scientists

Cell-mediated TCR/CD3-depletion of CRISPR/Cas-edited, TCR-to-CAR replaced T cells for the generation of ultra-pure off-the-shelf CAR T cell products

T lymphocytes are immune cells that defend the body from pathogens and cancer. They can be genetically modified to express designed receptors such as Chimeric Antigen Receptors (CARs), enabling them to recognize antigens of choice. Such CAR T cells have been employed in the treatment of several oncological disorders, the best clinical example being CD19-specific CAR T cells used to treat patients suffering from B cell leukaemia and lymphomas.

We recently improved a method that allows for efficient manufacturing of T cell receptor (TCR) replaced CAR T cells.  We employ CRISPR-Cas9 to introduce a CAR transgene into the TCR alpha chain (TRAC) gene locus. To this end, sgRNA/Cas9 complexes and DNA molecules carrying a CAR-transgene are co-transfected into T cells. By exploitation of Homology-directed DNA Repair, the CAR-transgene can be introduced under the control of the TRAC promoter, which furthermore prevents formation of functional TCRs.

This method mainly produces TCR-replaced CAR T cells carrying the CAR as their only specificity-determining receptor, and TCR-/CAR- double negative T cells, incapable of any antigen-specific action. While these cells would be fit for third-party use, a small fraction of unedited or CAR+/TCR+ double positive T cells remains, which, if transfused in an allogeneic setting, might cause Graft-vs-Host disease. This project aims to eradicate all remaining TCR+ contaminants in order to generate off-the-shelf CAR T cell products.

Team: Jonas Kath, Stefania Martini, Magdi Elsalab, Weijie Du

Funding: Einstein Kickbox - Young Scientists

D-Lactate in diabetes: a secondary clinical manifestation or a perpetrator of the disease?

Ketone bodies and their derivates in Type 1 Diabetes (T1D): a secondary clinical manifestation or a perpetrator of the disease?

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of b-pancreatic cells by infiltrating CD8+ T cells into the pancreas. Under homeostatic conditions, b-pancreatic cells produce the necessary amount of insulin to promote GLUT(1-4) trafficking to the plasma membrane enabling the cells to uptake glucose and use it as a source of energy. However, during the onset of T1D, the autoimmune response that takes place in the pancreas results in decreased insulin production. Suboptimal insulin levels are not enough to keep the glucose uptake required to fulfill the metabolic demands of the organism. As a result, the liver starts oxidizing fatty acids (FAO) in order to provide alternative metabolites for the cells. The metabolic intermediates released to the bloodstream by the increased rates of FAO augment the concentration of ketone bodies (KBs) and their derivates compared to homeostatic conditions. In this study, we want to address whether the high concentrations of KBs and derivates are harmless or if it is enhancing the T1D autoimmune process. For that purpose, we will examine patient cohorts with different KBs plasma levels. We will ex vivo assess different compartments of the immune response that have been shown to be regulated by KBs in in vitro experiments.

Team: Adrian Madrigal, Valerie Plajer, Mikie Phan

Funding: Einstein Kickbox - Young Scientists

Establishment of a bone marrow on the chip model to analyze the effects of stem cell transplantation on the bone marrow and its components

Most malignant hematological diseases are treated with hematopoietic stem cell transplantations (HSCT). On the other hand, the allogeneic transplantation is based on transplants from non-related donors whereby the chance of rejection is high when the matching of antigens is not absolute. Because of decreased development of severe side effects syngeneic stem cell transplantation is better tolerated but on the other hand less effective. The reason is the anti-tumor immune effect of allogeneic HSCT. After HSCT patients suffer from delayed immune reconstitution as well as a higher fracture rate. On the one hand, the syngeneic transplantation uses transplants from either twins or previously harvested and stimulated cells from the patient itself. This transplantation has less risk of rejections or reactions of the immune system.

The molecular basis of changes in properties and delay of reconstruction of the bone structure are sparsely examined in patients as well as experimental setups.

With the planned model we will simulate the process of stem cell transplantation in a 3D in vitro model by using human cells and investigate its effects on the bone marrow and immune cells of the model. Our long-term perspective is to develop possibilities to reduce severe side effects on the bone in HSCT patients. With a successful establishment of the 3D model, we will be able to study changes in the course of HSCTs in vitro and predict possible effects when applied to patients.

Team: Radost Anika Saß, Melanie-Jasmin Ort, Lisa-Marie Burkhardt

Funding: Einstein Kickbox - Young Scientists

Pyruvate dehydrogenase kinases as a novel target to treat osteoarthritis: From aggressive to quiescent fibroblast-like synoviocytes

Osteoarthritis (OA) is the leading cause of joint pain and age-related disability worldwide, due to the increase of life expectancy and an active, aging population. Unlike rheumatoid arthritis, therapies used in OA are still limited to pain control. Therefore, novel targets for the development of disease-modifying drugs are required.

In recent years, fibroblast-like synoviocytes (FLS), which maintain the structural and dynamic integrity of joints physiologically, were identified as key drivers of cartilage degradation. FLS can be divided into two major populations. The destructive phenotype which is restricted to the synovial lining layer stimulates osteoclastogenesis, thereby promoting bone erosion. On the other hand, FLS of the sublining layer are classified as invasive effector cells driving synovitis. Recent research has revealed that glucose metabolism shapes the FLS phenotype, promoting disease progression. Among them, the proliferative and metabolically active FLS are marked by an increased glycolysis compared to the non-invasive, quiescent FLS. Inhibition of glycolytic enzymes already attenuated the severity of cartilage damage in vivo. Our data suggest that pyruvate dehydrogenase kinase (PDK) 3 may play a crucial role in the pathogenesis.

Therefore, we hypothesize that shifting the metabolism of FLS from glycolysis to mitochondrial respiration via PDK3 that regulates the mitochondrial flux might be a novel target for the development of disease-modifying drugs.

Team: Alexandra Damerau, Nayar Alejandro Durán Hernández, Tazio Maleitzke

Funding: Einstein Kickbox - Young Scientists

A study about the influence of magnesium implants on pseudarthroses in a mouse model

If the bone fails to restore a bony continuity over more than six months after a fracture or bony reconstruction, we speak of a pseudarthrosis. For the reconstruction of critical size defects in long bones and the mandible, autologous osteomyocutanous transplants are used. Bony healing between transplant and original bone often fails. In mandible reconstructions, metal removal and following dental rehabilitation is delayed or impeded if a non-union situation occurs. There are many possible reasons for non-unions, including insufficient callus formation and a lack of vascularization.

For non-unions, the revision and the treatment with autologous cancellous bone are considered the gold standard. In larger defects, an autologous bone block is needed to fill the defect, involving the possible creation of a donor site morbidity.

Magnesium holds promising attributes for the use as a resorbable implant material. We hypothesize that, by supporting angiogenesis and its osteostimulative properties, magnesium implants are suitable for treating non-union situations.

In this project, we want to create a mouse model for treating non-union situations to evaluate magnesium implants' influence on existing pseudarthroses. This study aims to find a new treatment option for situations where increased bone formation is needed, for example, in patients suffering from non-unions after fractures and for oncological patients who have lost parts of their bone due to cancer treatment.

Team: Heilwig Fischer, Katharina Schmidt-Bleek, Carsten Rendenbach, Agnes Ellinghaus, Sabine Stumpp

Funding: Einstein Kickbox - Advanced Scientists

EDITCAPS – A new assay for unbiased detection of chromosomal rearrangements and off-targets in gene-edited cell products

Gene editing, the targeted modification of DNA, allows the controlled repair of mutations to fix genetic diseases. The most important safety concern for the translation of gene-edited cell products is the risk of off-target events. Off-target editing could introduce mutations or translocations with oncogenic potential. Standard assays for the unbiased identification of potential offtargets are currently performed in surrogate cell lines or using in vitro digested genomic DNA. Here, we propose a new simplified approach that allows the cost-efficient off-target identification directly from the DNA of the gene-edited cells. To this end, we plan to adapt existing workflows of targeted enrichment sequencing for the quick and sensitive identification of structural variants and off-target integration of DNA donor templates during gene editing procedures.

Team: Dimitrios Wagner, Björn Fischer-Zirnsak, Dominik Seelow

Funding: Einstein Kickbox - Advanced Scientists

Patient-derived lung cancer organoids as a 3D platform for personalized therapy

The heterogeneity of lung cancer is a major cause of its high mortality and personalized treatment strategies are urgently needed to overcome this fatal disease. Patient-derived lung cancer organoids (PDLCOs) are capable of modeling individual tumor biology and could serve as platforms to investigate the effect of therapies. However, so far, the low efficacy and reproducibility in generating PDLCOs hinders their use in preclinical research and diagnostics. Additionally, the current tumor organoids lack immune components, which are critical for the evaluation of anticancer therapies. In our project, we strive to establish a robust protocol for the generation of PDLCOs from surgically resected tissue to create a biobank for the in vitro evaluation of personalized antitumor therapies and immunotherapies in particular. We will evaluate different culture protocols to unravel niche factor dependencies of tumor stem cells, which are suggested to be essential for proliferation. Stringent testing of PDLCOs and the primary tissue they derived from will be performed to ensure their in vivo comparability. Furthermore, the capacities of including tumor microenvironment components into the respective cultures and their immunogenicity will be evaluated. Ultimately, the generated organoids could be installed as prediction models of therapeutic efficacy and could be valuable tools in personalized medicine.

Team: Lukas Ehlen, Regina Stark, Birgit Sawitzki, Janine Arndt

Funding: Einstein Kickbox - Advanced Scientists

ECRT Consumable Grants - Förderperiode 2021

A virus-free approach to generate safe and effective CAR-T cell products for third-party use in patients with solid tumours

T cells are a major part of our adaptive immunity, being able to eliminate infected or transformed target cells and are increasingly exploited as powerful tools in oncology. They can be redirected to recognize and eliminate cancer cells using Chimeric Antigen Receptors (CARs), fusion proteins of antibodies for antigen recognition and T cell-specific signaling domains. While already effective in patients suffering from certain blood cancers, the generation of an individual autologous CAR-T-Cell product for a single patient is a difficult and costly endeavor. Treatment delay due to production processes and high costs hinder their broad application in the clinic. This kickbox application aims to test a novel approach to create CAR-T-Cell product from healthy donors that are unable to attack the patients cells via their endogenous T cell receptors in an allogenic treatment setting of solid tumors. Therefore, this "next generation" of CAR-T cells may enable the generation of potent but cost-effective treatments of solid tumours.

Team: Dimitrios L. Wagner, Jonas Kath, Lennart Ostendorf

Funding: ECRT Consumable Grant

hiPSC-Derived Cardiac Progenitors: Who Are You?

