Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

CAR-T cells and TRUCKs that recognize an EBNA-3C-derived epitope presented on HLA-B*35 control Epstein-Barr virus-associated lymphoproliferation.

BACKGROUND: Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact. METHODS: TÜ165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B*35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells. RESULTS: After co-cultivation with EBV target cells, TÜ165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-γ and tumor necrosis factor-α). Moreover, TÜ165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines. CONCLUSIONS: Our results demonstrate that TÜ165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. TÜ165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation.

From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment.

Success in cancer treatment over the last four decades has ranged from improvements in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven beneficial for the treatment of inflammatory and autoimmune diseases. In this review, we report on challenging examples that bridge between treatment of cancer and immune-mediated diseases, addressing mechanisms and experimental models as well as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. However, new developments using human ex vivo approaches modeling cancer, for example in microfluidic human organs-on-chips, promise to identify key molecular, cellular and immunological features of human cancer progression in a fully human setting. Classical drugs which bridge the gap, for instance, include cytotoxic drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals developed for cancer therapy have also shown efficacy in the treatment of autoimmune diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have achieved spectacular remissions in refractory B cell leukemia and lymphoma and are currently under development for tolerance induction using cell-based therapies such as CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from bridging cancer and autoimmune diseases as well as tolerance induction.

Analysis of the therapeutic potential of different administration routes and frequencies of human mesenchymal stromal cells in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.

Multidimensional Analysis Integrating Human T-Cell Signatures in Lymphatic Tissues with Sex of Humanized Mice for Prediction of Responses after Dendritic Cell Immunization.

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo. Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8+ T cell responses in NOD.Cg-Rag1 tm1Mom -Il2rγ tm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8+ memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo. A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Biohybrid cochlear implants in human neurosensory restoration.

BACKGROUND: The success of cochlear implantation may be further improved by minimizing implantation trauma. The physical trauma of implantation and subsequent immunological sequelae can affect residual hearing and the viability of the spiral ganglion. An ideal electrode should therefore decrease post-implantation trauma and provide support to the residual spiral ganglion population. Combining a flexible electrode with cells producing and releasing protective factors could present a potential means to achieve this. Mononuclear cells obtained from bone marrow (BM-MNC) consist of mesenchymal and hematopoietic progenitor cells. They possess the innate capacity to induce repair of traumatized tissue and to modulate immunological reactions. METHODS: Human bone marrow was obtained from the patients that received treatment with biohybrid electrodes. Autologous mononuclear cells were isolated from bone marrow (BM-MNC) by centrifugation using the Regenlab™ THT-centrifugation tubes. Isolated BM-MNC were characterised using flow cytometry. In addition, the release of cytokines was analysed and their biological effect tested on spiral ganglion neurons isolated from neonatal rats. Fibrin adhesive (Tisseal™) was used for the coating of silicone-based cochlear implant electrode arrays for human use in order to generate biohybrid electrodes. Toxicity of the fibrin adhesive and influence on insertion, as well on the cell coating, was investigated. Furthermore, biohybrid electrodes were implanted in three patients. RESULTS: Human BM-MNC release cytokines, chemokines, and growth factors that exert anti-inflammatory and neuroprotective effects. Using fibrin adhesive as a carrier for BM-MNC, a simple and effective cell coating procedure for cochlear implant electrodes was developed that can be utilised on-site in the operating room for the generation of biohybrid electrodes for intracochlear cell-based drug delivery. A safety study demonstrated the feasibility of autologous progenitor cell transplantation in humans as an adjuvant to cochlear implantation for neurosensory restoration. CONCLUSION: This is the first report of the use of autologous cell transplantation to the human inner ear. Due to the simplicity of this procedure, we hope to initiate its widespread utilization in various fields.

Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms.

BACKGROUND AND AIMS: The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation or de novo infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System® either with the well-established CliniMACS® Plus (Plus) device or with its more versatile successor CliniMACS Prodigy® (Prodigy). METHODS: Manufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8-1 × 109 leukocytes collected by lymphapheresis (n = 3) and using the MACS GMP PepTivator® HCMVpp65 for antigenic restimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-γ), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators. RESULTS: Both devices produced largely similar results for target cell viabilities: 37.2-52.2% (Prodigy) vs. 51.1-62.1% (Plus) CD45+/7-AAD- cells. Absolute numbers of isolated target cells were 0.1-3.8 × 106 viable IFN-γ+ CD3+ T-cells. The corresponding proportions of IFN-γ+ CD3+ T-cells ranged between 19.2 and 95.1% among total CD3+ T-cells and represented recoveries of 41.9-87.6%. Within two parallel processes, predominantly IFN-γ+ CD3+CD8+ cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ+ CD3+CD4+ helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-γ- T-cells (3.6-20.8%) compared to the Plus products (19.9-80.0%). CONCLUSION: The manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-γ- T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft-versus-host disease.

Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids.

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-ß1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-ß1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 µg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor surveillance in relapsed HNSCC patients could be reversed by the re-establishment of ADCC-mediated effector cell activity, thus supporting NK cell-based immunotherapy in combination with antineoplastic monoclonal mAbs.

Increased sMICA and TGFß1 levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells.

Disseminated head-and-neck squamous cell carcinoma (HNSCC) escapes immune surveillance and thus frequently manifests as fatal disease. Here, we report on the distribution of distinct immune cell subpopulations, natural killer (NK) cell cytotoxicity and tumor immune escape mechanisms (TIEMs) in 55 HNSCC patients, either at initial diagnosis or present with tumor relapse. Compared to healthy controls, the regulatory NK cells and the ratio of pro/anti-inflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor ß1 (TGFß1) plasma levels were markedly elevated. Increased sMICA and TGFß1 concentrations correlated with tumor progression and staging characteristics in 7 follow-up HNSCC patients, with significantly elevated levels of both soluble factors from the time of initial diagnosis to that of relapse. Patient plasma containing elevated sMICA and TGFß1 markedly impaired NKG2D-dependent cytotoxicity against HNSCC cells upon incubation with patient-derived and IL-2 activated NK cells vs. those derived from healthy donors. Decreased antitumor recognition was accompanied by reduced NKG2D expression on the NK cell surface and an enhanced caspase-3 activity. In-vitro blocking and neutralization experiments demonstrated a synergistic negative impact of sMICA and TGFß1 on NK cell functionality. Although we previously showed the feasibility and safety of transfer of allogeneic donor NK cells in a prior clinical study encompassing various leukemia and tumor patients, our present results suggest the need for caution regarding the sole use of adoptive NK cell transfer. The presence of soluble NKG2D ligands in the plasma of HNSCC patients and the decreased NK cell cytotoxicity due to several factors, especially TGFß1, indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy.

Mechanisms of tumor and viral immune escape from natural killer cell-mediated surveillance.

Human natural killer (NK) cells recognize and efficiently eliminate MHC class I low or negative malignant targets and virally infected host cells, without requirement for prior sensitization. However, viruses and various tumor cells display elaborate adaptations to evade and overcome immunosurveillance. The current review focuses on escape mechanisms of viruses and malignantly transformed 'stressed' cells to evade from NK cell cytotoxicity. A general overview of recent clinical studies using allogeneic donor NK cells is given, summarizing first data about a possible benefit for patients suffering from high-risk leukemia and solid tumors. Finally, the review discusses the future perspectives and hypotheses aiming to improve therapeutic NK cell strategies against tumor immune escape mechanisms.

Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation.

BACKGROUND: Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and has a poor prognosis. Here we assessed the capability of ex vivo expanded cytokine-induced killer cells to lyse both alveolar and embryonic rhabdomyosarcoma cell lines and investigated the mechanisms involved. DESIGN AND METHODS: Peripheral blood mononuclear cells from six healthy donors were used to generate and expand cytokine-induced killer cells. The phenotype and composition of these cells were determined by multiparameter flow cytometry, while their cytotoxic effect against rhabdomyosarcoma cells was evaluated by a europium release assay. RESULTS: Cytokine-induced killer cells efficiently lysed cells from both rhabdomyosarcoma cell lines. Antibody-mediated masking of either NKG2D molecule on cytokine-induced killer cells or its ligands on rhabdomyosarcoma cells (major histocompatibility antigen related chain A and B and UL16 binding protein 2) diminished this effect by 50%, suggesting a major role for the NKG2D molecule in rhabdomyosarcoma cell killing. No effect was observed after blocking CD11a, CD3 or TCRalphabeta molecules on cytokine-induced killer cells or CD1d on rhabdomyosar-coma cells. Remarkably, cytokine-induced killer cells used tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to activate caspase-3, as the main caspase responsible for the execution of apoptosis. Accordingly, blocking TRAIL receptors on embryonic rhabdomyosarcoma cell lines significantly reduced the anti-tumor effect of cytokine-induced killer cells. About 50% of T cells within the cytokine-induced killer population had an effector memory phenotype, 20% had a naïve phenotype and approximately 30% of the cells had a central memory phenotype. In addition, cytokine-induced killer cells expressed low levels of activation-induced markers CD69 and CD137 and demonstrated a low alloreactive potential. CONCLUSIONS: Our data suggest that cytokine-induced killer cells may be used as a novel adoptive immunotherapy for the treatment of patients with rhabdomyosarcoma after allogeneic stem cell transplantation.

A novel five-colour flow cytometric assay to determine NK cell cytotoxicity against neuroblastoma and other adherent tumour cells.

For the evaluation of novel therapies, and for initial in vitro testing of potential in vivo graft-versus-tumour-effects (GvT), cytotoxicity of effector cells against target tumour cells needs to be determined in a reliable fashion. Recently Zimmermann et al. [Zimmermann, S.Y., Esser, R., Rohrbach, E., Klingebiel, T., Koehl, U., 2005. A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemia cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J. Immunol. Methods. 296(1-2), 63-76] introduced a single platform, no-wash flow cytometric assay to quantify natural killer (NK) cell cytotoxicity against leukaemia cells. Here we have optimised this method introducing a novel five-colour flow cytometric assay for the evaluation of NK cell activity against adherent tumour cells, in particular neuroblastoma cells (NB cells). Beside an enhanced cytotoxic activity corresponding to increasing effector/target (E:T) ratios, we could demonstrate an increasing cytotoxicity in a time-dependent manner over a time period of 8 h. The usefulness of this novel method was also confirmed with human tumour cells lines of various other origin including breast and ovarian carcinoma and Wilms tumour cells freshly isolated from a patient after surgery. In addition to flow cytometric analysis, we monitored NK-cell-mediated induction of target cell apoptosis via the caspase cascade in attacked NB cells by fluorescence microscopy after immunofluorescence staining of activated Caspase-3 (Casp-3) in combination with detection of CD45(+) and CD9(+) for discrimination between NK and NB cells. In summary, this novel flow cytometric cytotoxicity assay enables efficient quantification of the phenotype of both, effector and adherent target tumour cells, and therefore represents a useful tool for research on immunotherapies that rely on cytotoxic effector cells.

Human-antigen R (HuR) expression in hypertension: downregulation of the mRNA stabilizing protein HuR in genetic hypertension.

In aged spontaneously hypertensive rats (SHR), vasorelaxant responses to NO are attenuated compared with normotensive control rats (Wistar-Kyoto [WKY]) because of a decreased expression of the important NO receptor soluble guanylyl cyclase (sGC). Because the expression of sGC subunits alpha1 and beta1 is controlled at the post-transcriptional level by the mRNA-binding protein human-antigen R (HuR), we now assessed whether or not altered expression of HuR could account for downregulation of sGCalpha1 and sGCbeta1 in genetic hypertension. The expression of HuR (and sGCalpha1 and sGCbeta1) in aortas from aged SHR was significantly decreased at the mRNA and protein level compared with age-matched WKY rats, whereas expression of HuR was not different in prehypertensive young (2 months of age) SHR and age-matched WKY rats. The mRNA-binding activity of HuR in native aortic protein extracts from aged SHR was markedly reduced compared with normotensive WKY rats, as detected by RNA electrophoretic mobility shift analysis, using biotin-labeled adenine and uracil (AU)-rich element (ARE)-containing RNA probes from the 3'-untranslated region of sGCalpha1 and sGCbeta1. In contrast, ARE-binding activity was not different between prehypertensive young SHR and young WKY rats. In vitro RNA degradation assays using the same AU-rich sGC mRNA probes revealed an accelerated sGCalpha1 and sGCbeta1 mRNA decay in the presence of native protein extract from hypertensive SHR, which was less rapid with aortic protein from normotensive WKY rats. These findings suggest that in this animal model of genetic hypertension, the reduced expression of sGC subunits is mediated by downregulation of the sGC mRNA-stabilizing protein HuR.

Down-regulation of soluble guanylyl cyclase expression by cyclic AMP is mediated by mRNA-stabilizing protein HuR.

We analyzed whether the cyclic AMP induced down-regulation of the nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is mediated by the mRNA-protecting protein HuR. Exposure (up to 24 h) of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin (10 microM), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Spisomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N6-phenyl-cAMP (10 microM), strongly reduced sGCalpha1beta1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 microM)-sensitive fashion. In vitro degradation of sGCalpha1 and beta1 poly(A)+ mRNA by native rat aortic protein was markedly increased by pretreatment of intact aortas with forskolin. Native protein extract from rat aorta shifted the electrophoretic mobility of biotin-labeled riboprobes from the 3'-untranslated region of sGCalpha1 and beta1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGCalpha1 and beta1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In cultured smooth muscle cells from rat aorta, forskolin induced a rapid increase in Fos/p-Fos protein levels and activator protein 1 (AP-1) binding activity. Inhibition of this transcription factor by an AP-1 decoy prevented the forskolin-induced down-regulation of HuR. We conclude that forskolin/cAMP decrease the expression of heterodimeric sGC in rat aortic smooth muscle cells via activation of Fos/AP-1, which decreases the expression of HuR and thus destabilizes the sGCalpha1 and beta1 mRNA.

Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta.

We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.

Dual control of subtilin biosynthesis and immunity in Bacillus subtilis.

The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and beta-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. beta-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.