[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Recently, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have been suggested as an alternative to MSCs for the treatment of various inflammatory disorders. However, while a first case report observed beneficial therapeutic effects of repeated MSC EV infusions in a patient with therapy refractory graft versus host disease (GvHD), in vitro findings revealed that MSC-EVs were significantly less immunosuppressive than their parental cells. Here, we compared the immunosuppressive potency of MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT MSCs), with their secreted EVs in a standardized lymphocyte proliferation assay (LPA). Both BM MSCs and AT-MSCs exhibited a remarkable inhibition of lymphocyte proliferation (LP) (88.1 ± 1.5% and 75.5 ± 1.5%, respectively), while isolated EVs derived from them failed to suppress LP at dose levels up to 100 µg/ml. Thus, our data further substantiate previous reports suggesting that cell-cell contact plays an important role on the immunosuppressive potential mediated by MSCs. Hence, MSC-EVs are still a matter of debate and might not be a reasonable substitute for MSCs with regard to the immunosuppressive function. Collectively, these contrasting findings may also reflect the importance of relevant translational aspects when designing new studies. Standardization of MSC culture conditions before EV collection, as well as isolation and characterization methods with regard to EV purity are urged. Moreover, prior to the clinical use, dose-finding studies evaluating MSC-EV preparations in suitable preclinical models are warranted.
Stem Cells and Development 03/2015; 24(11). DOI:10.1089/scd.2014.0563 · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) essentially contribute to the induction and regulation of innate and adaptive immunity. Based on these important properties, DCs may profoundly influence tumor progression in patients. However, little is known about the role of distinct human DC subsets in primary tumors and their impact on clinical outcome. In the present study, we investigated the characteristics of human 6-sulfo LacNAc (slan) DCs in clear cell renal cell carcinoma (ccRCC). slanDCs have been shown to display various tumor-directed properties and to accumulate in tumor-draining lymph nodes from patients. When evaluating 263 ccRCC and 227 tumor-free tissue samples, we found increased frequencies of slanDCs in ccRCC tissues compared to tumor-free tissues. slanDCs were also detectable in the majority of 24 metastatic lymph nodes and 67 distant metastases from ccRCC patients. Remarkably, a higher density of slanDCs was significantly associated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Tumor-infiltrating slanDCs displayed an immature phenotype expressing interleukin-10. ccRCC cells efficiently impaired slanDC-induced T-cell proliferation and programming as well as natural killer (NK) cell activation. In conclusion, these findings indicate that higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The induction of a tolerogenic phenotype in slanDCs leading to an insufficient activation of innate and adaptive antitumor immunity may represent a novel immune escape mechanism of ccRCC. These observations may have implications for the design of therapeutic strategies that harness tumor-directed functional properties of DCs against ccRCC.
[Show abstract][Hide abstract] ABSTRACT: To examine the potential role of 6-sulfo LacNAc(+) (slan) dendritic cells (DCs) displaying pronounced proinflammatory properties in the pathogenesis of multiple sclerosis (MS).
[Show abstract][Hide abstract] ABSTRACT: Background aims
Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity.
Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 1010 leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting.
A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 106 CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection.
Clinical-scale isolation of “minimally manipulated” CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.
[Show abstract][Hide abstract] ABSTRACT: In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft-versus-host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow-derived MSCs (BM-MSCs) were gamma-irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)-assay, Annexin V-staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non-irradiated BM-MSCs. Notably, irradiated BM-MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM-MSCs in vitro and thus might increase the safety of MSC-based cell products in clinical applications.
Journal of Cellular and Molecular Medicine 03/2014; 18(6). DOI:10.1111/jcmm.12264 · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) play a crucial role in the development of cell-mediated immunotherapy due to their ability to induce and maintain strong immune responses. In our study, we evaluated a biocompatible Ni(II)-NTA-modified poly(ethylene imine) dendritic glycopolymer (Ni(II)-NTA-DG) as new carrier system to increase the antigen uptake into iDCs for future DC based immunotherapy. Ni(II)-NTA-DG led to an increase in His6-Gp160 uptake in monocytes and iDCs, where His6-Gp160 is localised in the early endosomal and lysosomal compartments. Ni(II)-NTA-DG and the formed polyplexes induced an activation of iDCs, showing an increasing expression of costimulatory molecules CD86, CD80 and proinflammatory cytokines IL-6 and IL-8. Beside no influencing effect of Ni(II)-NTA-DG and polyplexes on the maturation of antigen-bearing DCs, the mature peptide bearing DCs remained their ability to migrate along a gradient of CCR7 ligands. Thus, Ni(II)-NTA-DG with advancing biological properties is a promising carrier system for the future application in DC-based immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c(+) DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c(+) DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.
[Show abstract][Hide abstract] ABSTRACT: We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Adoptive transfer of human bone marrow mesenchymal stromal cells (BM-MSCs) in the context of post-transplant GvHD relies on their immunosuppressive abilities, which are still not fully characterized. Recently, an important role for the CD39-CD73-adenosine pathway in mediating immunosuppression was demonstrated in Tregs. Within this pathway extracellular nucleotides are phosphohydrolised to generate immunosuppressive adenosine. Since BM-MSCs express CD73 and recent data indicate that BM-MSCs express CD39, we decided to further investigate the CD39 expression in BM-MSCs and check whether inhibition of extracellular nucleotide phosphohydrolysis by POM-1 would impact not only on immunosuppressive capacity, but also on colony forming ability and differentiation potential.
Methods: Expression of CD39 in BM-MSC lines was assessed by flow cytometry, immunocytochemistry and western blotting. With the aim to check whether CD39 would influence immunosuppressive properties of MSCs, anti- CD39 monoclonal antibody OREG-103/BY40 (CD39mAb) and a selective inhibitor of ENTPD 1 (CD39), 2 and 3, POM-1, were used within standardized immunosuppressive assays, such as the lymphocyte proliferation assay (LPA) and mixed lymphocyte reaction (MLR). For the colony forming unit (CFU-F) capacity, and in vitro differentiation potential into osteoblasts and adipocytes, POM-1 was used in 2 different concentrations (2 and 20µM).
Results: All tested BM-MSC lines (n=20) expressed CD39 (19.2±3.3% CD39+ cells among BM-MSCs). Flow cytometric results were confirmed by immunocytochemistry as well as western blotting. Notably, addition of POM-1 significantly impaired the immunosuppressive capacity of BM-MSCs (at the concentration of 20 µM for the LPA and 2 µM for the MLR) in both assays (n=6, mean inhibition (MI) of 24.28±8.3% for the LPA; n=4, MI of 42.28±9.33% for the MLR) as compared to the non-treated BM-MSCs (MI of 70.2±2.7% for the LPA, p<0.0001; MI of 78.1±3.11% for the MLR, p=0.0015). Preliminary results using the CD39mAb indicate that the immunosuppressive capacity of BM-MSCs is reduced, confirming our results with POM-1. Furthermore, inhibition of ENTPDases by addition of POM-1 to the cultures hampered the colony formation ability of BM-MSCs (n=3). Interestingly, we also demonstrated that POM-1-mediated inhibition of ENTPdases resulted in compromised BM-MSCs differentiation potential.
Conclusions: Here, we demonstrated that chemical inhibition of ENTPDases impairs the immunosuppression mediated by MSCs. Regarding the blockade of CD39 using a monoclonal antibody, we could observe preliminary effects that should be further analyzed. Moreover, our results suggested for the first time that ENTPDases has an important function in colony formation ability as well as in differentiation potential of MSCs. These novel findings improve our understanding of the mechanistic role of ENTPDases in MSC function and thus can contribute to the amelioration of MSC-based therapies for the treatment of autoimmune diseases as well as their use in regenerative medicine.
Annual Meeting of the German Society for Transfusion Medicine and Immunohematology (DGTI), Münster, Germany; 09/2013
[Show abstract][Hide abstract] ABSTRACT: Due to their clinical success there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform which consists of a universal effector and individual target modules. Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (e.g. the extracellular CD137 ligand domain) to the target module the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector to target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.Leukemia accepted article preview online, 20 August 2013. doi:10.1038/leu.2013.243.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 08/2013; 28(1). DOI:10.1038/leu.2013.243 · 9.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversially discussed. Therefore, the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome were analyzed in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Further, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha (SDF-1α) in patients' cells culture supernatants were almost 2-fold lower (p<0.01) compared to controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Cocultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area forming-cells and fewer colony forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of SDF-1α secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.
[Show abstract][Hide abstract] ABSTRACT: Chemotherapy is an important treatment modality for many patients with advanced cancer. Recent data revealed that certain chemotherapeutic agents differentially affect maturation, cytokine production and T cell-stimulatory capacity of dendritic cells (DCs), which play a crucial role in the induction of antitumor immunity. Whereas most reports are based on mouse or human monocyte-derived DCs, studies investigating the direct effect of chemotherapeutic drugs on native human DCs are rather limited. Here, we evaluated the impact of various chemotherapeutic drugs on the immunostimulatory properties of 6-sulfo LacNAc(+) (slan) DCs, representing a major subpopulation of human blood DCs. Due to their various antitumor effects, slanDCs may essentially contribute to the immune defence against tumors. We demonstrated that doxorubicin and vinblastine significantly impair the release of TNF-α, IL-6, and IL-12 by slanDCs. Functional data revealed that both drugs inhibit slanDC-mediated proliferation of T lymphocytes and their capacity to differentiate naive CD4(+) T cells into proinflammatory T helper type I cells. Furthermore, these agents markedly suppressed the ability of slanDCs to stimulate interferon-γ secretion by natural killer (NK) cells. In contrast, paclitaxel, mitomycin C, and methotrexate sustained the ability of slanDCs to produce proinflammatory cytokines and their potential to activate T lymphocytes and NK cells. These results indicate that doxorubicin and vinblastine impair the ability of native human DCs to stimulate important immune effector cells, whereas methotrexate, mitomycin C, and paclitaxel maintain their immunostimulatory properties. These novel findings may have implications for the design of treatment modalities for tumor patients combining immunotherapeutic strategies and chemotherapy.
International Journal of Cancer 03/2013; 132(6). DOI:10.1002/ijc.27786 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding and uptake of immune complexes (ICs) via low-affinity Fc gamma receptors on dendritic cells (DCs) is well known as a booster of immune responses. It can be helpful when stimulating immunity against pathogenic microbes but may be harmful when antibodies form complexes with autologous antigens. No human DC subtype specialized in handling ICs has been identified to date. Incubating human blood mononuclear cells with ICs and studying their cellular binding, we identified 6-sulfo LacNAc-expressing DCs (slanDCs) as having an outstanding capacity to bind ICs compared to other myeloid DCs, plasmacytoid DCs or monocytes. Using selective blocking of different Fc gamma receptors, we identified CD16 (FcγRIII) as the major IC-binding structure on slanDCs. In addition, CD16 proved critical for phagocytosis of IgG-coated erythrocytes and CD16-targeted antigen led to a more efficient proliferation of CD4+ T cells than CD32 (FcγRII)-targeted antigen. Interestingly, these CD16-mediated functions are short lived and restricted to the immature stage of slanDCs in blood. We show that CD16 is rapidly shed from the surface of maturing slanDCs, resulting from the combined action of the metalloproteinases ADAM10 and ADAM17. In conclusion, these data provide strong evidence that slanDCs play an important role in IC-driven immune responses.
[Show abstract][Hide abstract] ABSTRACT: Imiquimod and resiquimod represent Toll-like receptor (TLR) 7 and 8 agonists, which emerged as attractive candidates for tumor therapy. To elucidate immune cells, which mainly contribute to TLR7/8-mediated antitumoral activity, we investigated the impact of imiquimod and resiquimod on native human 6-sulfo LacNAc (slan) dendritic cells (DCs). We found that both TLR7/8 agonists significantly improve the release of various proinflammatory cytokines by slanDCs and promote their tumor-directed cytotoxic activity. Furthermore, resiquimod efficiently augmented the ability of slanDCs to stimulate T cells and natural killer cells. These results indicate that imiquimod and resiquimod trigger various immunostimulatory properties of slanDCs, which may contribute to their antitumor effects.
Cancer letters 02/2013; 335(1). DOI:10.1016/j.canlet.2013.02.003 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nuclear autoantigen La can be detected on the surface of dying cells. Here we present an assay which enables us to show that La protein is not limited to the surface of dying cells but will be released upon stress-induced cell death. As released La protein tightly binds to the surface of neighboring intact cells we asked the question whether or not La protein could serve as a stress-inducible target e.g. for redirecting of regulatory T cells (Tregs) into damaged tissues to downregulate an immune response. In order to provide first proof of concept we developed a novel fully humanized single-chain bispecific antibody (bsAb) which on the one hand is directed to the La antigen and on the other hand to the CD3 complex of T cells. A cross-linkage of Tregs with La-decorated target cells mediated by this bsAb resulted indeed in the activation of the Tregs in a target-dependent manner. Moreover, such bsAb activated Tregs displayed a potent suppressive capacity and negatively influenced proliferation, expansion and cytokine production of autologous CD4(+) and CD8(+) Teff cells.
Journal of Autoimmunity 01/2013; 42. DOI:10.1016/j.jaut.2013.01.002 · 7.02 Impact Factor