ECRT Projects

The ECRT aims to facilitate particularly early career scientists to form small teams to develop their ideas into research projects. The ECRT provides the Einstein Kickbox which offers seed money to carry out initial experiments to validate the novel research ideas, and collaboration support for their team work. The ECRT also offers research and consumable grants to further develop the concept projects into fully-fledged research. 

You are here:

Currently running projects funded by the ECRT

Einstein Kickbox - Funding Period 2020

Extracellular matrix formation in pro- versus anti- inflammatory milieus – a key modulator in bone regeneration?

Team: Sophie Görlitz, Lisa-Marie Burkhardt, Aaron Herrera, Julia Berkmann, Christian Bucher

Funding: Einstein Kickbox - Young Scientists

The outset of all regenerative processes is defined by an inflammatory reaction. Inflammatory cells remove necrotic tissue, release inflammatory and proangiogenic factors and exert chemotactic effects. A tight regulation of the immune reaction is a prerequisite for an undisturbed healing. A prolonged pro-inflammatory reaction and an imbalance towards pro-inflammatory T cells impaired bone healing, while the transfer of anti-inflammatory T cells supported healing. Consequently, a bivalent influence of pro-/ anti- inflammatory T cells on fracture healing is suggested. Besides the known chemotactic and angiogenic functions, recent work describes a novel role of T cells in regulating extracellular matrix formation during bone healing. It was suggested that T cells help to organize the collagenous matrix and slow down the process of cartilage mineralization. Here we hypothesize that negative or positive effects of pro- or anti- inflammatory T cells, respectively, are at least partially related to modulations of ECM deposition. The link between T cells and ECM formation is however still poorly understood.

Hence, in this study we are planning to further dissect the influence of pro-/anti-inflammatory cytokines on extracellular matrix formation in a well-characterized in vitro 3D tissue formation model.  We aim to identify target cytokines which hinder or promote ECM formation. The expected insights are of high relevance for a better understanding of the crosstalk between the immune system and early ECM formation processes in bone healing and might contribute to develop future therapeutic concepts.

Pharmacologic inhibition of CRLR as a novel approach to treat obesity

Team: Jessica Appelt, George Soultoukis, Denise Jahn, Paul Köhli

Funding: Einstein Kickbox - Young Scientists

Obesity is a worldwide epidemic that has developed into a major global health problem affecting all ages and socioeconomic groups. This condition is often associated with an increased risk of stroke, diabetes mellitus type 2 as well as a reduced healing capacity. Interestingly, a weight reduction of only 5-10% can circumvent severe health conditions such as diabetes. It can be achieved through changes in diet, exercise or bariatric surgeries. However, adjustments in lifestyle often fail and surgeries are associated with increased risks for obese patients. Additionally, to date only a small number of drugs have been approved for clinical use. Thus, there is a demand for alternative strategies such as the development of efficient and safe anti-obesity drugs.

In this regard, a group of small peptides derived from the Calca gene may be of interest as targets for anti-obesity therapies – Procalcitonin (PCT) and alpha Calcitonin gene related peptide (αCGRP). Recently, we showed that mice lacking PCT and αCGRP kept on a high fat diet have a significantly improved health status compared to wild-type controls. Furthermore, both peptides are suggested to bind to the calcitonin receptor like receptor (CRLR), a G-protein coupled receptor and therefore an excellent drug target.

Hence, in our study we are planning to analyse the eligibility of CRLR antagonists as possible strategy for the fight against obesity, associated comorbidities and the related drop in regeneration capacity.

Are they in contact? - Image evaluation of the interfaces between different materials

Team: Ana Prates Soares, Andreia Sousa da Silveira, Heilwig Hinzmann

Funding: Einstein Kickbox - Young Scientists

In biomedical sciences, the interaction between organism and biomaterials is defined at the interface. When body and material enter in contact, they react to each other. As a result, rejection, absorption, oxidation, bonding, integration, failure or success can occur. The amount of contact between materials can be a good parameter for evaluating how stable their attachment is. Our aim is to create an image-computational tool that can calculate and display the contact surfaces between materials and tissues. In the present project, non-destructive imaging data from classical X-rays, Cone Beam Computed Tomography (CBCT), Micro-CT and Phase contrast-enhanced micro-CT (PCE-CT) will be used. We will develop an image-computational tool to assess the contact at the interface between materials from grayscale images to obtain a statistical quantification of interfacial contact. The output of the proposed approach will reveal detailed information about the superficial area of contact between the biomaterial and hard tissue. In the future, the use of our tool may help getting better insights about the correlation between interfacial contact and biomechanical properties. Thus supporting the development of new materials and design optimization.

iPSC-Derived Cardiac Progenitors - Who Are You?

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

Funding: Einstein Kickbox - Young Scientists

The regenerative potential of the heart is not sufficient to repair damage caused by myocardial infarction. Thus, there is an urgent need for approaches that reverse cardiac remodeling, e.g. by providing a pro-regenerative environment or generating de novo heart muscle cells. Research on multipotent cardiac progenitor cells (CPCs) has gained traction as they can differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo, potentially overcoming the limitations of current cell therapy strategies. In humans, various populations of CPCs, including Sca-1+, have been shown to improve cardiac function in preclinical models of myocardial infarction, contributing to the regeneration of the infarcted area. However, limitations associated with these cells, particularly poor accessibility and ethical concerns, hamper clinical translatability. Human induced pluripotent stem cells (hiPSCs) represent a readily available source of autologous fetal-like CPCs. We found that Sca-1+ cells isolated from hiPSCs during cardiomyocyte differentiation exhibited a stable phenotype through in vitro cultivation and were similar to human fetal CPCs in terms of morphology and gene expression of cardiac markers such as GATA4 and Nkx2.5. Our aim is to further characterize Sca-1+ cells to gain a better understanding of different Sca-1+ subpopulations and to identify GMP-compliant isolation and expansion strategies using single-cell RNA sequencing and cell surface marker screening.

The influence of Short Chain Fatty acids and other metabolites produced by commensal bacteria on macrophage biology – a high throughput screening approach

Team: Elena von Coburg, Caitlin Jukes, Philipp Burt, Liviana Ricci

Funding: Einstein Kickbox - Young Scientists

Commensal bacteria play a hugely important role in maintaining gut health by producing essential vitamins and breaking down non-digestive carbohydrates. Previous studies have shown that the microbial communities and their metabolites provide crucial signals upon inflammatory insults that alter the immune response of the host. Of note, the microbiome itself and its metabolites can be altered in human diseases such as inflammatory bowel disease (IBD). It is thought that these metabolic changes can impact the phenotype of immune cells within the gut. Intestinal macrophages are highly sensitive immune cells reacting to environmental cues. They adjust their pro-inflammatory and anti-inflammatory properties to modulate immune responses and maintain mucosal homeostasis. Moreover, numerous studies have shown the anti-microbial impact of bacterial metabolites on macrophages and their pro-inflammatory properties in IBD patients. To date, it is unknown which bacterial species are able to drive this impact and whether this effect can be recapitulated in vivo. In our project, we aim to identify a common molecular signature that drives an anti-microbial phenotype in macrophages by combining functional bacteria killing assays with high throughput techniques for gene expression and cytokine secretion. This will allow us to rapidly screen bacteria, which have regenerative therapeutic potential based on their ability to ameliorate the pro-inflammatory macrophage phenotype found in inflammatory diseases.

Unravel fibronectin tensional state – how does 3D physiological microenvironment impact early ECM formation?

Team: Matthias Kollert, Georgios Kotsaris, Julia Mehl

Funding: Einstein Kickbox - Young Scientists

The interplay of cells with their surrounding matrix is essential in homeostasis, regeneration and diseases. Apart from cell morphology being affected by mechanical cues from their microenvironment, it was found that early native extracellular matrix (ECM) development affects cell function. The interactions of structural ECM components were shown to be mechanoregulated as cells mediate the remodeling of fibronectin (Fn) matrix into a predominantly collagen matrix.

Fn is one of the most abundant ECM proteins and exhibits different conformational states depending on local strains in early ECM development which directly affects ECM formation. For example, in vitro experiments in 2D systems revealed that the tensional state of Fn regulates the nucleation of other ECM proteins such as collagen I. Here, we want to use a physiological 3D microenvironment with viscoelastic material properties to investigate early ECM formation in vitro.

ECM is a dynamic network undergoing constant modifications during regeneration. Understanding the mechanoregulation of early ECM formation by elucidating the spatiotemporal tensional states of the Fn matrix could answer why Fn is crucial for the assembly of other ECM proteins and how it is affected by tissue mechanical properties. We propose a novel strategy combining 3D alginate hydrogels with Fn visualization techniques which will be beneficial to regenerative therapies and tissue engineering and could be extrapolated to model degenerative diseases.

Next generation CAR-T – A novel virus-free approach to generate safe and effective CAR-T cell products for third-party use

Team: Dimitrios L. Wagner, Jonas Kath und Leila Amini

Funding: Einstein Kickbox - Young Scientists

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. Therefore, this "next generation" of CAR-T cells may enable the generation of potent but cost-effective treatments of cancers. 

Photo-crosslinkable cytokine-release system for enhanced bone regeneration

Team: Norman Drzeniek, Paul Köhli

Funding: Einstein Kickbox - Young Scientists

The only commercially available drug for the treatment of non-union of long bone fracture, local recombinant BMP-7, was taken off the market due to serious side effects, leaving orthopedic surgeons without any further pharmacological solutions for a challenging pathology.

Since then, IGF-1 has been investigated among other cytokines as a promising alternative for bone healing. However, the key limitation of cytokine therapy remains the short in vivo half life of just a few minutes and so far no alternative to BMP-7 made it to clinical application.

To address this issue we want to design a novel type of "in vivo drug factory" that would prolong the release of IGF-1 in vivo but could also be easily assembled during surgery and injected into the fracture gap. Our strategy involves the combination of mRNA-transfected hMSC as a transplantable source of IGF-1 with a biomaterial vehicle that would interact with cell-secreted cytokines and modulate their release kinetics.An additional engineering challenge lies in combining cell transfection with cell encapsulation and material injection into a simple procedure that respects the clinical setting of use.

The role of the circadian clock in bone healing

Team: Denise Jahn, Serafim Tsitsilonis, Achim Kramer, Johannes Keller

Funding: Einstein Kickbox - Advanced Scientists

Impaired bone healing occurs in up to 10 percent of fractures and leads to pain, a long-term reduction in the quality of life and high socio-economic costs. More than 50 years ago, clinicians observed the phenomenon of traumatic brain injury (TBI) accelerating fracture healing. Until now, the underlying pathophysiology remained unknown, but we previously found compelling evidence that the stimulatory effect of TBI on fracture healing is transmitted through an increased adrenergic signaling, caused by an interrupted circadian clock. Several studies have shown a circadian rhythm in bone metabolism, affecting osteoclast activity, osteocytes and osteoblast function. Furthermore, the circadian clock regulates the body physiology through the sympathetic nervous system, which has a strong impact on bone tissue. Although there are a number of studies investigating the influence of the circadian clock on bone metabolism, the impact on fracture healing is largely unknown. In our project, we would like to investigate the role of the circadian clock in bone healing. Therefore, we will use a mouse line with a disrupted circadian clock in our standard osteotomy model. In addition, we will monitor the circadian rhythm of these mice as well as the released catecholamine. The understanding of the role of the circadian clock in fracture healing holds great potential to aid in the development of new drugs to improve fracture healing.

Developing menstrual blood–derived mesenchymal stem cell transplantation as an anti-inflammatory and proregenerative therapy for osteoarthritis by silencing TLR2 expression

Team: Ping Shen, Max Löhning, Hans-Dieter Volk, Tobias Winkler, Tazio Maleitzke, Lisa Grunwald

Funding: Einstein Kickbox - Advanced Scientists

Osteoarthritis (OA) is a chronic joint disease featured by cartilage deterioration and chronic pain. Regardless of the complexity of the initial causes, chondrocytes residing in cartilage are exposed to endogenous TLR agonists that are generated during cartilage breakdown. For instance, the 32-mer and 29-kDa fibronectin fragment, enzymatic products of matrix proteins, have been detected in the synovial fluid of OA patients and revealed an anti-anabolic, pro-catabolic and pro-inflammatory function by activating TLR2-mediated signalling pathways. Mesenchymal stem cells (MSC) hold great promise as treatment for the inflammation-accelerated degenerative disease OA, as they simultaneously carry regeneration potency and immunomodulatory capacity. On the other hand, MSCs are sensitive and respond to the inflammatory and degenerative cues, such as TLR agonists in the diseased microenvironment, to secrete inflammatory cytokine IL-6, IL-8 and G-CSF. We plan to overcome this limitation by endowing MSCs with a resistance to the local TLR2 agonists by silencing TLR2 expression. We will use menstrual blood–derived mesenchymal stem cells (MenSC) which hold, among other benefits, the advantage of non-invasive and periodical acquisition. Thus, we will transplant TLR2ko MenSCs to the knee joints of the OA-diseased guinea pigs and evaluate whether the knockout of TLR2 will endow MenSCs with an advanced therapeutic effects comparing to wild type MenSCs.

Phenotype and transcriptional landscape of T cells in acute and chronic joint inflammation

Team: Caroline Peine, Nayar Durán, Tazio Maleitzke, Philipp Burt

Funding: Einstein Kickbox - Advanced Scientists

Osteoarthritis (OA) is a severe disease with a very high prevalence world-wide. Only recently, the involvement of an inflammatory component in OA was recognized. Inflammatory cell infiltrates are frequently found in the synovium and the adjacent infrapatellar fat pad of the affected joints. In addition, also joint traumata are associated with a phase of synovial inflammation and it is generally accepted that patients that have experienced such a trauma have an increased likelihood to develop OA in the respective joint later in their life. Intraarticular soft tissue inflammation can be detected very early in the course of OA and is correlated with more rapid cartilage destruction. Besides macrophages and neutrophils, infiltrates of T cells are found in OA-associated joint inflammation. Mouse models have shown a substantial contribution of CD4+ and CD8+ T cells to the development of posttraumatic OA. Hence, using flow cytometry, we will study which T cell subtypes are present in the inflamed intraarticular soft tissues early after traumatic events such as meniscal tears compared with those in late-stage OA patients undergoing knee-replacement surgery. Thus, we can potentially identify specific pathogenic T cell subpopulations that are present both in acute and chronic joint inflammation and that could present a potential therapeutic target for the early treatment of posttraumatic OA.

Models deciphering mechanical force-induced tenocyte-immune cell signaling

Team: Franka Klatte-Schulz, Birgit Sawitzki, Sara Checa Esteban, Georg Duda, Gäbel Christiane, Serafim Tsitsilonis

Funding: Einstein Kickbox - Advanced Scientists

Tendon pathologies are very common and represent a significant burden for the patient. The pathomechanisms of chronic tendon pathologies (Tendinopathy) as well as failed tendon regeneration after acute rupture are not fully understood. Therefore, to date there exist no treatment option to support tendon healing, or to prevent the development of tendinopathy.

Excessive mechanical forces can cause acute ruptures and recurrent mechanical overload induces micro ruptures that lead to chronic conditions. Furthermore, inflammation is crucial for tendon healing and the development of tendon pathologies. In autoimmune-driven Achilles tendon enthesitis, IL23 Receptor (IL23R) expressing γδ T-cells that secrete IL17 have a driving pathogenic role. Previously, we showed that IL17/IL23R signaling is also present in non-autoimmune tendon pathologies. We hypothesize that mechanical strain together with inflammatory signals can modify the tenocyte-immune cell communication.

We will use an ex-vivo model of mechanical force-induced tenocyte-immune cell communication and gather information on factors influencing the IL17/IL23R signaling. In addition to varying mechanical strain or time, we will test different inflammatory triggers and cellular compositions on the secretion of IL6/IL23/IL17 associated cytokines and MMP classes. The obtained results will be used to develop a mechano-biochemical mathematical model, which aims on investigating the spatio-temporal regulation of IL6/IL23/IL17 signaling under mechanical and inflammatory stimulation.

Clinically Usable Allograft Cell-based Assay for the Assessment of Alloreactive T cells in Kidney Transplant Patients

Team: Constantin Thieme, Nina Babel, Toralf Roch

Funding: Einstein Kickbox - Advanced Scientists

Suppressing unfavorable immunity towards the allograft and the complications that this immune suppression entails are main challenges in organ transplantation. Several immunosuppressive agents and protocols are currently in use and applied according to a rough risk assessment using the HLA-mismatch, the level of antibodies against alloantigens, and individual experience of the treating clinician. Thus, the initial treatment can be adjusted with the onset of complications due to too strong or too weak immunosuppression. However, complications harming the allograft are currently only detected when the graft function is already impaired, often leading to irreversible graft damage. This deteriorates the long-term graft survival. Therefore, there is an urgent clinical need for guidance of immunosuppressive therapy. The research project that we will execute with support of the Einstein Kickbox grand is based on a method established and patented in our group that uses kidney transplant cells collected and cultivated from urine of kidney transplant patients. We envision redefining our protocol in order to make it more simple and applicable in a clinical context. The Einstein Kickbox grant with the BioThinking support will enable us to generate ideas and hypothesis that facilitate the assay optimization under consideration of the clinical and diagnostic demands. It also will help to find support from funding institutions and potential collaboration partners in the industry.

ECRT Research Grants - Funding Period 2020-2022

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 Stachelscheidt, Erik Brauer

Funding: ECRT Research Garnt

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, Petra Reinke, Stephan Schlickeiser, Ola Winqvist, Amir Sherif

Funding: ECRT Reserch 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

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

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, Uwe Kornak, Sven Geißler

Funding: ECRT Research Grant

ECRT Research Grants - Funding Period 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, in press). 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

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: Birgitt Sawitzki, Britt Wildemann, Franka Klatte, Anja Kühl, Uta Syrbe, Martina Seifert

Funding: Einstein Kickbox - Advanced Sciensists & 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

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

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant