A consistent picture?

In times of declining party identification, political parties need to persuade and mobilize their voters from election to election. Setting topics in such a way that voters are convinced to cast their vote has become an essential prerequisite for success in modern election campaigns. Social media are suitable for this, as parties can set their own topics or highlight the topics most important to the voters and communicate them to a large audience in organic posts or target specific voter groups with ads. While tendencies of issue ownership in posts on Facebook are repeatedly shown empirically, there is a lack of studies investigating which strategies parties follow in their investment decisions on Facebook ads. Based on theoretical expectations derived from the literature about digital political marketing and issue prioritization in election campaigns, this article investigates whether parties communicated consistently on Facebook with regard to the issues they set in organic posts, sponsored posts, and ads during the 2021 German federal election campaign. The results of a manual quantitative content analysis (n = 1,029 posts, n = 1,197 sponsored posts, n = 2,643 ads) show that parties focused on issue ownership in their posts. Still, their investments in sponsored posts and ads followed different strategies. Here, most parties highlighted social policy, contradicting issue ownership for some parties. The article provides novel insights into digital campaigning and discusses the extent to which parties can engage audiences beyond their organic reach within party-affiliated audiences

Missed opportunity to connect with European citizens? Europarties’ communication on Facebook during the 2019 European election campaign

The digitization of political communication and major transformations in the European Union (EU) have altered the conditions for European election campaigns. Whereas national political parties remain highly visible political actors in the EU, Europarties attract relatively little attention from the media and citizens. Social media could provide Europarties with an opportunity to raise awareness among European citizens. In our study, we investigated the social media campaign strategies of Europarties by conducting a manual quantitative content analysis comparing their Facebook posts with the posts of national parties from 12 European countries, focusing on the communication elements used to inform and mobilize citizens, especially in relation to the lead candidates. Our results revealed that some Europarties employed the concept of European lead candidates by emphasizing their candidates in their Facebook posts. However, in their relative inactivity on Facebook compared with national parties, Europarties did not seem to counteract the oft-cited lack of a European public sphere

Keep Them Engaged! Investigating the Effects of Self-centered Social Media Communication Style on User Engagement in 12 European Countries

On Facebook, patterns of user engagement largely shape what types of political contents citizens can see on the platform. Higher engagement leads to higher visibility. Therefore, one of the major goals of political actors' Facebook communication is to produce content with the potential to provoke user engagement, and thereby increase their own visibility. This study introduces the concept of self-centered social media communication style which focuses on 'salient' and 'owned' issues with populist and negative appeals and investigates how user engagement is related to its main elements. We also explore how users' receptivity to these content-related factors is shaped by country context. More specifically, we hypothesize that users are more likely to react, comment on and share posts focusing on salient topics or issues 'owned' by parties rather than more permanent policy issues, and posts including populist appeals and negativity. Further, we test how these effects are moderated by geographical regions and the level of party system polarization. We manually coded 9,703 Facebook posts of 68 parties from 12 European countries in the context of the 2019 European elections. Our findings show that users are more likely to engage with immigration-related, domestic, populist and negative posts, but react less to posts dealing with environmental or economic issues. While issue ownership does not play a significant role for user engagement, country context plays a minor role. However, some populist appeals are more effective in more polarized countries

Inhibition of SDF-1-induced migration of oncogene-driven myeloid leukemia by the L-RNA aptamer (Spiegelmer), NOX-A12, and potentiation of tyrosine kinase inhibition

Resistance to targeted tyrosine kinase inhibitors (TKI) remains a challenge for the treatment of myeloid leukemias. Following treatment with TKIs, the bone marrow microenvironment has been found to harbor a small pool of surviving leukemic CD34+ progenitor cells. The long-term survival of these leukemic cells has been attributed, at least in part, to the protective effects of bone marrow stroma. We found that the NOX-A12 'Spiegelmer', an L-enantiomeric RNA oligonucleotide that inhibits SDF-1α, showed in vitro and in vivo activity against BCR-ABL- and FLT3-ITD-dependent leukemia cells. NOX-A12 was sufficient to suppress SDF-1-induced migration in vitro. The combination of NOX-A12 with TKIs reduced cell migration in the same in vitro model of SDF-1-induced chemotaxis to a greater extent than either drug alone, suggesting positive cooperativity as a result of the SDF-1 blocking function of NOX-A12 and cytotoxicity resulting from targeted oncogenic kinase inhibition. These results are consistent with our in vivo findings using a functional pre-clinical mouse model of chronic myeloid leukemia (CML), whereby we demonstrated the ability of NOX-A12, combined with the ABL kinase inhibitor, nilotinib, to reduce the leukemia burden in mice to a greater extent than either agent alone. Overall, the data support the idea of using SDF-1 inhibition in combination with targeted kinase inhibition to override drug resistance in oncogene-driven leukemia to significantly diminish or eradicate residual leukemic disease

