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The ability of human γδ T cells from healthy donors to kill pancreatic ductal adenocarcinoma (PDAC) in vitro and in vivo in immunocompromised mice requires the addition of γδ T-cell–stimulating antigens. In this study, we demonstrate that γδ T cells isolated from patients with PDAC tumor infiltrates lyse pancreatic tumor cells after selective stimulation with phosphorylated antigens. We determined the absolute numbers of γδ T-cell subsets in patient whole blood and applied a real-time cell analyzer to measure their cytotoxic effector function over prolonged time periods. Because phosphorylated antigens did not optimally enhance γδ T-cell cytotoxicity, we designed bispecific antibodies that bind CD3 or Vγ9 on γδ T cells and Her2/neu (ERBB2) expressed by pancreatic tumor cells. Both antibodies enhanced γδ T-cell cytotoxicity with the Her2/Vγ9 antibody also selectively enhancing release of granzyme B and perforin. Supporting these observations, adoptive transfer of γδ T cells with the Her2/Vγ9 antibody reduced growth of pancreatic tumors grafted into SCID-Beige immunocompromised mice. Taken together, our results show how bispecific antibodies that selectively recruit γδ T cells to tumor antigens expressed by cancer cells illustrate the tractable use of endogenous γδ T cells for immunotherapy. Cancer Res; 74(5); 1349–60. ©2014 AACR.

PDF file - 2448K, Phenotype of established gamma delta T-cells and PDAC-cell lines (S1); Design and purification of the recombinant Her2xCD3 bsscFv (S2); Her2xCD3 bsscFv enhances the gamma delta T-cell lysis of Panc89 cells (S3); Design and purification of the recombinant (Her2)2xVγ9 tribody (S4); (Her2)2xVγ9 does not mediate lysis of antigen-negative lymphoma cells (S5); Increase of gamma delta T-cell cytotoxicity against Panc89 cells by tribody (Her2)2xVγ9 (S6); Tribody (Her2)2xVγ9 enhances CD107a expression on gamma delta Tcell lines co-cultured with PancTu-I cells (S7).

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All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells(1). Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci(2). Consequently, MYC is deregulated, resulting in massive perturbation of gene expression(3). Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma(4). In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

During therapeutic application, mesenchymal stem cells (MSCs) may interact with their environment via their expressed toll-like-receptors (TLRs) leading to pro- or anti-inflammatory immune responses. The present study aimed to describe the gingival margin-derived stem/progenitor cells' (G-MSCs) TLR-induced immune regulatory response to specific TLR agonists. Gingival cells were obtained, immunomagnetically sorted via anti-STRO-1 antibodies and seeded out to achieve colony forming units (CFUs). G-MSCs were investigated for stem cell characteristics and TLR expression. Specific TLR agonists were applied and m-RNA expression of pro- and anti-inflammatory factors was analyzed via real-time polymerase chain reaction. G-MSCs showed all characteristics of stem/progenitor cells. All TLR agonists induced pro-inflammatory cytokines, except for the TLR3 agonist, which significantly promoted the anti-inflammatory response. (p⩽0.05, Wilcoxon-Signed-Ranks-Test). TLR-induced immunomodulation by G-MSCs could impact their therapeutic potential in vivo. Two distinctive pro-inflammatory and an anti-inflammatory TLR-induced phenotypes of G-MSCs become noticeable in this study.

Physical activity can impact the immune system in different ways, e.g. by alteration of the humoral and cellular immune response. Physical activity at medium intensity enhances numbers of cytotoxic T cells, NK cells and macrophages in healthy people. The aim of this study was to compare the effects of endurance and resistance training on the immune system in breast cancer patients during adjuvant chemotherapy. In a prospective, controlled and randomized intervention exploratory trial, 12-week supervised endurance or resistance training were compared with usual care twice a week. Endpoints were the absolute numbers of the immune cells such as CD3+ T lymphocytes including CD4+- and CD8+, αβ T cells, γδT cells, CD3−/CD16+/56+ NK cells and CD19+ B cells, before and after 12 weeks of treatment. Cell numbers were analyzed using fluorescence-activated cell sorting. Despite different physical interventions in all groups immune cell count decreased in CD3 T cells including TCR αβ and CD4 T cells, NK cells and CD19 B cells 12 weeks after initiation of chemotherapy and start of the physical intervention program, while the reduction of γδ T cells and CD8 T cells is less prominent in the RT and UC group. Chemotherapy led to a decrease in nearly all measured immune cells. In this study, physical intervention with endurance or resistance training did not suppress cellular immunity any further. Larger multicenter trials are needed to evaluate the exact impact of sports intervention on immune cell subpopulations.

Background Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. Material and Methods Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs’ incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. Results G-MSCs showed all stem/progenitor cells’ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). Conclusions The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions. Key words:Stem cells, TLR, gingiva, polymerase chain reaction, FACS.

Background 18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro , secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. Materials and methods Respiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2–6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. Results Within 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-β1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. Conclusions We conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.

The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum. We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells. We could show that the combination of both medicines in the preclinical experiment produces a working advantage.

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

Natural killer (NK) cells are innate immune cells involved in antiviral defence and tumour surveillance. To fulfil these tasks, NK cells make use of two major effector functions, cytokine and chemokine release and cytotoxicity. In addition, NK cells proliferate in response to cytokines such as IL-2. NK cells possess a large array of activating and inhibitory receptors and their activation demands a complex crosstalk between those receptors. The signalling pathways leading to NK-cell activation are a field of intensive research. The first clue for signal specificity was provided by studies showing that a pathway leading to NF-κB activation selectively induces cytokine release, but is dispensable for cytotoxicity. Here, we demonstrate that in human NK cells caspase activity is required for the upregulation of select activation markers and IFN-γ and TNF production, but not for cytotoxicity. Interestingly, caspases have previously been linked in T cells to the same mechanism of NF-κB induction that is active in NK cells. Moreover, we provide evidence that caspases are involved in IL-2-induced proliferation. Thus, our data provide the basis for a novel approach using caspase inhibitors to generate cytotoxic NK cells, while simultaneously suppressing cytokine release.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1.

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/ DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.

Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132--a specific inhibitor of the proteasome--blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation.

Background/Objectives Pancreatic ductal adenocarcinoma (PDAC) still has a poor prognosis and current treatments including immunotherapy often fail. This might be due to the pronounced immunosuppressive milieu impairing infiltration and function of immune effector cells. This study aimed at a comprehensive analysis of immune cells in PDAC patients by determining absolute and relative peripheral blood cell numbers of immune cell subsets along with their functional capacity. Methods Whole blood cells or isolated peripheral blood mononuclear cells were characterized by flow cytometry. PDAC tissues were analyzed by immunohistochemistry. Anti-tumor activity of immune effector cells was determined by RTCA system. Results Our data demonstrate that relative CD4 + memory- and regulatory T cell numbers were enhanced, whereas determination of absolute cell numbers revealed generally lower immune cell numbers in PDAC patients compared to healthy controls. γδ T cells accumulated at higher numbers compared to αβ T cells in the malignant ductal epithelium of PDAC tissues indicating that γδ T cells infiltrate into the tumor. Cytotoxicity against tumor cells of even small numbers of T- and NK cells could be induced by a bispecific antibody targeting CD3 + T cells to human epidermal growth factor receptor (HER)2 expressing PDAC cells or Trastuzumab. Importantly, a critical number of γδ T cells was required for significant tumor cell killing by a bispecific antibody engaging the γδ T cell receptor on γδ T cells and HER2 on tumor cells. Conclusion Monitoring immune cells along with the determination of their functional capacity provides a comprehensive assessment as a prerequisite for a personalized immunotherapeutic PDAC treatment.

Aims Down syndrome-associated dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) is a ubiquitously expressed protein kinase. Up to date a variety of targets have been identified, establishing a key role for Dyrk1a in selected signalling pathways. In cardiomyocytes, Dyrk1a acts as a negative regulator of hypertrophy by phosphorylating transcription factors of the NFAT family, but its mechanistic function in the heart remains poorly understood. This study was designed to investigate a potential protective role of Dyrk1a in cardiac hypertrophy in vivo . Methods and results We generated transgenic mice with cardiac-specific overexpression of Dyrk1a. Counterintuitively, these mice developed severe dilated cardiomyopathy associated with congestive heart failure and premature death. In search for the cause of this unexpected phenotype, we found that Dyrk1a interacts with all members of the D-cyclin family and represses their protein levels in vitro and in vivo . Particularly, forced expression of Dyrk1a leads to increased phosphorylation of Ccnd2 on Thr280 and promotes its subsequent proteasomal degradation. Accordingly, cardiomyocytes overexpressing Dyrk1a display hypo-phosphorylated Rb1, suppression of Rb/E2f-signalling, and reduced expression of E2f-target genes, which ultimately results in impaired cell cycle progression. Conclusions We identified Dyrk1a as a novel negative regulator of D-cyclin-mediated Rb/E2f-signalling. As dysregulation of this pathway with impaired cardiomyocyte proliferation leads to cardiomyopathy, dose-specific Dyrk1a expression and activity appears to be critical for the hyperplastic and hypertrophic growth of the developing heart.

Cysteine-proteases belonging to the family of caspases have been described as central executioners of apoptotic signals. The relevance of apoptosis in T cell homeostasis is primarily defined by the necessity to quickly and efficiently eliminate effector T lymphocytes subsequent to clonal expansion and differentiation. This process is executed through the activation of caspases. Apoptosis can be initiated by interaction of the death receptor Fas with its ligand, triggering a signaling cascade comprising Fas-associated death domain protein (FADD), caspase-8 and caspase-3, culminating in cell death. Surprisingly, emerging evidence suggests a contribution of these molecules also in non-apoptotic and even proliferative signals. The potentially dual role of caspases does not seem to be restricted to T cells but is observed in other cells of the hematopoietic lineage as well. A block of the FADD/caspase-signaling pathway, either by inserting mutations or by using pharmacological inhibitors, not only leads to resistance of cells to apoptotic stimuli, but also negatively affects growth responses. Despite large research effort, the exact molecular mechanism how caspases mediate proliferation remains to be clarified. This review summarizes facts and hypotheses about potential junctions, coupling the opposing signals leading to death or proliferation and discusses, under which circumstances active caspases might confer to either pathway.

The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF.

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.

The activation of caspases cleaving a plethora of specific substrates is pivotal for initiation as well as execution of apoptosis. The recognition motif for caspases is a tetrapeptide sequence containing an essential aspartic acid residue at the fourth position (often DXXD). Here, we report that the caspase cleavage sites of most identified substrates show a high degree of conservation between different species. However, we have identified differences in the cleavage sites of five substrates between murine and human proteins leading to either select processing in only one species or to different cleavage patterns. Finally, we provide evidence that murine c-Abl but not its human homolog serves as efficient substrate during apoptosis.

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.

The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients’ γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.

The ability of human γδ T cells from healthy donors to kill pancreatic ductal adenocarcinoma (PDAC) in vitro and in vivo in immunocompromised mice requires the addition of γδ T-cell–stimulating antigens. In this study, we demonstrate that γδ T cells isolated from patients with PDAC tumor infiltrates lyse pancreatic tumor cells after selective stimulation with phosphorylated antigens. We determined the absolute numbers of γδ T-cell subsets in patient whole blood and applied a real-time cell analyzer to measure their cytotoxic effector function over prolonged time periods. Because phosphorylated antigens did not optimally enhance γδ T-cell cytotoxicity, we designed bispecific antibodies that bind CD3 or Vγ9 on γδ T cells and Her2/neu (ERBB2) expressed by pancreatic tumor cells. Both antibodies enhanced γδ T-cell cytotoxicity with the Her2/Vγ9 antibody also selectively enhancing release of granzyme B and perforin. Supporting these observations, adoptive transfer of γδ T cells with the Her2/Vγ9 antibody reduced growth of pancreatic tumors grafted into SCID-Beige immunocompromised mice. Taken together, our results show how bispecific antibodies that selectively recruit γδ T cells to tumor antigens expressed by cancer cells illustrate the tractable use of endogenous γδ T cells for immunotherapy. Cancer Res; 74(5); 1349–60. ©2014 AACR.

L’interaction du TNF avec son recepteur de 55 kDa (TNF-RI) joue un role majeur dans le declenchement des voies de signalisation conduisant a l’apoptose. Un des mecanismes maintenant bien connu est le recrutement, par l’intermediaire du domaine de mort du TNF-RI, de proteines adaptatrices qui elles-memes recrutent la pro-caspase 8. Plus recemment, il a ete montre que le TNF-RI interagit avec une autre proteine, FAN, qui active une sphingomyelinase neutre, declenchant ainsi une autre voie de signalisation, la voie sphingomyeline-ceramide. Nous demontrons maintenant que la proteine FAN est impliquee dans l’apoptose induite par les recepteurs TNF-RI et CD40.

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an “atypical apoptosis” characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.

The generation of proinflammatory eicosanoids in response to tumor necrosis factor (TNF) involves the activation of cytosolic phospholipase A(2) (cPLA(2)), presumably by phosphorylation through extracellular signal-regulated kinases (ERK). Earlier results had suggested that a pathway involving the p55 TNF receptor (TNF-R55), neutral sphingomyelinase (N-SMase), and c-Raf-1 activates ERK and cPLA(2). We have previously shown that a cytoplasmic region of TNF-R55 distinct from the death domain regulates the activation of N-SMase through binding of the adapter protein FAN. Analysis of embryonal fibroblasts from FAN knockout mice revealed that TNF-induced activation of both ERK and cPLA(2) occurs without involvement of FAN. Furthermore, we provide evidence that the TNF-dependent activation of ERK and cPLA(2) requires the intact death domain of TNF-R55. Finally, we demonstrate that in murine fibroblasts cPLA(2) is phosphorylated in response to TNF solely by ERK, but not by p38 mitogen-activated protein kinase, suggesting a signaling pathway from TNF-R55 via the death domain to ERK and cPLA(2).

Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD.fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor κB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G2/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G1 phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G2/M phase augmented it.

The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-κB (NF-κB) induction. Notably, the two proapoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-κB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.

The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor–receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding motif of TNF-R55. This region has been previously recognized as a distinct functional domain that is both required and sufficient for the activation of neutral sphingomyelinase (N-SMase). Overexpression of full-length FAN enhanced N-SMase activity in TNF–treated cells, while truncated mutants of FAN produced dominant negative effects. The data suggest that FAN regulates ceramide production by N-SMase, which is a crucial step in TNF signaling.

The human p55 tumor necrosis factor (TNF) receptor (TR55) initiates at least two independent signaling cascades. The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C, an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors such as NF-kappaB. The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, proline-directed protein kinases, as well as phospholipase A2 and appears critical for the inflammatory responses induced by TNF. While the domain of TR55 that induces A-SMase is probably identical to the death domain, the exact location and extent of a putative N-SMase activation domain are still unknown. Structure-function analysis of TR55 deletion mutants revealed a novel region of 11 amino acids at position 309-319 that is both necessary and sufficient for activation of N-SMase. The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-kappaB, and cytotoxicity. Taken together, our results suggest that a functionally independent region of TR55 is responsible for selectively initiating the N-SMase pathway that couples to an important inflammatory signaling cascade.

Previous studies have characterized phenotypic and functional alterations within T-cell receptor αβ-expressing T cells in patients with granulomatosis with polyangiitis (GPA). We analyzed the frequency, subset composition and in vitro activation of blood γδ T cells in GPA patients. We observed a significant reduction of γδ T cell numbers, due to the selective depletion of the Vδ2 subset which remained consistent over time upon repeated analysis. The loss of Vδ2 T cells was not due to migration into the inflamed lesions as very few γδ T cells were detected in inflammatory infiltrates. The memory subset distribution did not differ among Vδ2 T cells from healthy donors and GPA patients. Importantly, the remaining Vδ2 T cells were capable of responding to phosphoantigen stimulation in vitro. The marked depletion of blood Vδ2 T cells in GPA suggests cellular exhaustion, possibly due to chronic exposure to and continuous overstimulation by microbial phosphoantigens.

Poly(I:C) mediated NFκB - and IRF7-activation is stronger than co-stimulatory anti-CD28 mAb, thereby increasing antiviral chemokine and granzyme B release in CD4 T cells. dsRNA is frequently associated with viral replication. Here, we compared the costimulatory effect of the synthetic analog of dsRNA, poly(I:C), and the agonistic anti-CD28 mAb on anti-CD3 mAb-activated, freshly isolated human CD4 T cells. We tested the hyphothesis that poly(I:C) and anti-CD28 mAb costimulation differ in their effect on the CD4 T cell immune response. Our study shows that costimulation of CD4 T cells by poly(I:C) enhanced CD3-induced production of IP-10, MIP1-α/β, RANTES, and granzyme B involved in antiviral activity more than anti-CD28 mAb. poly(I:C) stimulation, on its own, activated the transcription of IRF7 in human CD4 T cells. Combined CD3 and poly(I:C) stimulation significantly enhanced the transcription of IRF7 and additionally, NF-κBp65 phosphorylation, which might be involved in the induction of antiviral chemokines and the enhanced cytotoxic activity of poly(I:C)-treated CD4 T cells. In comparison with poly(I:C), anti-CD28 mAb as a costimulus induced a stronger proinflammatory response, as indicated by enhanced TNF-α secretion. poly(I:C) had a costimulatory effect on Akt phosphorylation, whereas anti-CD28 mAb only slightly enhanced Akt phosphorylation. In contrast to poly(I:C), anti-CD28 mAb was essential for proliferation of anti-CD3-stimulated CD4 T cells; however, poly(I:C) further increased the anti-CD28/anti-CD3-mediated proliferation. These results indicate that poly(I:C)- and anti-CD28 mAb-induced signaling differ in their costimulatory effect on the CD3-driven, antiviral chemokine release and proinflammatory cytokine secretion in freshly isolated human CD4 T cells.

D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-β1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.

The inhibitor of κB kinase e (IKKe) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKe involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKe, designated IKKe-sv1 and IKKe-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type- and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKe (IKKe-wt) leads to the activation of NF-κB- as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKe-sv1 still activates NF-κB, whereas IKKe-sv2 is also defective in NF-κB activation. Both splice variants form dimers with IKKe-wt and inhibit IKKe-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKe.

gammadelta T-cells are a numerically small subset of T-cells with distinct features. They recognise antigens that are not seen by other immune cells. At the functional level, gammadelta T-cells share some features with alphabeta T-cells but also exert functions that are otherwise performed by specialised subsets of alphabeta T-cells (e.g. IL-17 production, regulatory activity). We discuss the potential role of gammadelta T-cells in various clinical forms of vasculitis.

Monomorphic MHC class II determinants are attractive targets for immunomodulation. HLA-DR ligation on antigen-presenting cells (APCs) can dramatically alter their function or induce cell death. In monocytes, HLA-DR triggering diminishes their capacity to stimulate T cell proliferation. To further investigate this monocyte-dependent T cell inhibition, we activated human T cells +/- HLA-DR triggering on APCs and tested whether this can induce T cell anergy. Only anti-HLA-DR, but not anti-proliferative control agent anti-CD45, could modulate monocytes in primary cultures with stimulated T cells, so that T cells were hyporesponsive during re-stimulation. Cell separation studies demonstrated that HLA-DR ligation on monocytes is sufficient for mediating T cell anergy. Secretion of monokines was severely reduced after primary culture. Monocytes anergized independently of soluble factors. Extracellular signal-regulated kinase (ERK) phosphorylation occurred early with anti-HLA-DR, but late with anti-CD45 antibody. However, ERK inhibition did not reverse the T cell-anergizing potential of HLA-DR-ligated monocytes implicating other signaling pathways involved in tolerance induction. When analyzing the anergized T cells, they were refractory to exogenous IL-2 and characterized by defective secretion of various cytokines. Expression of CD25, CD28, intracellular CD3zeta and CTLA-4 was reduced. The hyporesponsive T cells up-regulated cell-cycle inhibitors p27(kip1) and p21(cip1) in correlation with human T cell anergy. In contrast, caspase-3 and -8, known to contribute to T cell proliferation, were equally decreased in anti-HLA-DR- and anti-CD45-inhibited cultures. In summary, anti-HLA-DR treatment can generate tolerogenic monocytes transmitting T cell anergy that may be exploited for future immunomodulatory strategies to treat immune-mediated disease states.

Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1–249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [ 3 H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.

The role of death receptors and caspases as mediators of programmed cell death is well established. This review focuses on new insights into alternative functions of these molecules in activation and proliferation of lymphocytes and other hematopoietic cell types. The involvement of the death receptor Fas and caspases in immunodeficiency and autoimmunity is discussed. Elucidation of the mechanisms that control the decision whether death receptors and caspases drive activation/proliferation or apoptosis may broaden our knowledge about the pathogenesis of numerous diseases and facilitate the development of novel therapeutic strategies.

Caspases have been described as proteases essential for the release of certain cytokines and for initiation as well as execution of apoptosis. Increasing evidence indicates, however, that caspase activity is also required for activation-induced proliferation of mature T lymphocytes. The molecular mechanism, how caspase activity facilitates T cell proliferation, is still controversially discussed. In this study, we show that proliferation of human T cells in response to a specific antigenic stimulus is completely prevented by caspase inhibition. In addition, we demonstrate that this lack of proliferation is due to a failure to initiate cell cycle progression, but not the result of increased T cell death. Our results demonstrate that caspase inhibition leads to strongly reduced IL-2 release, failure to up-regulate CD25, and a lack of proper regulation of cell cycle-associated proteins. Furthermore, T cell proliferation was partially rescued by addition of exogenous IL-2. Using Jurkat cells, we show that in the absence of caspase-8, the mitogen-induced activation of the transcription factor NF-κB is moderately diminished, while the activity of the composite element CD28 response element and NF-IL-2B AP-1 sites is strongly reduced. Finally, we provide evidence that caspase inhibition suppresses the activation of purified monocytes by bacterial Ags.

Human repp86 becomes detectable in the nucleoplasm of cycling cells at the G(1)-S boundary, condenses at the centrosomes with the onset of mitosis, during which it progressively locates to the mitotic spindle and to the midbody, and vanishes at the completion of cytokinesis. The repp86 cDNA was cloned and sequenced. Full-length repp86 and its COOH-terminal domain cosediment with polymerized microtubules, linking repp86 to the family of microtubule-associated proteins. During prophase and metaphase, repp86 interacts on the mitotic spindle with the putative motor protein Hklp2. Thus, repp86 may function in targeting Hklp2 to the microtubule minus ends, its activity being regulated by phosphorylation of serine/threonine residues. Exogenous overexpression of repp86 provokes accumulation of cells in G(2)-M phase and subsequent polyploidization, suggesting that excess repp86 may interfere with correct nuclear division.

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.

Ki-Mcm6, a New Monoclonal Antibody Specific to Mcm6: Comparison of the Distribution Profile of Mcm6 and the Ki-67 Antigen

TNF-α is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNFtriggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF. J. Clin. Invest. 108:143‐151 (2001). DOI:10.1172/JCI200111498.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.