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  • 1
    Abstract: High plasticity is a hallmark of mesenchymal stem cells (MSCs), and as such, their differentiation and activities may be shaped by factors of their microenvironment. Bones, tumors, and cardiomyopathy are examples of niches and conditions that contain MSCs and are enriched with tumor necrosis factor alpha (TNFalpha) and transforming growth factor beta1 (TGFbeta1). These two cytokines are generally considered as having opposing roles in regulating immunity and inflammation (pro- and anti-inflammatory, respectively). Here, we performed global gene expression analysis of human bone marrow-derived MSCs and identified overlap in half of the transcriptional programs that were modified by TNFalpha and TGFbeta1. The two cytokines elevated the mRNA expression of soluble factors, including mRNAs of pro-inflammatory mediators. Accordingly, the typical pro-inflammatory factor TNFalpha prominently induced the protein expression levels of the pro-inflammatory mediators CCL2, CXCL8 (IL-8), and cyclooxygenase-2 (Cox-2) in MSCs, through the NF-kappaB/p65 pathway. In parallel, TGFbeta1 did not elevate CXCL8 protein levels and induced the protein expression of CCL2 at much lower levels than TNFalpha; yet, TGFbeta1 readily induced Cox-2 and acted predominantly via the Smad3 pathway. Interestingly, combined stimulation of MSCs by TNFalpha + TGFbeta1 led to a cooperative induction of all three inflammatory mediators, indicating that TGFbeta1 functioned as a co-inflammatory cytokine in the presence of TNFalpha. The cooperative activities of TNFalpha + TGFbeta1 that have led to CCL2 and CXCL8 induction were almost exclusively dependent on p65 activation and were not regulated by Smad3 or by the upstream regulator TGFbeta-activated kinase 1 (TAK1). In contrast, the TNFalpha + TGFbeta1-induced cooperative elevation in Cox-2 was mostly dependent on Smad3 (demonstrating cooperativity with activated NF-kappaB) and was partly regulated by TAK1. Studies with MSCs activated by TNFalpha + TGFbeta1 revealed that they release factors that can affect other cells in their microenvironment and induce breast tumor cell elongation, migration, and scattering out of spheroid tumor masses. Thus, our findings demonstrate a TNFalpha + TGFbeta1-driven pro-inflammatory fate in MSCs, identify specific molecular mechanisms involved, and propose that TNFalpha + TGFbeta1-stimulated MSCs influence the tumor niche. These observations suggest key roles for the microenvironment in regulating MSC functions, which in turn may affect different health-related conditions.
    Type of Publication: Journal article published
    PubMed ID: 28553282
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  • 2
    Abstract: Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3zeta or composite signaling domains containing a costimulatory protein together with CD3zeta. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.
    Type of Publication: Journal article published
    PubMed ID: 28572802
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  • 3
    Keywords: IFN-GAMMA ; CENTRAL-NERVOUS-SYSTEM ; INTERFERON-GAMMA ; inflammation ; EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS ; IMMUNE PRIVILEGE ; INDOLEAMINE 2,3-DIOXYGENASE ; TRYPTOPHAN CATABOLISM ; EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS ; DEATH-1 PD-1 PATHWAY
    Abstract: The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naive Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNgamma-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNgamma-dependent manner. In turn, Dkk3 decreased IFNgamma activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4(+) and CD8(+) T-cell responses.
    Type of Publication: Journal article published
    PubMed ID: 25759692
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  • 4
    Abstract: gammadelta T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make gammadelta T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human gammadelta T cells with CD19 antibodies for immunotherapy of B-lineage ALL, gammadelta T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded gammadelta T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFalpha, and IFNgamma expression of primary gammadelta T cells were significantly increased and correlated with CD16-expression of gammadelta T cells. gammadelta T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded gammadelta T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring gammadelta T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of gammadelta T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of gammadelta T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded gammadelta T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation.
    Type of Publication: Journal article published
    PubMed ID: 25520723
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  • 5
    Abstract: The tryptophan (TRP) to kynurenine (KYN) metabolic pathway is now firmly established as a key regulator of innate and adaptive immunity. A plethora of preclinical models suggests that this immune tolerance pathway - driven by the key and rate-limiting enzymes indoleamine-2,3-dioxygenase and TRP-2,3-dioxygenase - is active in cancer immunity, autoimmunity, infection, transplant rejection, and allergy. Drugs targeting this pathway, specifically indoleamine-2,3-dioxygenase, are already in clinical trials with the aim at reverting cancer-induced immunosuppression. In the past years, there has been an increase in our understanding of the regulation and downstream mediators of TRP metabolism, such as the aryl hydrocarbon receptor as a receptor for KYN and kynurenic acid. This more detailed understanding will expand our opportunities to interfere with the pathway therapeutically on multiple levels. Here, we discuss the perspective of targeting TRP metabolism at these different levels based on reviewing recent insight into the regulation of TRP metabolism and its downstream effectors.
    Type of Publication: Journal article published
    PubMed ID: 25628622
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  • 6
    Abstract: The tryptophan (TRP) to kynurenine (KYN) metabolic pathway is now firmly established as a key regulator of innate and adaptive immunity. A plethora of preclinical models suggests that this immune tolerance pathway - driven by the key and rate-limiting enzymes indoleamine-2,3-dioxygenase and TRP-2,3-dioxygenase - is active in cancer immunity, autoimmunity, infection, transplant rejection, and allergy. Drugs targeting this pathway, specifically indoleamine-2,3-dioxygenase, are already in clinical trials with the aim at reverting cancer-induced immunosuppression. In the past years, there has been an increase in our understanding of the regulation and downstream mediators of TRP metabolism, such as the aryl hydrocarbon receptor as a receptor for KYN and kynurenic acid. This more detailed understanding will expand our opportunities to interfere with the pathway therapeutically on multiple levels. Here, we discuss the perspective of targeting TRP metabolism at these different levels based on reviewing recent insight into the regulation of TRP metabolism and its downstream effectors.
    Type of Publication: Journal article published
    PubMed ID: 25628622
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  • 7
    Abstract: Mesenchymal stem cells (MSCs) are known to limit immune responses in vivo by multiple soluble factors. Dickkopf-3 (DKK3), a secreted glycoprotein, has recently been identified as a novel immune modulator. Since DKK3 has been reported to be produced by MSCs, we investigated whether DKK3 contributes to the immune suppression of anti-tumor responses by MSCs. Whereas wild-type MSCs inhibited immune responses against two different transplantation tumors, DKK3-deficient MSCs did not affect the rejection process. Increased CD8(+) T cell and reduced M2-type macrophages infiltration was observed in tumors inoculated together with DKK3-deficient MSCs. Thus, DKK3 could alter the composition of the tumor stroma, thereby supporting the MSCs-mediated suppression of immune responses against these tumor transplants.
    Type of Publication: Journal article published
    PubMed ID: 26734010
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  • 8
    Abstract: Major histocompatibility complex (MHC) multimers are essential tools in T cell immunomonitoring, which are employed both in basic and clinical research, as well as for assessing clinical samples during therapy. The generation of MHC monomers loaded with synthetic peptides is an elaborate and time-consuming process. It would be beneficial to assess the quality of these monomers prior to downstream applications. In this technical note, we describe a novel flow cytometry-based, cell-free, quick, and robust assay to check the quality of MHC monomers directly after refolding or after long-term storage.
    Type of Publication: Journal article published
    PubMed ID: 28228758
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  • 9
    Keywords: tumor ; INHIBITION ; RESPONSES ; DENDRITIC CELLS ; REGULATORY T-CELLS ; CATABOLISM ; IDO ; INDOLEAMINE 2,3-DIOXYGENASE ACTIVITY ; ARYL-HYDROCARBON RECEPTOR ; L-TRYPTOPHAN TRANSPORT
    Abstract: The tryptophan (TRP) to kynurenine (KYN) metabolic pathway is now firmly established as a key regulator of innate and adaptive immunity. A plethora of preclinical models suggests that this immune tolerance pathway - driven by the key and rate-limiting enzymes indoleamine-2,3-dioxygenase and TRP-2,3-dioxygenase - is active in cancer immunity, autoimmunity, infection, transplant rejection, and allergy. Drugs targeting this pathway, specifically indoleamine-2,3-dioxygenase, are already in clinical trials with the aim at reverting cancer-induced immunosuppression. In the past years, there has been an increase in our understanding of the regulation and downstream mediators of TRP metabolism, such as the aryl hydrocarbon receptor as a receptor for KYN and kynurenic acid. This more detailed understanding will expand our opportunities to interfere with the pathway therapeutically on multiple levels. Here, we discuss the perspective of targeting TRP metabolism at these different levels based on reviewing recent insight into the regulation of TRP metabolism and its downstream effectors.
    Type of Publication: Journal article published
    PubMed ID: 25628622
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  • 10
    Keywords: DIFFERENTIATION ; TOLERANCE ; STEM-CELLS ; EPITHELIAL-CELLS ; MicroRNAs ; SELF-ANTIGEN ; LINEAGE COMMITMENT ; HISTONE ; DNA METHYLATION DYNAMICS ; AIRE PROMOTER
    Abstract: The induction of central tolerance in the course of T cell development crucially depends on promiscuous gene expression (pGE) in medullary thymic epithelial cells (mTECs). mTECs express a genome-wide variety of tissue-restricted antigens (TRAs), preventing the escape of autoreactive T cells to the periphery, and the development of severe autoimmunity. Most of our knowledge of how pGE is controlled comes from studies on the autoimmune regulator (Aire). Aire activates the expression of a large subset of TRAs by interacting with the general transcriptional machinery and promoting transcript elongation. However, further factors regulating Aire-independent TRAs must be at play. Recent studies demonstrated that pGE in general and the function of Aire in particular are controlled by epigenetic and post-transcriptional mechanisms. This mini-review summarizes current knowledge of the regulation of pGE by miRNA and epigenetic regulatory mechanisms such as DNA methylation, histone modifications, and chromosomal topology.
    Type of Publication: Journal article published
    PubMed ID: 25784915
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