Myocardial infarction represents a global health threat. Cell therapies employing cardiac progenitor cells (CPCs) are promising to boost the regenerative potential of the human heart. As CPC sources are either scarce and ethically-charged or bound to carry patient-associated comorbidities, we are investigating alternative sources of CPCs such as human induced pluripotent stem cells (hiPSCs). To that end, we sorted stem cell antigen-1- positive cells from hiPSCs differentiating into cardiomyocytes and obtained a highly proliferative CPC-like population (iCPCSca-1). This strategy is reproducibly used to isolate CPCs from the heart. However, the nature of the recognized epitope in humans is still highly controversial. To validate this strategy we analyzed iCPCsSca-1, the flowthrough cell fraction (FT cells), and human fetal Sca-1 cardiomyocyte progenitor cells (hCMPCs), the reference sample, using single cell RNA sequencing. Preliminary analysis revealed that hiPSC-derived cells are more closely related to each other than to hCMPCs, and FT cells were the most heterogeneous and distant sample compared with hCMPCs. Interestingly, hCMPCs consisted of three distinct subpopulations, of which only two expressed NKX2.5 – a transcription factor considered a CPC marker. We now seek to identify the phenotype of these subpopulations, assess reliability by analyzing cells derived from different fetal and hiPSC donors, and ultimately improve the sorting strategy by identifying novel surface markers to isolate specific CPC subpopulations.

Team: Ana Garcia Duran, Timo Nazari-Shafti, Sebastian Neuber, Andranik Ivanov

Funding: ECRT Consumable Grant

Unravel fibronectin tensional state in a 3D physiological microenvironment – the impact of inflammatory signaling on early ECM formation

In regeneration, the cellular deposition of ECM is crucially linked with properties of the surrounding microenvironment. However, in an injury situation the ECM secreting fibroblast-like cells are influenced not only by the mechanical cues in their 3D microenvironment, but also by the inflammatory response orchestrating the healing process. We here suggest a novel, more holistic approach to reflect the versatile stimuli acting through immunological and mechanical cues on fibroblast-like cells upon injury. We aim to define whether Fn deposition and tensional state are related to mechanical properties and immune signaling to better understand beneficial and adverse healing scenarios. Understanding this complex interplay of immune response, 3D microenvironment mechanics and cell function will provide further insight into modeling of degenerative diseases and benefit regenerative approaches involving cell delivery using biomaterials.

Team: Matthias Kollert, Georgios Kotsaris, Julia Mehl, Christian Bucher

Funding: ECRT Consumable Grant

ECRT Research Grants - Förderperiode 2021-2023

3D tissue models for preclinical testing of augmented CAR T cells

T cells have been identified as one of the main defense lines of the immune system against tumors. The scientific break throughs of the recent years with immunologic checkpoint inhibitors and adoptive T-cell therapy are offering novel treatment options when the body defense mechanisms fail and T cells become dysfunctional. T cells expressing a chimeric antigen receptor (CAR) are one of the recent advancements and combine the specificity of an antibody to target-specific antigens on tumor cells with the ability of T cells to kill tumor cells.

CAR T cells have shown remarkable effects in the treatment of late stage lymphoma patients. Unfortunately, CAR T cell therapy for solid tumors has not been nearly as successful and further research is needed to improve the existing treatment options. However, current used test systems for CAR T cells such as conventional 2D tumor cell cultures and murine xenograft models lack important features of human tumors in vivo.

In this project we want to establish novel test systems for the development and preclinical testing of CAR T cells that closer resemble the situation in the patient. For this we will employ iPSC derived organoids that will be generated in close collaboration with the BIH Core Facility Stem Cells as well as patient derived organoids. Introduction of fluorescent reporters will allow high throughput screening of CAR-T cell induced toxicity using the confocal high content imaging platform at the BCRT. The focus will be on generating a model for lung-cancer using overexpression of the epidermal growth factor receptor (EGFR), a very common tumor associated antigen with tumorigenic potential. Once the organoid models are established, we can test the different CAR T cell products that are currently used and further developed in the institute. Next to the direct tumoricidal activity, also homing and invasion in the established tissue models as well as the capacity of CAR T cells to initiate durable responses will be studied in static and microfluidic systems.

Team: Leila Amini, Regina Stark, Harald Stachelscheid, Hans-Dieter Volk, Ugarit Daher

Funding: ECRT Research Grant

Resolving the Activin A Receptor complex and its endothelial function in Fibrodysplasia Ossificans Progressiva (FOP)

The TGF-β superfamily member Activin A induces signal transduction via two type I (ALK4/7) and two type II (ACVR2A/B) receptors, which upon ligand binding form heterotetrameric receptor complexes and induce phosphorylation of SMAD2/3. Fibrodysplasia Ossificans Progressiva (FOP) is a rare, severely disabling disease characterized by gain-of-function mutations in the Bone morphogenetic protein (BMP) type I receptor ALK2, the most prominent of which is R206H. This arginine to histidine change turns a normally silent ALK2 signaling complex  responsive to Activin A, and finally results in aberrant SMAD1/5 signaling leading to heterotopic ossification (HO). While there is no approved treatment for FOP; several treatment options targeting Activin A or ALK2 are in research and development. Previous studies propose an involvement of the endothelium to the progression of FOP, as the aberrant Activin A signaling is recapitulated in FOP ECs. However, 1) the role of ECs in the formation of HO is not yet fully understood, 2) the composition of the receptor complex is not resolved and 3) the detailed molecular and cellular mechanism of Activin A binding to FOP-ALK2 remains to be explored.  Therefor the PhD candidate will perform (a): siRNA based approaches, in combination with co-immunoprecipitation experiments to determine the composition of the receptor complexes. Further the applicant will take advantage of an existing SNAP- and HALO-tagged BMP/TGFβ type I and II receptor library, and perform Stimulated Emission Depletion (STED)-based super resolution imaging. Finally the applicant (c) will set-up a new drug screening plat form for yet unapproached targeting strategies, to functionally interfere with both WT and FOP-ALK2 in ECs treated with Activin A and other ligands of the family. This project is particularly interesting for master students with an interest and/or background in signal transduction, imaging and translational research.

Team: Jerome Jatzlau, Petra Knaus, Holger Gerhardt, Wiktor Burdzinski

Funding: ECRT Research Grant

The search for the holy stromal cell - Identification and characterization of specialized and regenerative stromal cell populations to accelerate cartilage repair

Knee osteoarthritis (KOA) is one of the most common form of joint disease among elderly patients and remains a major orthopedic challenge since mature hyaline articular cartilage has only a limited capacity for intrinsic repair. Despite its prevalence, the molecular mechanisms triggering KOA are poorly characterized and there are currently no disease modifying therapies that can halt or revert KOA. Cell therapeutic approaches using autologous chondrocytes or mesenchymal stromal cells (MSCs) have been developed and tested in the last decade. MSCs can be found in bone marrow, adipose-tissue, synovium and cartilage itself, and are capable of differentiating into osteogenic, chondrogenic and adipogenic lineages. First attempts to inject MSCs in KOA patients have provided promising results during clinical trials, showing a significant decrease in inflammation and enhanced joint function. However, in most studies, regeneration of cartilage was not observed. Our preliminary results and current literature suggest that the MSCs inhibiting inflammation and the MSCs regenerating cartilage are not one and the same.

The overall aim of this project is to delineate the MSC subpopulations in the synovial membrane and the epiphyseal bone marrow area, to conceptualize a novel mixed MSC population therapeutic strategy for KOA. The project has both a wet-lab and dry-lab outcomes. The wet-lab outcomes will entail delineating MSC subpopulations using novel scRNA-seq techniques and perform functional assays to characterize the cells for their differentiation, regeneration, and immunomodulatory capacity. In the dry-lab, mathematical models will be derived from the data to explain the macro-level dynamics; attractants and surrounding factors that are required for maintenance and re-activation of chondrogenic MSCs during ageing.

Team: Annemarie Lang, Mir-Farzin Mashreghi, Christof Schütte, Vikram Sunkara, Sebastian Kurmies

Funding: ECRT Research Grant

ECRT Research Grants - Förderperiode 2020-2022

A chemokine system to unleash T cells against solid cancer - Regenerate immune responses against tumor

The immune system harbors intrinsic capacity to fight malignancy. Tumor-specific T cells play a crucial role in combatting cancer by recognizing and killing malignant cells. To enhance tumor-specific immune responses, T cells can be redirected using genetically engineered receptors against tumor antigens. Clinical success of engineered chimeric antigen receptor (CAR)-T cell has been achieved for hematological malignancies. However, success of CAR-T cell therapy in treating solid tumors has been limited, since transferred CAR-T cells could not infiltrate and persist in the hostile tumor environment. To improve adoptive transfer of CAR-T cells against solid tumors, we initiated a study to learn from intrinsic cues that promote T cell infiltration and persistence in human solid tumors.

We joined forces with Karolinska Institutet and Umeå University (Sweden) to study solid cancer disease. Here, we unraveled chemotactic mechanisms in muscle-invasive bladder cancer (BC). BC is a solid tumor disease with poor prognosis, yet, T cell infiltration into the BC can resuscitate patients to reject the tumor. We addressed the question which signals in BC favor T cell trafficking to the tumor and intratumor expansion. In this approach, we identified a distinct Th1 chemokine (C1, see notes) as main driver of T cell infiltration at the tumor site. In-vitro, we found that protective T cell subsets expressed a C1-specific receptor variant (R1*) and functionally, R1*+ T cells enriched when targeted by the ligand C1. Strikingly, analyzing the novel R1*-C1 axis in the tumor, we could predict overall survival and successful response to therapy in BC-patients. We hypothesize that R1*-C1 is a T cell-activating pathway in cancer with therapeutic potential.

In this project, we continue this approach within our European translational group. We pursue two aims:

1) Understanding how C1 improves anti-tumor T cell function.

2) Setting up a new therapy via R1*+ CAR-T cells.

1) We want to test the use of C1 for in-vitro culture and potential clinical application. For this, we will study signaling induced by the R1*-C1 pathway by applying i) our established flow cytometry method on phosphorylation targets (e.g. STAT-proteins) ii) multiplex-mass cytometry (in-house available in the cytometry core facility) and iii) a unique DIGI-West approach (with Tübingen in the EU-network Reshape).

2) We address to select natively R1*-expressing T cells from human PBMC as a starting population for CAR-T cell production (nR1-CAR-T cells). Further, we plan to endow ex-vivo generated tumor-specific T cells with the genetically engineered receptor R1* (eR1-CAR-T cells). In addition, we intend to access HER2-specific CAR-T cell products that were previously applied against mamma carcinoma (with Baylor College of Medicine Houston, TX, USA). Here, we seek to verify if low expression of R1* in products correlates with poor response rates following adoptive therapy.

In this project, we aim to gain insights into the newly described R1*-C1 axis in human solid cancer by native pre-selection of R1*-expressing cells or R1*-engineering for CAR-T cell generation. Thereby, we employ novel therapeutic approaches to boost the efficacy of tumor-specific CAR-T cells to reach and survive in the solid cancer microenvironment.

