Summary 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 β‐galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. β‐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.

BackgroundRhabdomyosarcoma 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 MethodsPeripheral 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. ResultsCytokine-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 TCRαb molecules on cytokine-induced killer cells or CD1d on rhabdomyosarcoma 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. ConclusionsOur 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.Key words: CIK, NKG2D, lysis, rhabdomyosarcoma.Citation: Kuçi S, Rettinger E, Voß B, Weber G, Stais M, Kreyenberg H, Willasch A, Kuçi Z, Koscielniak E, Klöss S, von Laer D, Klingebiel T, and Bader P Haematologica 2010;95(9):1579-1586. doi:10.3324/haematol.2009 This is an open-access paper. . Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation. Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation

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 (CD56CD3) was carried out with the CliniMACS Prodigy in a single process, starting with approximately 1.2 × 10 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 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-VIVO10, 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 CD56CD3 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 CD56CD16 and CD56CD16 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 CD123 AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor nec...

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.

Abbreviations: ADCC, antigen-dependent cellular cytotoxicity; HNSCC, head-and-neck squamous cell carcinoma; HSCT, haploidentical stem cell transplantation; KIR, killer cell immunoglobulin-like receptor; NCR, natural cytotoxicity receptor; NK, natural killer; NKG2D, natural-killer group 2, member D; vitsMICA/NKG2DL, soluble major histocompatibility complex Class I chain-related peptide A; TGFb1, transforming growth factor beta 1; TIEM, tumor immune escape mechanismDisseminated 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/antiinflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor b 1 (TGFb 1 ) plasma levels were markedly elevated. Increased sMICA and TGFb 1 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 TGFb 1 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 TGFb 1 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 TGFb 1 , indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy.

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 M), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole-3Ј,5Ј-cyclic monophosphorothioate, Sp-isomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N 6 -phenylcAMP (10 M), strongly reduced sGC␣ 1 ␤ 1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 M)-sensitive fashion. In vitro degradation of sGC␣ 1 and ␤ 1 poly(A) ϩ 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 3Ј-untranslated region of sGC␣ 1 and ␤ 1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGC␣ 1 and ␤ 1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3Ј,5Ј-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 sGC␣ 1 and ␤ 1 mRNA.

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-Rag1tm1Mom-Il2rγtm1Wj 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.

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), 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-β1 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.