Research achievements

In the 12-year funding period, the SFB was able to sharpen its profile as a unique consortium of basic scientists and clinicians joining to develop innovative approaches to control unwanted immune reactions. Promising results were obtained demonstrating that addressing the cellular machinery of the adaptive immune system can provide novel ways to treat or even cure autoimmunity and other chronic inflammatory diseases.

Our work could confirm the rational for founding the SFB 12 years ago - the ultimate goal, the cure of autoimmune or chronic inflammatory disease and induction of allograft tolerance, requires both elimination of pathogenic, chronically activated (long-lived) effector/memory cells, and strengthening the regulatory mechanisms providing long-term tolerance. This concept has to be embedded into a strategy for personalized therapy by implementing biomarkers for patient stratification. Fortunately, expertise for either field was represented in the SFB, and the diversity of different approaches targeting diverse cell populations and diverse mechanisms delivered several candidate concepts that finally may result in successful application in the clinic. Some of them could be proved in first clinical trials.

We see the SFB650 as a success story demonstrating how such a CRC structure supports interdisciplinarity which is in particular important for translational research. Many “spin-off” projects have been developed from the CRC consortium that supported now at individual grant level (e.g. ERC), or by forming new national and international consortia (e.g. EU grants initiated by CRC members). Moreover, the research success helped career development of CRC members as proven by several calls to prestigious local and external positions.

 

a)     Progress in utilizing the power of regulatory cell populations

Classical immunosuppression is able to achieve transient relieve from disease symptoms due to inhibition or elimination of effector cells or their products, yet permanent cure is only possible by a concomitant strengthening of the self-control mechanisms of the immune systems leading to a balanced, tolerant immune system. For these reasons, major efforts in the SFB650 was targeting immunoregulatory components of the immune system, notably regulatory T and B cells but also suppressive Th2 cells and regulatory macrophages.

 

TP1 Hamann: Induction of stable Foxp3+ regulatory T cells demonstrated the demonstrated the impact of differential homing on the functional specialization and efficacy of Tregs. It contributed to the generation and characterization of a Foxp3-reporter mouse ("DEREG") used worldwide now. The role of epigenetic modification of a novel region (Treg-specific demethylation region "TSDR") in the Foxp3-locus for a stable phenotype of nTregs were discovered and the involved transcription factor bindings sites identified. The demethylation of the TSDR already occurs in the thymus and proceeds via active demethylation. It could be shown that this epigenetic feature is a valid biomarker for stable Tregs in mouse and man. Furthermore, it was demonstrated that coupling of antigenic peptides to synthetic carriers or nanobeads improves their tolerogenic efficacy and protects from developing EAE. In addition, it was shown that the inhibitory cytokine IL-27 has a dampening role in acute inflammation and infection and recombinant IL-27 is able to reduce immunopathology and death in an influenza model.

 

TP2 Fillatreau: Engineering resting B cells for the suppression of unwanted immunity developed a novel approach to genetically engineer naïve resting B cells while maintaining them in a quiescent state in terms of expression of cell surface receptors. The approach enables the expression of complete antigen-coding sequences in resting B cells. This allows to capitalize on the tolerogenic properties of antigen-presenting resting B cells to inhibit the development of undesirable immune responses. We have used this technology to suppress the development of disease in a pre-clinical model of multiple sclerosis (EAE). We found that the adoptive transfer of such naïve resting B cells genetically engineered to express disease-relevant autoantigen resulted in the suppression of antigen-specific CD4+ and CD8+ T cell and B cell responses in recipient mice. To optimize this approach, we have delineated the mechanisms controlling the pathogenic and protective functions of B cells in autoimmune diseases. IL-6 production is a major mechanism of B cell-mediated pro-inflammatory function. Importantly, IL-6 producing B cells additionally secrete large amounts of GM-CSF in the human autoimmune disease multiple sclerosis. It is therefore critical to exclude any B cell with the capacity to produce IL-6 from our genetically modified B cell preparation. Our protocol used for genetic engineering does not trigger IL-6 expression by B cells in vitro. With the further identification of novel mechanisms implicated in the suppressive functions of B cells, such as their production of IL-35, we have provided novel opportunities to develop next generation of suppressive therapeutic B cells, once the challenge of expressing IL-35 is solved.

 

TP4 Baumgrass: Regulatory T cell induction by manipulation of transcription factors mainly investigated the molecular mechanisms and therapeutic potential of impaired TCR signaling and modulated expression/ activation of transcription factors (TFs) in T helper (Th) cells for strengthening tolerance. Thereby, we focused on manipulation of two crucial processes - induction of Treg cells and regulation of IL-2 expression by Th cells. Using in vitro and in vivo approaches in men and mouse we asked: i) Which roles do certain transcription factors and signaling pathways play in Treg cell induction and function? ii) Can low-dose CsA therapy imitate impaired TcR signaling and induce Treg cells in AD patients? iii) How does the regulatory network of IL-2 expression work?

The main results can be summarized as follow: 1) Discovery of in vivo induction of Treg cells by low-dose cyclosporine A therapy in AD patients. The therapeutic effect of low-dose CsA therapy in AD patients is not just mediated by a moderate immunosuppression of T cell hyperactivity but also by immunomodulation;

2) Identification of c-fos and NFATc2 as limiting factors of IL-2 production during initial Th-cell activation;

3) Demonstration that low Foxp3 level and concomitant high levels of NFATc2, c-Jun, c-Fos, and NF-kBp65 determine the anergic phenotype of Treg cells; 4) Discovery of the transcription factor TGIF1 as a repressor of peripheral Treg cell induction and IFN-γ expression of Th1 cells. Our data suggest that TGIF1 is associated with glycolysis and caspase 8-mediated apoptosis; 5) Identification of overrepresented DNA binding motif-pairs Runx-NFAT in Treg specific genes using in silico analysis with Herzel (ITB Berlin) and Runx as an interaction partner of NFATc2 by SILAC-based mass spectrometry; 6) Establishment of a “bioassay” to determine specific CsA effects in human whole blood samples by quantitative measurement of NFATc1/IL-2 in T cells using flow cytometry. This assay can be used to predict individual CsA efficacy and toxicity; 7) Demonstration that only a binary IL-2 expression ensures a wide linear antigen response range for Teff and Treg cells under real spatiotemporal conditions (in vivo). At low antigen concentrations binary IL-2 expression safeguards by its spatial distribution selective STAT5 activation only of closely adjacent Treg cells regardless of their antigen specificity. Our data show that the mode of IL-2 secretion is critical to tailor the adaptive immune response to the antigen amount.

 

TP6 Hartmann: Application of helminth-derived concepts to treat inflammatory diseases uncovered several aspects of nematode-induced modulation of host responses.

Worm infections are often chronic in nature, whereby an initial strong and protective Th2 response is followed by downregulation of effector molecules and induction of a regulatory response (Breg, Treg). Helminths release immunomodulatory molecules and target specific cells to ensure their survival within the host. These strategies also affect unrelated inflammatory diseases such as allergic airway inflammation and intestinal inflammation. Within the CRC project TP6 we showed that a specific helminth immune modulator, filarial cystatin elicited a distinct population of regulatory macrophages (Mreg). The nematode cystatin-induced Mreg stimulated IL-10 in CD4+ T cells and was able to suppress asthma and colitis in mice in a IL-10-dependent manner. Such regulatory monocytes could also be detected in a patient cohort in India suffering from filariasis. Only immunosuppressed patients which carried larval stages in the blood showed regulatory monocytes in their blood which had the capability to interfere with T cell responses. A translational approach was pursued by the establishment of a novel therapy via a transgenic probiotic secreting nematode-cystatin for site-directed treatment of gut inflammation. Furthermore, in enteric nematode infections we revealed novel regulatory roles of prominent innate immune cells involved in the helminth host interaction. Thereby, mast cells were shown to orchestrate the early immune response by induction of tissue derived cytokines and eosinophils were illustrated to exhibit regulatory roles by suppressing Th2 responses in favor of maintaining IgA class switch and thus gut homeostasis. Finally, we identified highly functional tissue resident Th2 memory cells in non-lymphoid compartments and could show that exclusively Th2 memory cells residing in the peritoneal cavity show innate effector function in response to tissue alarmins.

 

TP10 Babel, (Riemekasten): Evaluation of the therapeutic potential of regulatory T cells in human and murine lupus by application of low-dose IL-2 in vivo.

During the funding period, we were able to develop two major concepts from basic research to clinical applicability. First, we observed in a murine SLE model that Treg cells develop a homeostatic disturbance due to an acquired deficiency of IL-2 and that treatment with IL-2 corrects these Treg defects and prolongs the life-span of these mice. In a pilot clinical study, we translated this concept into the clinic and treated 12 refractory SLE patients with recombinant IL-2 within a phase I/IIa clinical trial. In these patients, we observed a quite selective expansion of the Treg cell population and importantly most patients responded clinically to the IL-2 treatment with only minimal side effects.

Interestingly, T cell receptor repertoire analyses by NGS for monitoring T cell repertoire at clonal level in follow-up revealed no clonal expansion under IL-2 treatment suggesting a polyclonal Treg expansion by IL-2 therapy.

Second, following our hypothesis that T cells play a pivotal role in the pathogenesis of Lupus nephritis (LN), we were able to establish flow cytometry of the urine to directly analyze kidney derived T cells and other immune cells in patients with LN. Interestingly the urinary T cell counts precisely separate patients with active LN form those with non-active LN or without renal involvement. This observation could be confirmed in a large cohort of 147 SLE patients and showed also usefulness for therapy monitoring. Presently urinary T cells are one of the most precise novel biomarkers for LN that are under development.

 

TP14 Sawitzki, Pascher: Influence of the early immune monitoring profile on outcome (rejection/induction of tolerance) after liver transplantation.

Within the CRC650 we have contributed to a better understanding of tolerance induction especially towards allogeneic solid organ transplants in preclinical models as well as patients. We could show that preformed antigen-reactive but also heterologous e.g. CMV-reactive memory T cells as well as conventional immunosuppressive drugs are a barrier to tolerance induction. However, adoptive transfer of CD4+CD25highFoxp3+ regulatory T cells enables tolerance induction and maintenance even under challenging conditions if combined with T-cell targeting. The mechanisms behind are most likely by inducing a gene expression pattern, which is linked with expansion of regulatory B cells. In addition, we have identified a transcriptional profile specific for more differentiated and inflammatory human effector memory and terminally differentiated effector memory T cells, which will be the basis for the development of novel therapeutic approaches in chronic inflammatory diseases.   

 

TP20 Romagnani, Uharek: Exploiting NK cells for suppression of unwanted immune responses in transplantation

Natural Killer (NK) cells are innate immune cells which play an important role in the early defence against pathogens, in particular Cytomegalovirus (CMV) infection and tumours. NK cells display the ability to kill allogeneic hematopoietic cells and infected cells but are unable to induce graft versus host disease (GvHD). In pre-clinical models, it has been shown that allo-reactive NK cells can prevent GvHD and graft rejection, which represent the most frequent and severe unwanted immune responses and complications during allogeneic hematopoietic stem cell transplantation (HSCT). In order to test immunoregulatory properties of NK cells, in the two previous funding periods we have performed adoptive transfer of human NK cells after haploidentical HSCT in a Phase I/II clinical trial and analysed the impact of the treatment on survival, GvHD and graft rejection. Importantly, by tracking donor NK cells in the recipients, we identified a novel population of polyfunctional NK cells, representing an intermediate stage of NK cell differentiation, which combines the ability to expand in vivo and to display effector functions. Moreover, the analysis of selective expansions of NK cell subsets in response to CMV infection/reactivation occurring in recipients of HSCT prompted us to investigate their phenotypic, molecular and functional properties, leading us to show for the first time that CMV-induced NK cell expansions undergo a global epigenetic reprogramming and acquire stable imprinting of the IFNG locus, similar to memory CD8+ T cells and Th1 cells. Finally, due to the expertise acquired during this funding period in NK cell recognition and differentiation, we have applied this knowledge to study other recently described populations of NK-like innate lymphoid cells (ILCs) and enabled the identification of triggers of ILC activation and differentiation.

 

TP26 Fillatreau, Scheffold, Uckert: Redirecting the antigen-specificity of polyclonal regulatory T cells to optimize their therapeutic efficacy and safety have accomplished major steps forward for monitoring antigen-specific Treg in human, for the genetic engineering of such cells, and for their optimal utilization in the clinic.

We have developed tools allowing the direct analysis of Foxp3 Treg reacting against certain antigens of interest. This technology has been used for the first comprehensive description of Treg reactions in human against pathogens and potential tolerogens [Bacher et al Cell (in press)]. The technology will provide the basis for selecting ex vivo Treg with relevant antigen-specificty for subsequent in vitro expansion and generation of an antigen-specific Treg transplant. We have also tested activation marker combinations allowing to isolate stable and molecularly defined polyclonal Treg ex vivo or even from expanded Treg cultures. This was used to select stable Treg, e.g. following CAR transduction in vitro, and provides a unique tool for the generation of stable genetically engineered Treg with defined (re-directed) target specificity with therapeutic relevance. To capitalize on this approach, we have addressed in a mouse model of T cell-mediated autoimmunity (EAE) how the antigen recognition properties of the T cell receptor (TCR) expressed by Treg influence their protective function. We found that the functional avidity of the TCR for the disease-relevant autoantigen critically determined the protective value of Treg in adoptive cell therapy. Thus, Treg expressing a TCR of high functional avidity for the target autoantigen almost completely protected mice from disease, while Treg expressing a TCR of low functional avidity had little effect on the disease course. Novel protocols to improve the genetic engineering of Treg were established.

 

TP28 Löhning: Suppression of autoreactive Tcell responses by Th2 cells

Studying the stability of Th1- and Th2-polarized T helper cells, we found out that stable Th2-polarized T cells such as T-bet–deficient (Tbx21-/-) Th2 cells produced high amounts of IL-10. Therefore, we hypothesized that these stable Th2 cells could suppress inflammatory T cell responses. The principal aim of our project was to investigate the mechanisms underlying the immunosuppression of T cells responses by virus-specific Tbx21-/- Th2 cells during Th1-promoting lymphocytic choriomeningitis virus (LCMV) infection. The main results can be summarized as follow: 1) T-bet–deficient Th2 cells suppress endogenous LCMV-specific T cell responses and modify their effector phenotype in vivo. The data indicate that adoptively transferred Tbx21-/- Th2 cells strongly suppress the expansion of virus-specific CD8 and CD4 T cells and in parallel modify their effector phenotype towards a memory-like phenotype during the effector phase of LCMV infection; 2) T-bet–deficient Th2 cells suppress not only naïve but also memory LCMV-specific CD8 T cells in vivo. We demonstrated that LCMV-specific Tbx21-/- Th2 cells strongly suppressed the expansion of co-transferred LCMV-specific memory CD8 T cells in vivo at the peak of infection. Moreover, memory CD8 T cells produced lower amounts of IFN-γ in Tbx21-/- Th2 cell recipient mice compared to control mice at the peak of infection; 3)T-bet–deficient Th2 cells protect mice from type I diabetes development. Tbx21-/- Th2 cells were able to completely protect RIP-GP mice from the development of type I diabetes, whereas all control mice became diabetic at day 10 post infection. Tbx21-/- Th2 cell recipients maintained blood glucose levels around 100mg/dL, whereas control mice displayed levels over 400mg/dL at day 8 post infection. We showed that Tbx21-/- Th2 cells were able to protect RIP-GP mice from diabetes development by suppressing the number of LCMV-specific CD8 T cells and their IFN-γ production; 4) Tbx21-/- Th2 cells downregulate costimulatory molecules expression on dendritic cells after LCMV infection in vitro. We identified that Tbx21-/- Th2 cells impair two crucial events during the activation of dendritic cells: the upregulation of their costimulatory molecules and their IL-12 and IFN-β production; 5) Using our LCMV infection model, we demonstrate that IL-10 but nor IL-4 or IL-13 production of Tbx21-/- Th2 cells was necessary and indispensable for the downregulation of costimulatory molecules on both lymphoid and myeloid dendritic cell subsets as well as their IL-12 and IL-1β production in vitro; 6) Lastly, we could show that IL-10 produced by Tbx21-/- Th2 cells mediates LCMV-specific CD8 T cell immune-suppression via downregulation of costimulatory molecules on dendritic cells in vivo.

These findings indicate that Tbx21-/- Th2 cells, by means of their high and stable IL-10 expression, possess a strong immunoregulatory potential for the suppression of unwanted inflammatory responses.

 

b)    Progress in targeting pathogenic memory/effector cells

 

TP5 Worm, Heine: Immune modulation of allergic diseases by nuclear hormone receptor ligands established perspectives of the function and therapeutic potential of vitamin D in type I allergies from bench to bedside based on data from cell biologic and molecular biologic approaches, validation in preclinical models leading to clinical translation into 3 clinical pilot trials.

We identified that human B cells can produce active vitamin D metabolite, calcitriol or chem. 1alpha,25-dihydroxy vitamin D3 and in consequence enhance IL-10 expression. Beyond, we clarified the mechanism how vitamin D receptors inhibit IgE-class switch, namely by a transrepressive DNA-complex to the IgE switch promoter, but also by interference with NF-kappaB activation. We validated our findings in preclinical models and proved that VDR inhibitis IgE in vivo, using a low-calcemic synthetic analogue. However, the synthetic ligand did not reach the clinical stage for safety reasons. But as we had shown that activated immune cells can actively synthesize calcitriol from its precursor 25-hydroxyvitamin D (25(OH)D we investigated, whether precursor vitamin D supplementation may target immune cells in vivo. In summary, during the funding period of this CRC we established a cell-biologic mode of action of vitamin D, which we confirmed as proof-of-principle in a preclinical model. We identified endogenous calcitriol synthesis in human cells and established a controlled setting for clinical investigation in patients by learning from two investigator-initiated trials.

We then raised the hypothesis that endogenous calcitriol can target specifically activated immune cells, if the precursor 25(OH)D is provided together with the antigen in a sufficient manner. In consequence, the specific IgE-response should be blocked and the IgE-dependent allergic response should be reduced. As new immune cells are generated and programmed towards a non-IgE response, the impact of vitamin D should induce a long-lasting tolerance. To prove this hypothesis, we investigated the impact additional provitamin D together with the allergen specific immunotherapy (SIT) in a preclinical model (Heine et al., 2014b). We found that 25(OH)D controls IgE sensitization and established a novel SIT-protocol in mice allowing to determine long-term efficacy. Finally and most importantly our data show that 25(OH)D enhances the SIT efficacy regarding both, block of the IgE-response and the allergic airway inflammation after repetitive allergen challenge. We translated our findings from the clinical pilot trial according AMG and GCP-criteria with authorization of the BfARM, Paul-Ehrlich-Institute and the Ethikkommission des Lageso Berlin (“ProGIT”: Provitamin D in Grass-pollen specific Immunotherapy, NCT NCT01466465, clinical trials.gov). In this trial we investigated vitamin D-deficient patients with grass pollen allergy regarding the response to a grasspollen-specific immunotherapy who received at the same time 5000 IU vitamin D or placebo in a double-blind randomized manner over 3 years. The last patient was treated and the data analysis is ongoing. Preliminary data support our hypotheses that a) activated immune cells actively respond to provitamin D due to endogenous calcitriol synthesis and b) the allergic IgE response can be blocked by vitamin D. The data will allow the power calculation for a randomized multi-center trial to prove this hypothesis regarding efficacy.

 

TP12 Alexander, Hiepe, Arnold: Development of tailored protocols for tolerance induction by immunoablation and hematopoietic stem cell transplantation in autoimmune diseases

 

Our previous studies during early funding period of CRC provided the proof-of-concept that an autoreactive immune system can be reset into a naive and self-tolerant state by eradicating the immune system with antithymocyte globulin (ATG) followed by transplantation of hematopoietic stem cells (HSCT) to regenerate the immune system, i.e. immune reset. In the last funding period we continued our investigator-initiated trial of immune reset vs. „standard of care“ in SLE (ASSIST, NCT00750971) with 7 patients include so far. By further analyzing the immune reconstitution after immune reset, we could demonstrate that remissions observed after immune reset were associated with a regeneration of Helios-expressing Foxp3+ regulatory T cells (Treg) with increased markers of thymic origin, such as CD45RA and CD31 and normalized TCR repertoire, suggesting that thymic output generates a new pool of natural Treg. In addition, by performing gene expression analysis of FACS-sorted CD14+ monocytes, we could demonstrate that the up-regulation of type-I interferon regulated genes, the so called IFN-signature, is completely abrogated after HSCT in SLE, indicating that immune reset disrupts the chronic activation of the innate immune system with reprogramming into a self-tolerant state. Furthermore, by analyzing the fertility status and pregnancy outcomes in 15 patients with autoimmune diseases treated with immune reset, we could show that four women had five pregnancies and six healthy offsprings during follow-up, and no miscarriages were observed, demonstrating that HSCT offers the opportunity to conceive during treatment-free remissions with favorable pregnancy outcomes.

 

TP16 Dörner: New generation anti-B cell therapy

SLE is a systemic autoimmune disease with different clinical manifestations and characterized by a loss of B cell tolerance reflected by the production of anti-nuclear antibodies and immune abnormalities. Immunotherapy targeting B-cells is therefore of current interest. In the project, CD22 expression in autoimmunity and the molecular effects of the humanized anti-CD22 antibody epratuzumab have been studied. CD22 as inhibitory co-receptor of the B cell receptor is expressed only on B cells and plays a crucial role during B cell activation by regulating the BCR threshold. After BCR activation, CD22 gets activated leading to the recruitment of the phosphatase SHP-1 which leads to the dephosphorylation of the key kinase Syk. This dampens the signaling strength. In addition, CD22 acts as an adhesion molecule, for the homing of re-circulated is differentially expressed during B cell differentiation.

We could show, that naïve B cells express higher levels of CD22 compared to antigen experienced B cells and is absent on plasma cells and the overall CD22 expression in SLE is higher than in controls. Furthermore, B cells treated with epratuzumab showed a decreased cell surface expression of CD62L and β7 integrin, while the expression of β1 integrin was enhanced. Additionally, CD22 activation by epratuzumab on B cells led to an enhanced spontaneous migration and migration towards the chemokine CXCL12. Further experiments highlighted that epratuzumab binding to CD22 induced an enhanced co-localization of CD22 with the BCR and SHP-1 with CD22 as well as a reduced calcium influx and phosphorylation of Syk and PLC-g2 after BCR activation. In vitro analysis could show a reduced production of the pro-inflammatory cytokines IL-6 and TNF-α, but not the regulatory cytokine IL-10 by B cells after activation of CD22 by Epratuzumab.

While all initial experiments were done using healthy donor B cells, the higher expression of CD22 on CD27- naïve B cells of SLE patients did not induce enhanced inhibition of BCR signaling reflected by the phosphorylation of Syk, PLC-g2 and Btk, as well as a reduced calcium influx. Furthermore, an enhanced survival and increased tyrosine phosphatase activity of SLE B cells compared to HD were found.

The overall data indicate that B cells of SLE patients are in a post-activation status with diminished BCR responses and reduced cytokine production while strikingly up-regulating CD22. The lack of clinical efficacy of epratuzumab in the two EMBODY™ Phase 3 clinical studies in Systemic Lupus Erythematosus (SLE) is consistent with our mechanism-of-action findings.

A key conclusion of our studies is that better understanding of the different B cell functions in SLE including the regulatory principles of BCR signaling and other functions, such as antigen presentation, check-point molecule expression is required to provide improved rationales to target B cells in SLE. However, our comprehensive study with epratuzumab targeting CD22 revealed a novel and promising clinical target for the treatment of SLE, the activation and balance of phosphatases especially the CD22 associated phosphatase SHP-1

 

TP17 Hiepe, Radbruch, Burmester: Targeting of memory plasma cells in autoimmune disease demonstrated that two different plasma cells compartments (short-lived plasmablasts and long-lived memory plasma cells) contribute to autoimmune pathology in autoimmune diseases and are therefore a promising therapeutic target.

Whereas short-lived plasmablasts reflect B cell hyperactivity, long-lived plasma cells secrete autoantibodies independently of B cell activation, T cell help and antigen contact. We demonstrated that long-lived plasma cells, which reside in niches in the bone marrow and inflamed tissues are refractory to conventional immunosuppressive drugs and therapies targeting B cells. They can be only depleted by immunoablation with ATG and proteasome inhibitors. Based on these data, we introduced the treatment with the proteasome inhibitor bortezomib in patients with refractory autoimmune diseases such as SLE, anti-NMDA receptor encephalitis and myasthenia gravis. In addition, we developed the strategy of antigen-specific plasma cell targeting and showed promising in vitro results.

 

TP23 Chang, Mashreghi, Radbruch: Targeting of pathogenic T helper cells in chronic rheumatic inflammation have identified several functional biomarkers, which are selectively expressed in pathogenic proinflammatory T helper memory cells, including the transcription factor Twist1, the transcriptional co-factor Hopx as well as the microRNAs miR-182 and miR-148a. Twist1 limits the Th1 mediated inflammation and indirectly promotes the survival of proinflammatory Th1 by transcriptionally regulating miR-148a expression. We could show that miR-148a promotes the survival of Th1 cells by targeting the pro-apoptotic factor Bim. Hopx which is also induced by T-bet, on the one hand protects Th1 cells from Fas-induced apoptosis, and on the other hand apparently regulates the metabolism of Th1 cells by interacting with factors involved in glucose transport and fatty acid synthesis. We could show that miR-182 is essential for proper clonal expansion of activated T helper cells by targeting Foxo1. Innovative therapeutic approaches using therapeutic oligonucleotides, so called Antagomirs, inhibited miR-182 expression, repressed clonal expansion and finally ameliorated in the transfer model of ovalbumin induced arthritis.  We further are evaluated the miRNA-148a as a target for the selective elimination of proinflammatory Th1 cells in T helper cell mediated transfer colitis model. Inhibition of miR-148a by systemic intravenous delivery of Antagomirs, reduced the number of proinflammatory Th1 cells in the inflamed colon and ameliorated inflammation. We could show that memory Th cells generated in protective and vaccine-induced immune responses against systemic pathogens, are preferentially located in the bone marrow, rest in terms of proliferation and express selective biomarkers including Ly6c (only murine cells), CD69 and CD49b. Of note, preliminary data show that systemic application of Antagomirs against miR-148a does not eliminate memory Th cells of the bone marrow, showing that miR-148a is a promising therapeutic target for the specific elimination of inflamed tissue resident memory Th1 cells

 

c)     Progress in technology support by Z-projects

 

Z2 Reinke, Volk: Coordination of clinical trials and development of GMP procedures for cell-based therapeutic approaches

The main goal of Z2 project was to develop and offer an infrastructure for translating new ideas into clinical biomarker and therapy studies. The following key elements can be defined for translating new therapeutic approaches into clinical investigator-initiated trials (IIT´s):

       -Establishing GMP manufactoring process for ATMPs

       -Manufactoring authorization for specific ATMPs by LaGeSo  

       -Study design and approval of study documents (IMPD) by the relevant regulatory authorities (PEI for ATMP) and the IRB

       -Establishment and validation of a biomarker portfolio for patient stratification, therapy monitoring, and mechanistic side studies

       -Performance of high-quality clinical trial (GCP)

       -Highly standardized biomarker analyses (GLP)

We established a broad portfolio of biomarkers for immune monitoring studies (Flowcytometry, functional assays, TCR analyses, gene expression, multiligand assays…) in collaboration with several groups (esp. TP14) and core units at BCRT. All test platforms mentioned are applicable and have been tested for feasibility in clinical studies. Many of the tests could be accredidated and has been audited for FDA/EMA compatibility (Core unit “immunocheck” at BCRT – spin-off of Z2). It was applied in several clinical trials performed by CRC members.

We developed, validated, and implemented into trials and Charité routine a robust IFNg-Elispot for detecting alloreactive (and other antigen specific) memory/effector T cells. Using this test, we could stratify kidney transplant patients perioperatively regarding their individual risk and guided for first time immunosuppression accordingly. After a promising pilot trial, under our guidance a European multicentre trialwas designed, approved and started at 2016 (personalised immunosuppression).

Furthermore, we developed a novel therapy approach to selectively target recently activated pathogenic effector T cells by a combination of short-term partial T-cell depletion and mTNF targeting at d2. This scheme resulted in excellent graft and patient survival in high risk patients (2/3rd graft, PRA+/PRT+) which were even long-term comparable with normal risk patients at standard treatment. Moreover, this scheme allows in normal risk patients for first time a monotherapy as early as d3 in almost all patients. This approach is presently tested in a multicentre trial guided by us.  

To allow low-dose immunosuppression in all patients, including those with enhanced pretransplant frequencies on donor-reactive memory/effector T cells, selective targeting of post-transplant rechallenged donor-reactive memory/effector T cells would be a novel approach. Our preclinical data showed that anti-TNF given 2-3 days post rechallenge could be a feasible option to reach this goal. First, we treated patient at enhanced risk (2nd/3rd graft, panel reactive antibodies and/or immune-mediated rejection within 1 year of previous graft) with the new induction protocol based on T-cell depletion (anti-CD3 or ATG) and one shot anti-TNF mAb at d2. 

Finally, we are able to develop a new, very efficient protocol to isolate and expand human nTreg for clinical use (from 50 ml blood) and the approved product was tested in a first-in-human study in kidney transplant patients (One study). The preparation was funded by CRC, the clinical trial by EU grant. A follow-up study (ProTreg) is almost ready for submission.

 

 

Z3 Kühl, Heppner: Histomorphological evaluation and in-situ analysis of regulatory and effector cells

The main focus of the central service project TPZ3 was the histopathology of regulatory and effector cells in health and disease. Standardized criteria for the evaluation of histomorphological changes following inflammation, infection or tumor from a wide variety of animal models and human samples were developed, refined and consistently applied e.g., for murine models of experimental autoimmune encephalitis development (TP2), graft-versus-host disease of the skin or within the colon (TP20), colitis (TP4, TP6) and intestinal symptoms of helminth infection (TP6) as well as arthritis (TP23). The systematic approach of the TPZ3 provided comparability of the histopathological evaluation within all research projects of the CRC650. A comprehensive guideline summarized the experiences for the histomorphological evaluation of intestinal inflammation in mouse models. First hand it offers help in assigning models to a specific category and scoring scheme to every researcher interested in established and new models of inflammation of the intestines. But it also exemplifies main steps in assessing any relevant mouse model of inflammation. Within the spirit of the CRC650 specifically translating findings from model systems to human disease, the TPZ3 challenged the relevance of about 90 mouse models of intestinal inflammation by extensively comparing its histomorphologies to human Crohn’s disease and ulcerative colitis.

Histochemistry, immunohistochemistry and immunofluorescence address regulatory and effector T cells within their context in situ that include other immune cells like eosinophilic granulocytes or plasma cells. These qualitative and quantitative analyses at single cell level revealed local interactions strongly supporting the more general histomorphological findings.

 

Z6 Grützkau, Häupl, Sawitzki: An integrated High-resolution immunophenotyping and computational biomarker discovery approach for monitoring clinical trials

The main objective of this Z-project was to provide an integrated hardware and computing platform to accompany clinical studies initiated within the CRC650 in terms of a comprehensive immunophenotyping of peripheral leukocyte subsets. Since several projects of the CRC consortium initiated clinical trials, in which cellular targets of the innate and adaptive immunity were therapeutically addressed, a global multiparametric immune monitoring approach by flow and mass cytometry was used to detect direct and indirect treatment effects at the single cell level of all blood cell compartments. So, this central technology platform helped to strengthen the translational activities to identify cellular biomarkers or signatures applicable for patient and therapy stratification.

Especially, mass cytometry (CyTOF-technology) was used since it allows a detailed fine-analysis of circulating leukocyte phenotypes by simultaneous monitoring of up to 100 parameters at the single cell level. This new technology just introduced in Germany for the first time at the DRFZ at the beginning of the new funding period of the CRC650 has been successfully established and implemented in several clinical studies.

We have developed and published the first OMIP (Optimized Multiparametric Immunophenotyping Protocol) applicable in mass cytometry. In addition, we have looked for alternative labeling options by introducing silver nanoparticles broadening the availability of heavy metal ion tags besides those of the lanthanide series. This labeling option is of special interest for the detection of weakly expressed antigens, since significant signal amplification is achieved by the application of nanoparticles of mean sizes of 10 nm, 40 nm and 100 nm. Perspectively, it is planned in collaboration with an industrial partner to enrich silver isotopes AM 107 and 109 to have additional tags available in mass cytometry. Another important technical development is the establishment of sample stabilizing protocols for whole blood samples. This allows the participation in multicentric clinical studies where blood samples were collected on site and subsequently sent as frozen samples to the center of analysis.

Mass cytometry and conventional cytometry-based multiparametric approaches generate huge amount of multidimensional data sets, which cannot be analyzed in a meaningful manner by manually created two-dimensional gating strategies. Therefore, an important aim was the development of an automated data clustering tool (immunoClust), which allows an unbiased way of data analysis in a hypothesis-generating manner. This tool has been also validated in frame of the so called FlowCap-competition where different software tools were compared with respect to identify features in high-dimensional flow cytometry data (e.g. cell populations) that most closely correlate with a clinical outcome.

In tight cooperation with our clinical partners of TP5, TP10, TP12 and TP17 we have focused on an in-depth phenotyping of blood cells of the following diseases: ankylosing spondylitis (AS), systemic lupus erythematosus (SLE) and atopic dermatitis (AD). We could identify NK cells in common and a subset expressing CD8 receptor in particular as biosensors for a successful therapy response to etanercept - a soluble TNF-a receptor in AS patients. The identified biomarker may help in clinical decisions regarding continuation of etanercept treatment or switching to another biological. Furthermore, the necessity of examining anti-TNF agents regarding their different modes of action is demonstrated here. In SLE we have focused on the identification of urinary biomarkers that can be used instead of taking renal biopsies to classify severity of lupus nephritis. In this context, we have aimed to define cell-based urinary abnormalities and signature. In this respect, mass cytometry was the method of choice since leukocytes present in the urine of active lupus patients were characterized by a high degree of autofluorescence. We have used our CyTOF-OMIP described before analyzing 29 surface markers in parallel at the single cell level. Most interestingly we could identify activated CD4 and CD8 effector lymphocytes characterized by the co-expression of CD38 and CD69 in urine samples only but not in corresponding blood samples. The frequency of these cells correlated with the disease activity of lupus patients and the severity grade of lupus nephritis. Furthermore, the promising blood-based biomarker Siglec1 (CD169) was characterized in more detail and has been now successfully established in the daily routine of lupus diagnostics at the Charité. Siglec1 is a mainly type I interferon-driven response protein cell-specifically expressed by peripheral monocytes. We could show that the expression correlates cross-sectionally and longitudinally over time with disease.