Team: Tino Vollmer, Michael Schmück-Henneresse, Hans-Dieter Volk, Leila Amini, Jacqueline Wendering, Sarah Schulenberg

Funding: ECRT Research Grant

Building a Platform for Fast Track Development and Characterization of Engineered Antigen Receptors

Figure 1) Flow cytometric Evaluation of CAR Signaling (FLECS) platform for inexpensive, standardized in vitro assessment of CAR function. In reporter cells, T cell receptor (TCR) or chimeric antigen receptor (CAR) signaling activates transcription of a TCR-inducible gene which is fused to a GFP reporter transgene via a P2A linker. Upon translation, GFP is freed by self-cleavage of the P2A linker; b) anti-CD3 antibody activates TCR, leading to GFP expression (pos. control); c) CD19-specific CAR is activated in CD19 antigen-coated well leading to GFP expression (pos. control); d) Upon binding their specific antigen (Ag), test CAR constructs may vary in signaling strength and consecutive GFP expression; e) test CARs in the absence of their specific antigen ideally show no/little basal GFP expression. CD19-CARs in absence of their antigen may serve as neg. controls; f) Hypothetical results of flow cytometric analysis of GFP expression for positive controls, test CARs 1-4 (with differing signaling properties) and a negative control. (Fig. generated on
Figure 2) Chimeric auto-antibody receptor design for Neuromyelitis Optica/Multiple Sclerosis: Chimeric auto-antibody receptors (CAARs) mediate recognition of pathogenic B cells by engineered CAAR-T cells through binding to surface-bound immunoglobulins (sIgG) specific for an immunogenic self-antigen. The CAAR consists of a peptide derived from the auto-antigen as its extracellular recognition domain which interacts with pathogenic auto-antibodies, a hinge domain for optimal intermembrane distance of the immunological synapse, a CD8 transmembrane (TM) domain to facilitate dimerization, a 4-1BB costimulatory domain and a CD3 zeta chain for signal transduction. (Fig. generated on

Redirecting T lymphocytes using chimeric antigen receptors (CARs) is a powerful tool in the emerging field of regenerative medicine. T cells reprogrammed to recognize and kill CD19 positive cancer cells dramatically improved leukemia and lymphoma treatment, whereas the transfer of CARs specific for allogeneic antigens (Ag) into regulatory T cells was shown to induce immune tolerance in transplantation models. Recent reports suggest CARs may facilitate innovative treatments for autoimmune diseases as novel receptor designs enable CAR-T mediated depletion of pathogenic auto-Ag specific B cells and prevent autoantibody (Ab) mediated diseases (1).

CARs are fusion proteins made of a target-specific extracellular domain (usually a single chain variable fragment derived from an Ab) and an intracellular signal transduction region (varying costimulatory domains and a CD3 zeta chain) which imitate T cell receptor (TCR) signaling. Optimal signaling strength of CARs is paramount for T cell activation and the clinical success, while binding affinity/avidity and minimal tonic signaling are also of importance (2). Surprisingly, careful evaluation of CAR signaling properties is rarely performed and no standardized assays have been described. Existing assays to predict in vivo CAR-T cell function are limited to tumor-specific CARs and rely on lengthy serial co-culture experiments. Thus, they are not practical for fast iterative optimization of CAR design.

We hypothesize that signaling properties of a given CAR can predict its in vivo performance. To this end, we plan to create a reporter cell line that generates an optical output in response to Ag-receptor signaling. In Jurkat cells, which are a standard model to study TCR signaling, the expression of a variety of genes is induced upon TCR ligation, some of which correlate exclusively and gradually with Ag-mediated TCR signaling strength.

To obtain a good reporter system, Jurkat cells will be genetically modified by inserting a reporter green fluorescent protein (GFP) transgene into a TCR-inducible gene via CRISPR/Cas9 (3). Ag-receptor (TCR or CAR) signaling will lead to the expression free GFP through the self-cleaving activity of a P2A linker (Fig. 1). CAR constructs to be tested can be readily transferred to the reporter cells via the delivery system of choice (retroviral, transposon, site-specific integration). After subsequent Ag stimulation, fluorescence intensity of CAR-mediated signaling will be detected by flow cytometry over multiple time points. Results from TCR/CD19 CAR signaling will serve as performance benchmarks.

We propose to name this platform FLECS as an acronym for Flow cytometric Evaluation of CAR Signaling. It will enable standardized mass-testing of new CAR constructs and ensure the selection of the most efficient CAR for any given Ag. It will be deposited in public cell banks and shared openly to advance CAR-redirected T cell therapy in regenerative medicine.

After establishing the FLECS platform, we will focus on the development of CARs for the severe, rapidly exacerbating, demyelinating, Multiple Sclerosis (MS)-like disease Neuromyelitis Optica (NMO). In NMO, auto-Abs against immunogenic self-Ag are thought to play a causative role. Similar to a recent report (1), we designed novel antigen receptors that incorporate the immunogenic epitope derived from the self-Ag to form a chimeric auto-Ab receptor (CAAR). Thus, cytotoxic T cells will be able to recognize B cells that display surface bound immunoglobulin (sIgG) specific for the auto-Ag (Fig.2). After FLECS analysis, the CAAR with best performance will be subjected to further functional analysis in vitro and in the mouse model. Eventually, targeted elimination of auto-Ag specific B cells may offer a novel therapeutic approach to prevent chronic inflammation through auto-Ab in NMO, while FLECS might facilitate the development of similar therapeutics for other autoimmune diseases.

Team: Dimitrios L. Wagner, Michael Schmück-Henneresse, Hans-Dieter Volk, Petra Reinke, Anja Hauser, Helena Radbruch, Jonas Kath

Funding: ECRT Research Grant

Cellular senescence in mechano-sensation and ECM organization

Aging is an irreversible, progressive process resulting in a decline of tissue functionality and its regenerative capacity. With an increasingly aged population in developed countries, understanding deviations in healing processes after injury in aged is key for developing novel treatment strategies. During life the body accumulates with cells which lack a proliferative capacity due to an irreversible cell cycle arrest which is termed cellular senescence. Senescent cells secrete a variety of pro-inflammatory cytokines which recruit immune cells for tissue clearance during development and to prevent cancer. Although this feature confers early-life benefits, the increasing pro-inflammatory environment contributes to late-life debility (Campisi 2013). This antagonistic function is even further emphasized by recent observations which not only show the accumulation of senescent cells within an injured tissue but that elimination of senescent cells delays wound healing in vivo (Demaria et al. 2014). While this investigation related the effect to the secretory phenotype, little is known how senescent cells directly contribute to tissue formation through ECM formation.

We investigated how the senescence program affects extracellular matrix deposition and tissue tension using a recently published in vitro wound healing model system (Brauer et al. 2019). We observed that cellular senescence strongly affected macroscopic tissue tension and fibrillar collagen secretion and assembly in an antagonistic manner. While senescence due to over-expression of cell cycle inhibitors (p16INK4/p21CIP) resulted in an increased contraction, DNA-damage-induced (Mitomycin C-treated) senescence strongly reduced tissue contraction. This indicates a direct and antagonistic role of cellular senescence in establishing tissue tension. Aside of the tensile state of the resulting ECM, cellular senescence also affected the biochemical composition via differential regulation of ECM proteins, e.g. collagen, fibronectin, decorin, and tenascin C. Furthermore, we found evidence that these macroscopic perturbations might be influenced by an altered mechanotransduction of senescent cells already on the single cell level (2D). The expression of integrins was altered correlating with an enlarged cell morphology and higher abundance of large focal adhesions particularly in cells that were driven into senescence by selective over-expression (p16/p21). How these two observations are linked remains so far unknown.

Based on these preliminary data, we propose a novel research question which we believe to be relevant and of high interest for a PhD project. Based on the understanding that senescent cells seem to strongly alter the extracellular niche we want to better understand the relevance of this finding for the in vivo tissue regeneration process. While the in vitro experiments focused on the behavior of a homogeneous population, senescent cells in vivo are found in relatively low abundance. We believe that senescent cells nevertheless impact the regeneration process and hypothesize that the ECM formed by senescent cells exhibits specific cell-instructive cues which influence cellular processes, e.g. migration, survival but also differentiation of surrounding stromal cells. A distinct matrix phenotype thereby might contribute to the impact senescent cells exert during tissue regeneration aside of their secretory phenotype. The PhD student will optimize a recently established procedure for decellularization in order to obtain cell-free ECM generated by senescent cells. The matrix will be used as a template for studying the cellular response both of fibroblasts and MSCs. Differences in ECM-dependent cellular behavior would support the hypothesis that an ECM created by senescent cells affects behavior of surrounding stromal cells. The student will further focus on the consequences of senescence-modulated cellular mechano-sensation on tissue formation. Starting from a basic description of cellular mechano-sensation, cellular organization will be studied on simplified geometries to describe differences in multi-scale cell organization (Werner et al. 2016). Finally collective cell organization, which leads to ECM formation, will be studied in more detail inside the established scaffold-based 3D tissue model to further understand the impact of senescent cells on early tissue regeneration.

Together, the results will contribute to a principle understanding of (i) how the resulting matrix properties steer the behavior of non-senescent through a distinct cell-instructive matrix code and (ii) how senescent cells sense and respond to their environment with regard of cell organization and tissue formation.

Team: Erik Brauer, Ansgar Petersen, Sven Geißler, Mina Sohrabi-Zadeh

Funding: ECRT Research Grant

Differentiation of induced pluripotent stem cells (iPSCs) into specific T cell subtypes using epigenetic editing

Immuno-suppressive CD4+ regulatory T cells (Tregs) are the main natural preventer of auto-immune diseases and chronic inflammation and are known to support tissue regeneration after injury and immuno-pathology. Therefore, Tregs are currently being extensively studied as "living drugs" in adoptive cellular therapy against pathogenic inflammation and to foster tissue regeneration.

Classical approaches of adoptive Treg therapy, which are based on the extensive in vitro expansion of pre-existing Treg populations, are currently being hampered by several obstacles:

1) the acquisition of proliferation-induced cellular senescence during in vitro expansion resulting in limited survival;

2) a functional instability of Tregs during inflammatory conditions;

3) the absence of pre-existing functional Treg populations in certain auto-immune disease patients and

4) a lack of pre-existing Treg populations displaying the required antigen-specificity or migration capacity, as the antigen-specific T cells in auto-immune patients have acquired a (pathogenic) pro-inflammatory phenotype.

Our group is trying to address these limitations from a new molecular angle: from epigenetics. We have identified several critical epigenetic control elements ('Epi-stabilizers') in T cells which are involved in maintaining a given T cell phenotype (Durek et al, Immunity 2016). The best characterized one is the so-called 'Treg-specific demethylated region – TSDR' in the FOXP3 gene, which is determining Treg function and cellular identity (Huehn et al, Nat Rev Immunol., 2009). To date, the demethylated state of the TSDR is the most reliable biomarker for human Tregs.

Previous work in the group established a CRISPR/Cas9 based system of 'epigenetic editing' allowing the targeted switching of methylation states at Epi-stabilizer elements. With this, Epi-stabilizers can be switched on (=demethylation) and off (=methylation) at will. While this system was successful to switch DNA methylation states and with that, regulate expression of the associated gene, the functional phenotype of the T cells was not completely switched probably due to the remaining pre-imprinted T cell epigenetic landscape.

This is why I now suggest to take a small detour in the functional re-programming of T cell phenotypes and include a re-programming step towards induced pluripotent stem cells (iPSCs). During this, the original T cells can be rejuvenated (addresses obstacle 1 of current Treg therapy, above), while the original TCR can be maintained (obstacle 4). This approach would allow Treg therapy even in Treg-deficient patients (obstacle 3). The resulting iPSCs can be grown in virtually unlimited numbers without senescence acquisition (obstacle 1), are easy to manipulate by epi-/genetic editing and can be stored for repetitive transfusions if needed (obstacle 1). In addition, iPSCs can also be generated from other cellular sources but still be re-differentiated into T cells. The epigenetic editing will be introduced during the T cell differentiation step from iPSC-derived hematopoietic precursor cells, which mimics the normal T cell development in the thymus (7) and thus, is a promising approach to induce stable functional T cell subsets (obstacle 2).

With this project, we expect to establish a system, with which large numbers of storable, fully functional T cell populations can be generated displaying defined advantageous characteristics (e.g. functional stabilization, re-juvenation, selected TCR-specificity). This system can be extended to induce features like a defined migration behavior (obstacle 4) or cytokine expression profile, since Epi-stabilizers in homing receptor (Szilagyi et al., Mucosal Immunol., 2017; Pink et al., J Immunol., 2016) and cytokine genes (Lee et al., Immunity, 2006) have also been identified. With this, we assume to break down several road blocks on the way towards a successful clinical application of adoptive T cell therapies.

Team: Christopher Kressler, Julia Polansky-Biskup, Harald Stachelscheid, Marcel Finke

Funding: ECRT Research Grant

Organoid disease models for spinal muscular atrophy

Team: Angélica Garciá Pérez, Mina Gouti, Markus Schülke, Lan Vi Nguyen

Funding: ECRT Research Grant

Role of human extracellular matrix properties in central nervous system regeneration

Oligodendrocytes constitute one of the four principal central nervous system (CNS) cell types - next to neurons, astrocytes and microglia. Oligodendrocytes form myelin sheaths around neuronal axons, ensuring efficient signal conduction in these axons. In Multiple Sclerosis (MS), a chronic immune-mediated demyelinating disease of unknown etiology, loss of myelin appears during both an initial relapsing-remitting phase and a subsequent secondary-progressive phase. The loss of myelin is associated with clinical disability, including pain, paralysis, vision loss and cognitive incline. Conversely, the generation of new myelinating oligodendrocytes and the repair of myelin, called oligodendrogenesis and remyelination respectively, are prerequisites for functional recovery. The pathological hallmark of MS is the presence of focal demyelinated lesions with partial axonal preservation and reactive astrogliosis. Focal demyelinated lesions can be partly or completely repaired by spontaneous remyelination. However, these regenerative processes are efficient only in a small subset of MS patients. Thus, for the development of highly effective remyelinating therapies it is particularly important to identify the factors that are suppressive or permissive of myelin regeneration.

While the determinants of lesion progression versus lesion repair in MS are still completely unknown, evidence points to the involvement of microenvironmental factors, including biomechanical and compositional extracellular matrix (ECM) properties. While past experimental approaches were often based on animal models, and have lead to the identification of a a few of individual ECM components as regulators of myelin repair. However, a fast growing body of conflicting data on the functional role of such singular components points to the necessity of a more holistic and human approach towards ECM-driven myelin regeneration:

In this project, we aim at identifying the correlative and causal relations between EMC mechanical as well as structural properties, ECM composition and the regenerative potential of human demyelinating and remyelinting MS lesions. Neuropathologically characterized human MS lesion tissue will be divided and subjected to 1) testing of biomechanical properties using microindentation together with structural characterization using label-free two-photon autofluorescence and second harmonic imaging and 2) decellularization of the ECM. The latter will allow us to study the lesion’s ECM as a whole without interference of cellular components. Isolated ECM will be again characterized for its biomechanical and structural properties and used as matrix for human myelinating stem cell cultures for the study of ECMmediated myelination efficiency. Stem cell fate and functional myelination studies will be performed in a microscopy based setting. These experiments will, for the first time, allow us to connect the biomechanical and structural properties of individual MS lesions and lesion-derived ECM with the efficiency of functional myelination in an ex vivo setting.

While we have broad insight into the promyelinating and myelin-supressive signaling effects of singular brain matrix components, nothing is known about the effect of the lesion ECM as a whole. Thus, in a next step, we aim at identifying the lesion type-specific ECM-mediated signaling pattern. Experimentally, we will look at phosphorylation patterns of multiple pro-oligodendrogenic and anti-myelinating signaling pathways on a single cell basis using high-contenct microscopy and advanced image analysis. Immunohistochemical and proteomic analysis of the individual decellularized lesion ECM will further enable us to characterize the different ECM components that underlie the individual biomechanical and regenerative properties. Lastly, through in vitro-mimicry studies, we aim at identifying which mechanical and biochemical properties may be used to functionally overcome brain ECM-associate inhibition of remyelination.

Taken together, through the integration of knowledge and methods from the fields of biomechanical engineering, biochemistry and regenerative cell biology, this project aims at characterizing key regulating brain ECM properties and identifying functional cellular regenerative mechanisms.

Team: Sarah-Christin Staroßom, Ansgar Petersen, Harald Stachelscheid, Erik Brauer, Juliana Campo Garcia, Roemel Jeusep Bueno

Funding: ECRT Research Garnt

ECRT Research Grants - Förderpeiode 2019-2021

Expansion of SpCas9-specific regulatory T cells as an approach to prevent hazardous inflammatory damage to CRISPR/Cas9-edited tissues

The field of gene therapy has been galvanized by the discovery of the highly efficient sitespecific nuclease system CRISPR/Cas9 from bacteria. Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues. Most applications aim to express the Cas9 nuclease in or deliver the protein directly into the target cell. Hence, intracellular protein degradation processes lead to presentation of Cas9 fragments on the cellular surface of gene-edited cells that can be recognized by T cells. Recently, we found a ubiquitous memory/effector T cell response directed toward the most popular Cas9 homolog from S. pyogenes (SpCas9) within healthy human subjects (Nature Medicine 2019). Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective effector T cell (TEFF) response indicating that SpCas9-reactive TREG cells may control respective TEFF responses. Consequently, SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo, especially in patients with low SpCas9-specific TREG/TEFF ratio. Therefore, we aim to test the enrichment and in vitro expansion of SpCas9-specific TREG ultimately aiming at their use in an adoptive immunotherapeutic approach.

Team: Michael Schmück-Henneresse, Sybille Landwehr-Kenzel, Petra Reinke, Hans-Dieter Volk, Dimitrios L. Wagner, Lena Peter

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant

Immunosuppressant-resistant anti-viral T cells for advanced adoptive T cell therapy

Due to the immunosuppressive therapy required for prevention of organ rejection after transplantation, some patients suffer from severe complications due to normally harmless chronic viral infections. Mostly potent anti-viral drugs can manage complications, however many of these bare toxicities, some patients are irresponsive or resistances can be developed. Thus, specific regeneration of the endogenous anti-viral immune response, without risking organ rejection by alloreactive T cells, by the means of adoptive anti-viral T cell therapy is an attractive alternative treatment. In some cases, our conventional approach of T cell therapy can only control the virus temporally, probably due to malfunctions of transferred T cells caused by the immunosuppressive therapy. Hence, we plan to generate T cells which are resistant to immunosuppressive treatment with widely used Tacrolimus for adoptive transfer. Tacrolimusresistant T cells shall be generated by knock out (k.o.) of the adapter protein FKBP12, which is required for the immunosuppressive function of Tacrolimus. The k.o. shall be achieved by a vector-free transfer of nucleoprotein complexes of the nuclease CRISPR associated protein 9 (Cas9) with a site-specific guide RNA by electroporation into virus-specific T cells. We plan to confirm specificity of the k.o. by sequencing of possible off target areas. Ultimately, we want to integrate resistance introduction into our GMP-conform protocol to facilitate clinical translation.

Team: Leila Amini, Dimitrios L. Wagner, Uta Rössler, Michael Schmück-Henneresse, Petra Reinke, Uwe Kornak, Daniel Kaiser, Andy Römhild, Ghazaleh Zarrinrad

Funding: Einstein Kickbox - Young Scientists & ECRT Research Grant

Serial high-resolution 3D-immunofluorescence bone imaging in patients undergoing hematopoietic stem cell transplantation

Little is known about the micro- and macro-structural changes in bone and their consequences on immune reconstitution in patients after hematopoietic stem cell transplantation (HSCT). We will study patients undergoing autologous (lymphoma/myeloma) and allogeneic (AML) HSCT. Both patient cohorts are treated with high-dose chemotherapy and are challenged with steroids, either before autologous HSCT as part of the standard treatment (lymphoma/ myeloma) or after allogeneic HSCT (AML) in case of graft-versus-host disease (GVHD). We have recently established high-resolution 3D-immunofluorescence bone imaging in experimental animal models. However, animal models of human tumors have important limitations and may not adequately reflect the clinical situation. Having immediate access to biopsies of HSCT patients, we are planning to apply the new imaging technique to bone biopsies from patients undergoing HSCT and to compare them to normal bone biopsies (e.g. from patients with malignant lymphoma at initial diagnosis and without BM manifestation). We will specifically investigate the bone micro-structure as well as the bone marrow niche for the physiological hematopoiesis and immune cells. In parallel, we will collect clinical data, such as cytopenia, chimerism, persistence of malignant cells, GVHD and infections. We will correlate the results of high-resolution 3D-immunofluorescence bone imaging with clinical characteristics, in order to identify clinically relevant structural changes in the bones.

Team: Il-Kang Na, Olaf Penack, Katarina Riesner, Sarah Mertlitz, Georg Duda, Katharina Schmidt-Bleek, Radost Anika Saß

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant

Tendon healing, scar formation or chronic inflammation – a matter of miscommunication between tenocytes and immune cells?

Tendon disorders can have diverse etiologies such as acute or chronic mechanical overload or enthesial autoimmunity & inflammation. Interestingly, although the causes are different the clinical outcome shows overlapping features with tendon degeneration and fibrosis. For autoimmune-mediated tendon disorders such as spondyloarthritis (SpA) the contribution of immune cell infiltration and activation is well accepted. For tendinopathies, due to chronic mechanical overload and tendon remodelling, infiltration of immune cells has been observed. Although the exact and especially individual pathology and the role of immune cells is far from being fully understood. With overlapping outcomes, it seems obvious that miscommunications between tenocytes and immune cells are shared pathomechanisms of these disorders. With the kick-box seed money we will start a comparative in depth analysis of immune cell composition and distribution applying multiplex immune histology. This will be continued within the three-year PhD project. Furthermore, we will dissect tenocyte & immune cell communications operational during healing upon acute injury as well as failed tendon regeneration in case of tendinopathy and enthesitis applying single cell RNA-seq. In future, elucidated signaling pathways and potential triggers driving tendon healing or degeneration will be investigated in tenocyte-immune cell co-cultures incorporating mechanical stimulation with the goal to identify novel therapeutic candidates.

Team: Birgit Sawitzki, Britt Wildemann, Franka Klatte-Schulz, Anja Kühl, Uta Syrbe, Martina Seifert, Christiane Gäbel

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant