Publications by authors named "Stephan Fricke"

36 Publications

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications.

Front Immunol 2021 3;12:658314. Epub 2021 May 3.

Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.
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http://dx.doi.org/10.3389/fimmu.2021.658314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127837PMC
May 2021

Reduction of Graft-versus-Host-Disease in NOD.Cg-Prkdc Il2rg/SzJ (NSG) Mice by Cotransplantation of Syngeneic Human Umbilical Cord-Derived Mesenchymal Stromal Cells.

Transplant Cell Ther 2021 May 5. Epub 2021 May 5.

Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

Graft-versus-host disease (GVHD) is one of the major complications following hematopoietic stem cell transplantation, which remains the sole curative therapy for many malignant diseases of the hematopoietic system. The immunomodulatory potential of mesenchymal stromal cells (MSCs) to treat GVHD is currently being tested in various preclinical and clinical trials. Because the results of the preclinical and clinical trials on the use of MSCs to treat GVHD have not been consistent, we analyzed the potential beneficial effects of syngeneic versus allogenic treatment, culture expansion of MSCs, and various MSC cell doses and time points of MSC transplantation in a murine GVHD model. We established the murine GVHD model based on the transplantation of umbilical cord blood-derived hematopoietic stem cells (UC-HSCs) and used this model to assess the therapeutic potential of umbilical cord blood-derived MSCs (UC-MSCs). The use of HSC and MSC populations derived from the same donor allowed us to exclude third-party cells and test the UC-HSCs and UC-MSCs in a matched setting. Moreover, we were able to compare various doses, transplantation time points, and the influence of culture expansion of MSCs on the impact of treatment. This resulted in 16 different treatment groups. The most efficient setting for treatment of UC-HSC-induced GVHD reactions was based on the simultaneous administration of 1 × 10 culture-expanded, syngeneically matched UC-MSCs. This therapy effectively reduced the number of CD8 T cells in the blood, protected the mice from weight loss, and prolonged their survival until the end of observation period. Taken together, our data show beneficial effects of (1) syngeneic over allogeneic UC-HSCs and UC-MSCs, (2) culture-expanded cells over freshly isolated primary cells, (3) simultaneous over sequential administration, and (4) high doses of UC-MSCs. The animal model of GVHD established here is now available for more detailed studies, including a comparative analysis of the efficacy of MSCs derived from alternative sources, such as adipose tissue and bone marrow.
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http://dx.doi.org/10.1016/j.jtct.2021.04.018DOI Listing
May 2021

Can flow cytometry outperform genetic testing in eosinophilia patients?

Cytometry A 2021 Feb 1. Epub 2021 Feb 1.

Department of GMP Process Development, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany.

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http://dx.doi.org/10.1002/cyto.a.24311DOI Listing
February 2021

Cancer Stem Cells-Origins and Biomarkers: Perspectives for Targeted Personalized Therapies.

Front Immunol 2020 7;11:1280. Epub 2020 Aug 7.

Department of Immunology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

The use of biomarkers in diagnosis, therapy and prognosis has gained increasing interest over the last decades. In particular, the analysis of biomarkers in cancer patients within the pre- and post-therapeutic period is required to identify several types of cells, which carry a risk for a disease progression and subsequent post-therapeutic relapse. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and can cause relapses. At the time point of tumor initiation, CSCs originate from either differentiated cells or adult tissue resident stem cells. Due to their importance, several biomarkers that characterize CSCs have been identified and correlated to diagnosis, therapy and prognosis. However, CSCs have been shown to display a high plasticity, which changes their phenotypic and functional appearance. Such changes are induced by chemo- and radiotherapeutics as well as senescent tumor cells, which cause alterations in the tumor microenvironment. Induction of senescence causes tumor shrinkage by modulating an anti-tumorigenic environment in which tumor cells undergo growth arrest and immune cells are attracted. Besides these positive effects after therapy, senescence can also have negative effects displayed post-therapeutically. These unfavorable effects can directly promote cancer stemness by increasing CSC plasticity phenotypes, by activating stemness pathways in non-CSCs, as well as by promoting senescence escape and subsequent activation of stemness pathways. At the end, all these effects can lead to tumor relapse and metastasis. This review provides an overview of the most frequently used CSC markers and their implementation as biomarkers by focussing on deadliest solid (lung, stomach, liver, breast and colorectal cancers) and hematological (acute myeloid leukemia, chronic myeloid leukemia) cancers. Furthermore, it gives examples on how the CSC markers might be influenced by therapeutics, such as chemo- and radiotherapy, and the tumor microenvironment. It points out, that it is crucial to identify and monitor residual CSCs, senescent tumor cells, and the pro-tumorigenic senescence-associated secretory phenotype in a therapy follow-up using specific biomarkers. As a future perspective, a targeted immune-mediated strategy using chimeric antigen receptor based approaches for the removal of remaining chemotherapy-resistant cells as well as CSCs in a personalized therapeutic approach are discussed.
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http://dx.doi.org/10.3389/fimmu.2020.01280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426526PMC
April 2021

Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities.

Sci Rep 2020 07 30;10(1):12786. Epub 2020 Jul 30.

Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstrasse 1, 04103, Leipzig, Germany.

Ionizing radiation is widely used to inactivate pathogens. It mainly acts by destroying nucleic acids but causes less damage to structural components like proteins. It is therefore highly suited for the sterilization of biological samples or the generation of inactivated vaccines. However, inactivation of viruses or bacteria requires relatively high doses and substantial amounts of radiation energy. Consequently, irradiation is restricted to shielded facilities-protecting personnel and the environment. We have previously shown that low energy electron irradiation (LEEI) has the same capacity to inactivate pathogens in liquids as current irradiation methods, but generates much less secondary X-ray radiation, which enables the use in normal laboratories by self-shielded irradiation equipment. Here, we present concepts for automated LEEI of liquids, in disposable bags or as a continuous process. As the electrons have a limited penetration depth, the liquid is transformed into a thin film. High concentrations of viruses (Influenza, Zika virus and Respiratory Syncytial Virus), bacteria (E. coli, B. cereus) and eukaryotic cells (NK-92 cell line) are efficiently inactivated by LEEI in a throughput suitable for various applications such as sterilization, vaccine manufacturing or cell therapy. Our results validate the premise that for pathogen and cell inactivation in liquids, LEEI represents a suitable and versatile irradiation method for standard biological research and production laboratories.
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http://dx.doi.org/10.1038/s41598-020-69347-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393095PMC
July 2020

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology.

Immun Inflamm Dis 2020 09 11;8(3):363-370. Epub 2020 Jun 11.

Department of Immunology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

Introduction: Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice.

Methods: In this study, we compared the use of CD34 HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR).

Results: It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdc IL2rγ /SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model.

Conclusion: From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells.
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http://dx.doi.org/10.1002/iid3.317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416029PMC
September 2020

CAR-Expressing Natural Killer Cells for Cancer Retargeting.

Transfus Med Hemother 2019 Feb 5;46(1):4-13. Epub 2019 Feb 5.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Since the approval in 2017 and the outstanding success of Kymriah® and Yescarta®, the number of clinical trials investigating the safety and efficacy of chimeric antigen receptor-modified autologous T cells has been constantly rising. Currently, more than 200 clinical trials are listed on clinicaltrial.gov. In contrast to CAR-T cells, natural killer (NK) cells can be used from allogeneic donors as an "off the shelf product" and provide alternative candidates for cancer retargeting. This review summarises preclinical results of CAR-engineered NK cells using both primary human NK cells and the cell line NK-92, and provides an overview about the first clinical CAR-NK cell studies targeting haematological malignancies and solid tumours, respectively.
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http://dx.doi.org/10.1159/000495771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558329PMC
February 2019

The Epitope-Specific Anti-human CD4 Antibody MAX.16H5 and Its Role in Immune Tolerance.

Front Immunol 2019 24;10:1035. Epub 2019 May 24.

Immune Tolerance Unit, Fraunhofer Institute of Cell Therapy and Immunology, Leipzig, Germany.

T cell modulation in the clinical background of autoimmune diseases or allogeneic cell and organ transplantations with concurrent preservation of their natural immunological functions (e.g., pathogen defense) is the major obstacle in immunology. An anti-human CD4 antibody (MAX.16H5) was applied intravenously in clinical trials for the treatment of autoimmune diseases (e.g., rheumatoid arthritis) and acute late-onset rejection after transplantation of a renal allograft. The response rates were remarkable and no critical allergic problems or side effects were obtained. During the treatment of autoimmune diseases with the murine MAX.16H5 IgG antibody its effector mechanisms with effects on lymphocytes, cytokines, laboratory and clinical parameters, adverse effects as well as pharmacodynamics and kinetics were studied in detail. However, as the possibility of developing immune reactions against the murine IgG Fc-part remains, the murine antibody was chimerized, inheriting CD4-directed variable domains of the MAX.16H5 IgG connected to a human IgG backbone. Both antibodies were studied and in specific humanized mouse transplantation models with a new scope. By incubation of an allogeneic immune cell transplant with MAX.16H5 a new therapy strategy has emerged for the first time enabling both the preservation of the graft-vs.-leukemia (GVL) effect and the permanent suppression of the acute graft-vs.-host disease (aGVHD) without conventional immunosuppression. In this review, we especially focus on experimental data and clinical trials obtained from the treatment of autoimmune diseases with the murine MAX.16H5 IgG antibody. Insights gained from these trials have paved the way to better understand the effects with the chimerized MAX.16H5 IgG as novel therapeutic approach in the context of GVHD prevention.
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http://dx.doi.org/10.3389/fimmu.2019.01035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543443PMC
August 2020

Incubation of Immune Cell Grafts With MAX.16H5 IgG1 Anti-Human CD4 Antibody Prolonged Survival After Hematopoietic Stem Cell Transplantation in a Mouse Model for Fms Like Tyrosine Kinase 3 Positive Acute Myeloid Leukemia.

Front Immunol 2018 22;9:2408. Epub 2018 Oct 22.

Immune Tolerance, Immunology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

Despite the constant development of innovative therapeutic options for hematological malignancies, the gold-standard therapy regimen for curative treatment often includes allogeneic hematopoietic stem cell transplantation (HSCT). The graft-vs.-leukemia effect (GVL) is one of the main therapeutic goals that arises from HSCT. On the other hand, graft-vs.-host disease (GVHD) is still one of the main and most serious complications following allogeneic HSCT. In acute myeloid leukemia (AML), HSCT together with high-dose chemotherapy is used as a treatment option. An aggressive progression of the disease, a decreased response to treatment, and a poor prognosis are connected to internal tandem duplication (ITD) mutations in the Fms like tyrosine kinase 3 (FLT3) gene, which affects around 30% of AML patients. In this study, C3H/HeN mice received an allogeneic graft together with 32D-FLT3 AML cells to induce acute GVHD and GVL. It was examined if pre-incubation of the graft with the anti-human cluster of differentiation (CD) 4 antibody MAX.16H5 IgG prevented the development of GVHD and whether the graft function was impaired. Animals receiving grafts pre-incubated with the antibody together with FLT3 AML cells survived significantly longer than mice receiving untreated grafts. The observed prolonged survival due to MAX.16H5 incubation of immune cell grafts prior to transplantation may allow an extended application of additional targeted strategies in the treatment of AML.
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http://dx.doi.org/10.3389/fimmu.2018.02408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204383PMC
October 2019

Effect of combined sublethal X-ray irradiation and cyclosporine A treatment in NOD scid gamma (NSG) mice.

Exp Anim 2019 Feb 3;68(1):1-11. Epub 2018 Aug 3.

Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany.

Cyclosporine A (CsA) is used in hematopoietic stem cell transplantations (HSCT) to prevent graft-versus-host disease (GvHD). GvHD is the most severe side effect of allogeneic HSCT and efficient therapies are lacking. Mouse models are an essential tool for assessing potential new therapeutic strategies. Our aim is to mimic a clinical setting as close as possible using CsA treatment after sublethal irradiation in NSG mice and thereby evaluate the feasibility of this mouse model for GvHD studies. The effect of CsA (7.5 mg/kg body weight) on sublethally X-ray irradiated (2 Gy) and non-irradiated NSG mice was tested. CsA was administered orally every twelve hours for nine days. Animals irradiated and treated with CsA showed a shorter survival (n=3/10) than irradiated animals treated with NaCl (n=10/10). Furthermore, combined therapy resulted in severe weight loss (82 ± 6% of initial weight, n=7, day 8), with weight recovery after the CsA application was ceased. A high number of apoptotic events in the liver was observed in these mice (0.431 ± 0.371 apoptotic cells/cm, n=2, compared to 0.027 ± 0.034 apoptotic cells/cm, n=5, in the non-irradiated group). Other adverse effects, including a decrease in white blood cell counts were non-CsA-specific manifestations of irradiation. The combination of CsA treatment with irradiation has a hepatotoxic and lethal effect on NSG mice, whereas the treatment without irradiation is tolerated. Therefore, when using in vivo models of GvHD in NSG mice, a combined treatment with CsA and X-ray irradiation should be avoided or carefully evaluated.
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http://dx.doi.org/10.1538/expanim.18-0056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389519PMC
February 2019

ABR, a novel inducer of transcription factor C/EBPα, contributes to myeloid differentiation and is a favorable prognostic factor in acute myeloid leukemia.

Oncotarget 2017 Nov 26;8(61):103626-103639. Epub 2017 Oct 26.

Division of Hematology and Oncology, University Hospital Leipzig, Leipzig, Germany.

Active related () gene deactivates ras-related C3 botulinum toxin substrate 1 (RAC1), which plays an essential role in regulating normal hematopoiesis and in leukemia. gene, closely related to ABR, acts as a tumor suppressor in chronic myeloid leukemia and has overlapping functions with . Evidence for a putative tumor suppressor role of has been shown in several solid tumors, in which deletion of ABR is present. Our results show downregulation of in AML. A block of ABR prevents myeloid differentiation and leads to repression of the myeloid transcription factor C/EBPα, a major regulator of myeloid differentiation and functionally impaired in leukemia. Conversely, stable overexpression of ABR enhances myeloid differentiation. Inactivation of the known ABR target RAC1 by treatment with the RAC1 inhibitor NSC23766 resulted in an increased expression of C/EBPα in primary AML samples and in AML cell lines U937 and MV4;11. Finally, AML patients with high expression at diagnosis showed a significant longer overall survival and patients who respond to azacitidine therapy showed a significant higher ABR expression. This is the first report showing that expression plays a critical role in both myelopoiesis and AML. Our data indicate the tumor suppressor potential of and underline its potential role in leukemia therapeutic strategies.
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http://dx.doi.org/10.18632/oncotarget.22093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732755PMC
November 2017

Disruption of the C/EBPα-miR-182 balance impairs granulocytic differentiation.

Nat Commun 2017 06 29;8(1):46. Epub 2017 Jun 29.

Division of Hematology and Oncology, Leipzig University Hospital, Johannisallee 32a, Leipzig, 04103, Germany.

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.
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http://dx.doi.org/10.1038/s41467-017-00032-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491528PMC
June 2017

Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo.

PLoS One 2017 2;12(5):e0176517. Epub 2017 May 2.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Introduction: One of the main obstacles in the widespread application of gene therapeutic approaches is the necessity for efficient and safe transfection methods. For the introduction of small oligonucleotide gene therapeutics into a target cell, nanoparticle-based methods have been shown to be highly effective and safe. While immune cells are a most interesting target for gene therapy, transfection might influence basic immune functions such as cytokine expression and proliferation, and thus positively or negatively affect therapeutic intervention. Therefore, we investigated the effects of nanoparticle-mediated transfection such as polyethylenimine (PEI) or magnetic beads on immune cell proliferation.

Methods: Human adherent and non-adherent PBMCs were transfected by various methods (e.g. PEI, Lipofectamine® 2000, magnetofection) and stimulated. Proliferation was measured by lymphocyte transformation test (LTT). Cell cycle stages as well as expression of proliferation relevant genes were analyzed. Additionally, the impact of nanoparticles was investigated in vivo in a murine model of the severe systemic immune disease GvHD (graft versus host disease).

Results: The proliferation of primary immune cells was influenced by nanoparticle-mediated transfection. In particular in the case of magnetic beads, proliferation inhibition coincided with short-term cell cycle arrest and reduced expression of genes relevant for immune cell proliferation. Notably, proliferation inhibition translated into beneficial effects in a murine GvHD model with animals treated with PEI-nanoparticles showing increased survival (pPEI = 0.002) most likely due to reduced inflammation.

Conclusion: This study shows for the first time that nanoparticles utilized for gene therapeutic transfection are able to alter proliferation of immune cells and that this effect depends on the type of nanoparticle. For magnetic beads, this was accompanied by temporary cell cycle arrest. Notably, in GvHD this nonspecific anti-proliferative effect might contribute to reduced inflammation and increased survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176517PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412997PMC
September 2017

Immunosuppression for in vivo research: state-of-the-art protocols and experimental approaches.

Cell Mol Immunol 2017 Feb 10;14(2):146-179. Epub 2016 Oct 10.

Fraunhofer-Institute for Cell Therapy and Immunology, Leipzig 04103, Germany.

Almost every experimental treatment strategy using non-autologous cell, tissue or organ transplantation is tested in small and large animal models before clinical translation. Because these strategies require immunosuppression in most cases, immunosuppressive protocols are a key element in transplantation experiments. However, standard immunosuppressive protocols are often applied without detailed knowledge regarding their efficacy within the particular experimental setting and in the chosen model species. Optimization of such protocols is pertinent to the translation of experimental results to human patients and thus warrants further investigation. This review summarizes current knowledge regarding immunosuppressive drug classes as well as their dosages and application regimens with consideration of species-specific drug metabolization and side effects. It also summarizes contemporary knowledge of novel immunomodulatory strategies, such as the use of mesenchymal stem cells or antibodies. Thus, this review is intended to serve as a state-of-the-art compendium for researchers to refine applied experimental immunosuppression and immunomodulation strategies to enhance the predictive value of preclinical transplantation studies.
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http://dx.doi.org/10.1038/cmi.2016.39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301156PMC
February 2017

Attenuation of graft-versus-host-disease in NOD scid IL-2Rγ(-/-) (NSG) mice by ex vivo modulation of human CD4(+) T cells.

Cytometry A 2016 09 25;89(9):803-15. Epub 2016 Aug 25.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-Host-Disease (GvHD) induced by human immune cells. We used a model of acute GvHD by transfer of human peripheral blood mononuclear cells (PBMCs) into NSG mice. The severity of GvHD was reflected by weight loss and was associated with engraftment of human cells and the expansion of leukocytes, particularly granulocytes and monocytes. Pre-treatment of PBMCs with the anti-human CD4 antibody MAX.16H5 IgG1 or IgG4 attenuated GvHD. The transplantation of 2 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 10(7) PBMCs pre-incubated with MAX.16H5 IgG1 or IgG4, GvHD development was reduced and survival was increased. Immune reconstitution was measured by flow cytometry and confirmed for human leukocytes (CD45), CD3(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) T helper cells. Human B cells (CD19) and monocytes (CD14) could not be detected. Histopathological analysis (TUNEL assay) of the gut of recipient animals showed significantly less apoptotic crypt cells in animals receiving a MAX.16H5 IgG1 pre-incubated graft. These findings indicate that pre-incubation of an allogeneic graft with an anti-human CD4 antibody may decrease the frequency and severity of GvHD after hematopoietic stem cell transplantation (HSCT) and the need of conventional immunosuppressive drugs. Moreover, this approach most probably provides a safer HSCT that must be confirmed in appropriate clinical trials in the future. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22930DOI Listing
September 2016

Cellular analyses in the monitoring of autoimmune diseases.

Autoimmun Rev 2016 09 5;15(9):883-9. Epub 2016 Jul 5.

Fraunhofer Institut für Zelltherapie und Immunologie, Perlickstraße 1, 04103, Leipzig, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.autrev.2016.07.010DOI Listing
September 2016

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Cytometry A 2015 Apr 24;87(4):334-45. Epub 2015 Feb 24.

Department of Immunology, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.
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http://dx.doi.org/10.1002/cyto.a.22619DOI Listing
April 2015

Characterization of the murine myeloid precursor cell line MuMac-E8.

PLoS One 2014 29;9(12):e113743. Epub 2014 Dec 29.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113743PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278753PMC
September 2015

Antibody- and aptamer-strategies for GvHD prevention.

J Cell Mol Med 2015 Jan 29;19(1):11-20. Epub 2014 Oct 29.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Prevention of Graft-versus-Host-Disease (GvHD) by preserved Graft-versus-Leukaemia (GvL) effect is one of the major obstacles following allogeneic haematopoietic stem cell transplantation. Currently used drugs are associated with side effects and were not able to separate GvHD from the GvL-effect because of general T-cell suppression. This review focuses on murine models for GvHD and currently available treatment options involving antibodies and applications for the therapeutic use of aptamers as well as strategies for targeting immune responses by allogenic antigens.
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http://dx.doi.org/10.1111/jcmm.12416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288345PMC
January 2015

Analysis of the tumoricidal and anti-cachectic potential of curcumin.

Anticancer Res 2014 Sep;34(9):4781-8

Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

Curcumin, the extract of the rhizome of Curcuma longa, is known for its health-promoting properties in traditional medicine. It has anti-inflammatory, antitumor and antioxidant properties and stimulates appetite. In the present study, we investigated the stability of curcumin and its effect on cytotoxicity, apoptosis and melanin content in melanoma cells and the effect on atrophic C2C12 muscle cells. Cytotoxicity of curcumin was dose-dependent and the EC50 for 24-h incubation was 69 μM. Saturation was reached at 30 μM for a 48-h incubation. The EC50 for 24-h incubation with degraded curcumin solution was 116 μM and that for 48-h was 94 μM. Curcumin induced a strong increase in caspase-3/7 activity at 30-40 μM. Electrical impedance measurements showed that sub-toxic doses of curcumin counteracted atrophy in an in vitro model system. These findings indicate not only the positive effects of curcumin on melanoma cells in vitro, but also that curcumin was able to considerably trigger anti-cachectic effects in vitro. However, the importance of the stability of curcumin and its tumoricidal and anti-cachectic potential might play a pivotal role in its use in the nutrition and health industrie since it degrades rapidly in aqueous solutions.
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September 2014

p8 Deficiency causes siderosis in spleens and lymphocyte apoptosis in acute pancreatitis.

Pancreas 2014 Nov;43(8):1277-85

From the *Division of Gastroenterology and Rheumatology, Department of Internal Medicine, Neurology, and Dermatology, Universitätsklinikum Leipzig AöR, Leipzig; †Institute of Anatomy, Martin-Luther-Universität Halle-Wittenberg AöR, Halle/Saale; ‡Institute of Anatomy, Universität Leipzig, Leipzig, Germany; §Instituto Gulbenkian de Ciência, Oeiras, Portugal; ∥Fraunhofer Institute for Cell Therapy and Immunology; ¶Institute of Clinical Immunology, Universitätsklinikum Leipzig; #Translational Center for Regenerative Medicine, Universität Leipzig; and **Division of Hematology and Oncology, Department of Internal Medicine, Neurology, and Dermatology, Universitätsklinikum Leipzig AöR, Leipzig, Germany.

Objectives: The gene p8 was initially described in pancreatic tissue during acute experimental pancreatitis, a disease that is characterized by a systemic immune response. Although early reports suggested that p8 affects leukocyte migration during acute pancreatitis (AP), no studies revealing its immune-modulatory effects have been performed.

Methods: We investigated the composition of the cellular immune system in naive p8 knockout (p8(−/−)) mice and compared with matched wild-type mice during pancreatitis.

Results: In young mice, there were no relevant differences in the composition of peripheral and splenic CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD11b(+)Gr-1(-), and Gr-1 cells. In mature p8(−/−) mice, increased splenic CD4CD25FoxP3 cells, spleen siderosis, and increased marginal zones in the splenic white pulp were found. During AP, peripheral and splenic CD3(+) and CD3CD4 declined stronger in the p8(−/−) mice. The spleen of the p8(−/−) mice showed severe hypoplasia of the white pulp and mild hyperplasia of the red pulp. This was associated with a significantly increased rate of apoptosis.

Conclusions: We conclude that p8 has no impact on the cellular composition of the adaptive and innate immune systems in noninflammatory conditions. However, it may limit apoptosis and maintain homeostasis of the immune reaction during AP.
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http://dx.doi.org/10.1097/MPA.0000000000000172DOI Listing
November 2014

Modelling hematological parameters after total body irradiation.

Int J Radiat Biol 2014 Jul 12;90(7):538-46. Epub 2014 Jun 12.

Fraunhofer Institute for Cell Therapy and Immunology (IZI) , Leipzig , Germany.

Purpose: The time- and dose-dependent reconstitution of hematopoiesis after radiation exposure is strongly related to the stem cell population and can be used to predict hematological parameters. These parameters allow further insight into the hematopoietic system and might lead to the development of novel stem cell transplantation models.

Materials And Methods: CD4-/- C57Bl/6 mice, transgenic for human CD4 and HLA-DR3, were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 × 1 Gy, 6 × 1.5 Gy, 6 × 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ and tissue damage after irradiation were examined by histopathology.

Results: The recovery curves for RBC, Hb, HCT and WBC showed the same velocity (< 1 week) for all radiation doses (3-12 Gy) starting at different, dose-dependent times. The only dose-dependent parameter was defined by the beginning of the recovery process (dose-dependent shift) and higher doses were related to a later recovery of the hematopoietic system. The RBC, Hb and HCT recovery was followed by a saturation curve reaching a final concentration independent of the radiation dose. Histological analysis of the bone marrow in the single dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities.

Conclusion: Specific functions were developed to describe the reconstitution of hematological parameters after total body irradiation.
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http://dx.doi.org/10.3109/09553002.2014.899443DOI Listing
July 2014

Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies.

Cytometry B Clin Cytom 2014 May 31;86(3):191-206. Epub 2014 Jan 31.

Institute of Clinical Immunology, Universität Leipzig, Medical Faculty, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), Universität Leipzig, Leipzig, Germany.

Background: The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations.

Methods: Peripheral blood samples from healthy adult volunteers (n = 25) were collected and split into eight panel fractions (100 µl each). Subsequently, premixed eight-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants (RTE), (vii) NK-cell subpopulations, and (viii) NK-cell activation markers. All samples were lysed, washed, and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, and percentile ranges).

Results: Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, nonswitched/switched, memory, (activated) CD21(low) , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/β- and γ/δ-T-cells, RTE in CD4/CD8 cells; (vii) immature/mature CD56(bright) , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46, and CD57+NK-cells. Clinical examples and quadrant statistics are provided.

Conclusion: The present study represents a practical approach to standardize the immunophenotyping of most T-, B-, and NK-cell subpopulations. That allows differentiating whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance.
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http://dx.doi.org/10.1002/cyto.b.21162DOI Listing
May 2014

Prevention of graft-versus-host-disease with preserved graft-versus-leukemia-effect by ex vivo and in vivo modulation of CD4(+) T-cells.

Cell Mol Life Sci 2014 Jun 26;71(11):2135-48. Epub 2013 Sep 26.

Fraunhofer Institute for Cell Therapy and Immunology (IZI), 04109, Leipzig, Germany,

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).
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http://dx.doi.org/10.1007/s00018-013-1476-0DOI Listing
June 2014

Low tumor burden is associated with early B-cell reconstitution and is a predictor of favorable outcome after non-myeloablative stem cell transplant for chronic lymphocytic leukemia.

Leuk Lymphoma 2014 Jun 3;55(6):1274-80. Epub 2013 Oct 3.

Department of Hematology/Oncology, University of Leipzig , Leipzig , Germany.

Abstract Reconstitution, engraftment kinetics and tumor cell clearance were analyzed after reduced intensity conditioning hematopoietic cell transplant (RIC-HCT) in patients with chronic lymphocytic leukemia (CLL). Patients were transplanted from unrelated (n = 40) or related (n = 10) donors after fludarabine and 2 Gy total body irradiation followed by cyclosporine and mycophenolate mofetil. The vast majority of patients (96%) engrafted with absolute neutrophil count (ANC) > 0.5 × 10(9)/L at day + 22. CLL cells decreased (median 2%, range 0-69%) within 28 days, but disappeared by day + 180 after HCT. Donor T-cell chimerism increased to > 95% at day 56 and donor B-cell chimerism to 94% at day + 360. Overall survival was 51 ± 8%, incidence of progression 37 ± 7% and non-relapse related mortality (NRM) 30 ± 7% at 4 years. The most common causes of NRM were graft-versus-host disease (GvHD) (14%) and sepsis (6%). Disease status at HCT was significantly associated with early B-cell reconstitution (p = 0.04) and with increased risk of relapse/progression in univariate and multivariate analysis (p = 0.022). Tumor cells were undetectable by day + 180, although B-cell reconstitution did not occur until 1.5 years after RIC-HCT. The best predictors for progression-free survival (PFS) and overall survival (OS) were complete response (CR) or first partial response (PR1) and the absence of bulky disease at transplant, respectively.
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http://dx.doi.org/10.3109/10428194.2013.836598DOI Listing
June 2014

Accreditation of flow cytometry in Europe.

Cytometry B Clin Cytom 2013 May 28;84(3):135-42. Epub 2013 Mar 28.

Universität Leipzig, Medizinische Fakultät, Institut für Klinische Immunologie, Johannisallee 30, Leipzig 04103, Germany.

ISO 15189 has been introduced to enable any clinical laboratory, irrespective of geographic location, to be accredited against internationally recognized standards and therefore facilitate direct international comparison of laboratories. Together with increasing use of ISO 15189 for standardization and competition purposes, often triggered by demands of patients and clinicians, clinical flow cytometry laboratories are becoming increasingly challenged to introduce compliant quality management systems. Whilst in most countries, ISO 15189 accreditation is not yet compulsory, there is increasing evidence to suggest that the implementation of this standard is growing. As a result, the European Society of Clinical Cell Analysis (ESCCA) has analysed the impact of accreditation in clinical flow cytometry laboratories. It found, through a discussion forum, that staff qualification, adaptation of multicolour antibody panels, and implementation of a comprehensive quality system (including quality assessment) have been identified as major challenges.
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http://dx.doi.org/10.1002/cyto.b.21079DOI Listing
May 2013

Differentiation of fungi using hybridization probes on the LightCycler(®).

Methods Mol Biol 2013 ;968:93-104

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

The polymerase chain reaction is a powerful molecular tool for the detection and analysis of very small amounts of DNA. Today, hybridization probes are often used in real-time PCR for more sensitive and specific detection of pathogens and for determination of gene regulation or mutation analysis instead of intercalating dyes like SYBR Green. Here, we describe how to generate suitable primers and hybridization probes for the specific detection of fungal DNA. Furthermore, we show the advantages of hybridization probes using the LightCycler-PCR for the detection of different Candida spp. and Aspergillus spp. in patient blood samples. The methods used to develop such PCR assays will also be presented in the following protocol.
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http://dx.doi.org/10.1007/978-1-62703-257-5_7DOI Listing
June 2013