Karin Schilbach

T-helper-1-cell cytokines drive cancer into senescence

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1−/− Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Human γδ T Lymphocytes Exert Natural and Il-2–induced Cytotoxicity to Neuroblastoma Cells

Human γδ T lymphocytes play an important role in nonadaptive reactions to infection and early tumor defense. This is the first report that freshly isolated, native γδ T cells of some healthy donors can kill human neuroblastoma cells to varying degrees. Their killing ability was increased and maintained during expansion and cultivation with interleukin-2 (IL-2; 400 IU/mL) for as long as 30 days (100% specific lysis at an effector-to-target cell (E:T) ratio of 20:1). γδ T lymphocytes without this spontaneous killing ability gained a specific cytolytic activity of 81% ± 10.4% SD after stimulation with IL-2 for 24 hours. γδ cells were isolated from peripheral blood by positive enrichment (using a magnetic cell sorting system; purity, 95.2% ± 3.2% SD, n = 21). High natural cytotoxic activity against human neuroblastoma cell lines (>50% specific lysis at an E:T ratio of 20:1) was exhibited by one of 11 donors, whereas two of 11 showed medium cytotoxicity (30% to 50% specific lysis). Eight of 11 donors showed very slight or no lytic activity against human neuroblastoma cells (<30% specific lysis). γδ T cells were also cytotoxic against Daudi (32.7% specific lysis at an E:T ratio of 20:1), Raji (10.3%), Colo 205 (23.1%), A 204 (54%), K 562 (100%), and SK-N-MC (100%) cells. Isolated γδ T cells were grown in Iscove modified Dulbecco medium with IL-2 (400 IU/mL). Increased cell proliferation (38.5% to 182%) was induced with phytohemagglutinin, IL-15, Clodronat, OKT3, or various combinations of these. Results of cold target inhibition assays suggest a natural killer-like activity of the γδ T-cell killing mechanism. Peptidase or papain render neuroblastoma cells unsusceptible to γδ T-cell killing, suggesting the involvement of antigen peptide(s) in the process of neuroblastoma cell killing. Treatment with acid phosphatase reduced specific lysis by 66.5% ± 34.1% SD, which suggests a binding to phosphorylated neuroblastoma cell-surface structures in the killing mechanism of γδ T cells. Heat shock did not affect the extent of neuroblastoma killing by γδ cells. Recognition of neuroblastoma cells by γδ cytotoxic T lymphocytes is negatively regulated by major histocompatibility complex I receptors. Evidence for natural and inducible cell cytotoxicity of γδ T cells against human neuroblastoma cells, easy propagation, purification, and missing alloreactivity in mixed lymphocytes cultures indicates a role for this subpopulation of T lymphocytes in adoptive immunotherapy.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Immune response of human propagated gammadelta-T-cells to neuroblastoma recommend the Vdelta1+ subset for gammadelta-T-cell-based immunotherapy.

Human peripheral γδ-T-cells are able to induce cytolysis of neuroblastoma (Nb) tumor cells. Besides innate effector functions against infected cells and tumors, γδ-T-cells are involved in T-helper 1/T-helper 2 (TH1/TH2) differentiation of αβ-T-cells. However, as different γδ-T-cell subsets vary considerably in their functional properties, the aim of the present study was to define repertoires of cytokines, chemokines, and angiogenic factors of in vitro expanded Vδ1+ and Vδ2+ T cells in response to Nb. After short-term culture, both subsets released TH1 [interleukin (IL)-2, interferon (IFN)-γ, IL-12, tumor necrosis factor (TNF)-α, TNF-β)] and TH2 cytokines (IL-4, -5, -6, -10, -13, Vδ1 also transforming growth factor (TGF)-β, chemokines (I-309, monocyte chemotactic protein (MCP)-1-3, regulated upon activation, normal T-cell expressed and secreted), ILs (IL-1, -8, -15), cytokines (leptin) as well as angiogenic growth factors [angiogenin (ANG), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), Insulin-like growth factor (IGF)-I]. These molecules were expressed at higher levels in Vδ2+ than Vδ1+ T cells. Nb challenge changed protein expression. TH2 cytokine and IFN-γ release was blocked in both γδ-T-cell subsets. In Vδ2+ γδ-T-cells, TH1 cytokines were down-regulated and tumor growth-promoting factors (ANG, VEGF, EGF, and IGF-I) were strongly up-regulated. In contrast, Vδ1+ γδ-T-cells stopped the release of tumor-supportive factors and tolerogenic TGF-β, and strongly up-regulated TNF-α, TNF-β, MCP-1 and -2 and maintained their IL-2 production. In summary, our data show that after being challenged with Nb cells, propagated Vδ1+rather than Vδ2+ T cells support antitumor responses by secretion of proinflammatory cytokines. Furthermore, in contrast to other cell types, Vδ1+ T cells do not sustain a growth-promoting or tolerogenic microenvironment. These data make Vδ1+ T cells an ideal candidate for upcoming immunotherapy trials in Nb.

BK K + channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells

Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

PD-1-PD-L1 pathway is involved in suppressing alloreactivity of heart infiltrating t cells during murine gvhd across minor histocompatibility antigen barriers.

Background. Graft-versus-host disease (GVHD) is still a major cause of morbidity and mortality after allogeneic stem cell transplantation. GVHD mainly affects skin, liver, and intestine, whereas other organs usually are spared. In the present study, we wanted to investigate whether local regulatory T cells (Treg) or differential expression of immunomodulatory molecules contribute to organ specificity of GVHD. Methods. In a murine B10.D2->BALB/c (both H-2d) model, GVHD was induced by transplantation of 1×107 bone marrow and 1×108 spleen cells. We compared expression of T-cell and dendritic cell markers, CD40-CD40L, various B7 family members, FoxP3, and Th1/Th2 cytokines between ileum (GVHD-target organ) and heart (nontarget organ). Results. GVHD was documented by an increase of CD4+ T cells with accompanying tissue destruction in ileum but not in heart. We found a significantly increased expression of PD-L1 in heart on day 14 and 21 as well as of CTLA-4 on day 21 after transplantation, whereas all other molecules were not different between heart and ileum. In heart, PD-L1 was expressed on lymphoid cells, endothelial cells, CD8&agr;+CD11c+DCs, and up-regulated during GVHD. In contrast, in the ileum only endothelial cells stained weekly positive for PD-L1. Furthermore, we could not find any evidence for the presence of Tregs in the heart. Conclusions. Our data indicate that immunomodulatory molecules such as PD-L1 rather than Tregs play pivotal roles in the tissue-specific regulation of alloresponses. Further studies are needed to refine the significance of the PD-L1 pathway in GVHD and its versatility for therapeutic intervention.

DNA Quantification and Fragmentation in Sputum after Inhalation of Recombinant Human Deoxyribonuclease

Background: Inhaled rhDNase may improve sputum viscosity and mucociliary clearance by cleavage of extracellular DNA derived for instance from dead leukocytes in purulent, highly viscous patient sputum. Methods: Here we established a method to quantify rhDNase-mediated DNA fragmentation in sputum using gel electrophoresis. Sputum of Pseudomonas aeruginosa colonized cystic fibrosis (CF) patients with (CF+) or without (CF-) rhDNase treatment or mechanically ventilated non-CF patients receiving rhDNase (non-CF+) or not (non-CF-) was analyzed. DNA measurements from T-lymphocytes served as controls. Absolute DNA content and the relative quantity within eight molecular mass ranges (12000 to 200 bp) was determined by gel electrophoresis and densitometric analysis. Results: Geometric mean sputum DNA concentrations were 0.41 mg/dl for CF- (n=54), 0.78 mg/dl for CF+ (n=60), 0.053 mg/dl for non-CF- (n=41) and 0.049 mg/dl for non-CF+ (n=28). Treatment with rhDNase resulted in fragmentation of DNA that was quantified by separation and densitometric analysis of the DNA on agarose gels. The new analysis method permits analysis of DNA cleavage with high accuracy. Conclusion: This new monitoring method facilitates DNA quantification and in vitro monitoring of rhDNase in sputum.

Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells

Stage-specific embryonic antigen-4 (SSEA-4) is a glycosphingolipid, which is overexpressed in some cancers and has been linked to disease progression. However, little is known about the functions of SSEA-4 and the characteristics of SSEA-4 expressing tumor cells. Our studies identified SSEA-4 expression on a subpopulation of cells in many solid tumor cell lines but not in leukemic cell lines. Fluorescence-activated cell sorting-sorted SSEA-4(+) prostate cancer cells formed fibroblast-like colonies with limited cell-cell contacts, whereas SSEA-4(-) cells formed cobblestone-like epithelial colonies. Only colonies derived from SSEA-4(+) cells were enriched for pluripotent embryonic stem cell markers. Moreover, major epithelial cell-associated markers Claudin-7, E-cadherin, ESRP1 and GRHL2 were down-regulated in the SSEA-4(+) fraction of DU145 and HCT-116 cells. Similar to cell lines, SSEA-4(+) primary prostate tumor cells also showed down-regulation of epithelial cell-associated markers. In addition, they showed up-regulation of epithelial-to-mesenchymal transition as well as mesenchymal markers. Furthermore, SSEA-4(+) cells escape from adhesive colonies spontaneously and form invadopodia-like migratory structures, in which SSEA-4, cortactin as well as active pPI3K, pAkt and pSrc are enriched and colocalized. Finally, SSEA-4(+) cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in decreased cellular adhesion to different extracellular matrices. In conclusion, we introduce SSEA-4 as a novel marker to identify heterogeneous, invasive subpopulations of tumor cells. Moreover, increased cell-surface SSEA-4 expression is associated with the loss of cell-cell interactions and the gain of a migratory phenotype, suggesting an important role of SSEA-4 in cancer invasion by influencing cellular adhesion to the extracellular matrix.

In melanoma, Hippo signaling is affected by copy number alterations and YAP1 overexpression impairs patient survival.

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org In melanoma, Hippo signaling is affected by copy number alterations and YAP1 overexpression impairs patient survival Moritz Menzel, Diana Meckbach, Benjamin Weide, Nora C. Toussaint, Karin Schilbach, Seema Noor, Thomas Eigentler, Kristian Ikenberg, Christian Busch, Leticia Quintanilla-Martinez, Ursula Kohlhofer, Antonia Göke, Friederike Göke, Rupert Handgretinger, Christian Ottmann, Boris C. Bastian, Claus Garbe, Martin Röcken, Sven Perner, Oliver Kohlbacher and Jürgen Bauer

Pre‐differentiated human committed T‐lymphoid progenitors promote peripheral T‐cell re‐constitution after stem cell transplantation in immunodeficient mice

T‐cell re‐constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34+ (huCD34+) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co‐transfer of in vitro‐pre‐differentiated committed T/NK‐lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34+ HSCs compared with CTLPs from a third‐party donor in a murine NOD‐scid IL2Rγnull model of humanised chimeric haematopoiesis. CTLPs (CD34+lin−CD45RA+CD7+) could be generated in vitro within 10 days upon co‐culture of huCD34+ or cord blood CD34+ (CB‐CD34) HSCs on murine OP9/N‐DLL‐1 stroma cells but not in a novel 3‐D cell‐culture matrix with DLL‐1low human stroma cells. In both in vitro systems, huCD34+ and CB‐CD34+ HSCs did not give rise to mature T cells. Upon transfer into 6‐wk‐old immune‐deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34+ HSCs resulted in rapid T‐cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34+ HSCs still expressed a T‐cell‐precursor phenotype (CD7+CD5+CD1a+/−). This strategy to enhance early T‐cell re‐constitution with ex vivo‐pre‐differentiated T‐lymphoid progenitors could bridge the gap until full T‐cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.