A role for gut-associated lymphoid tissue in shaping the human B cell repertoire

PMCID: PMC3754866Rockefeller University Press grants the public the non-exclusive right to copy, distribute, or display this Work under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode

Entwicklung antigen-spezifischer primärer humaner NK-Zellen zur Bekämpfung von HER-2-positiven Karcinomen

Table of contents Acknowledgements ....................................................................................................................4 Table of contents .......................................................................................................................5 1\. General introduction ...............................................................................................................9 1.1. Natural Killer cells............................................................................................................... 9 1.1.1. Discovery of Natural Killer cells ......................................................................................... 9 1.1.2. NK cell development and definition .................................................................................... 9 1.2. NK cell activation .............................................................................................................. 10 1.2.1. NK cell inhibitory receptors ............................................................................................. 11 1.2.2. Activating receptors ....................................................................................................... 12 1.2.3. NK cell licensing/education .............................................................................................. 12 1.3. NK cells in tumor therapy .................................................................................................. 13 1.4. HER-2 .............................................................................................................................. 15 1.4.1. HER-2 mediated cell signaling .......................................................................................... 15 1.4.2. Immunotherapy for HER-2 overexpressing tumors ............................................................ 16 1.5. Chimeric receptors ............................................................................................................ 17 1.5.1. Composition of a CR ....................................................................................................... 17 1.5.2. Advantages of a CR ........................................................................................................ 17 1.5.3. CRs in immunotherapy .................................................................................................... 18 1.6. Aim and outline of this study .............................................................................................. 20 2\. Materials and Methods .......................................................................................................... 22 2.1. Materials .......................................................................................................................... 22 2.1.1. Consumables and chemicals ............................................................................................ 22 2.1.2. Antibodies ...................................................................................................................... 22 2.1.3. Machines ........................................................................................................................ 23 2.1.4. Software ........................................................................................................................ 24 2.1.5. Statistics ........................................................................................................................ 24 2.2. Cell culture ........................................................................................................................ 24 2.2.1. Cell lines ......................................................................................................................... 24 2.2.2. Isolation of human PBMCs ................................................................................................ 25 2.3. Retroviral vectors .............................................................................................................. 25 2.4. Retrovirus production by 293T cells ..................................................................................... 26 2.5. Expansion of primary human NK cells .................................................................................. 26 2.6. Retroviral transduction ....................................................................................................... 27 2.6.1. RetroNectin assessed transduction protocol ....................................................................... 27 2.6.2. Spinocculation transduction ............................................................................................. 27 2.7. Antibody staining and FACS analysis ................................................................................... 27 2.8. T cell depletion ................................................................................................................. 28 2.9. Cytokine release assay (ELISA) ......................................................................................... 28 2.10. Pre-incubation with plate-bound recombinant MICA ............................................................ 28 2.11. Intracellular cytokine staining for IFN-γ ............................................................................ 29 2.12. IL-2 secretion assay ....................................................................................................... 29 2.13. CFSE labeling of NK cells ................................................................................................. 29 2.14. CD107a degranulation assay ............................................................................................ 29 2.15. 51Cr release assay ......................................................................................................... 30 2.16. Tumor cell challenge and in vivo imaging .......................................................................... 30 3\. Results ............................................................................................................................... 32 3.1. Expansion and transduction of primary human NK cells ........................................................ 32 3.1.1. Effect of PBL to feeder cell ratio on transduction efficacy ................................................... 32 3.1.2. Role of IL-2 in transduction efficacy ................................................................................. 34 3.1.3. Effect of timing and number of transductions on maximizing transduction efficacy ................ 35 3.1.4. Impact of transduction method on transduction efficacy ..................................................... 36 3.2. Genetically engineered NK cells express the transduced targeting receptor ............................. 38 3.3. Generation of pure NK cell cultures in order to investigate functionality ................................... 40 3.4. HER-2 expressing targets specifically activate genetically engineered NK cells ......................... 41 3.4.1. HER-2 expressing transfectant specifically stimulates CR-NK cells ........................................ 41 3.4.2. Classification of endogenously expressed HER-2 on carcinoma lines .................................... 42 3.4.3. CR-NK cells produce cytokines upon HER-2 recognition ...................................................... 43 3.4.4. All tested HER-2 positive carcinoma lines are specifically recognized by CR-NK cells ............. 44 3.4.5. MHC class I expression on tumor cells does not impact on tumor recognition ....................... 46 3.4.6. Analysis of IFN- γ production by CR-NK cells at single cell level .......................................... 47 3.4.7. IL-2 is specifically produced by CR-NK cells ...................................................................... 48 3.5. CR-NK cell cytotoxicity response towards HER-2 expressing targets ....................................... 48 3.6. CR-NK activation by autologous cells expressing HER-2 ......................................................... 51 3.7. Fresh tumor cells are recognized by both allogeneic and autologous CR-NK cells ..................... 52 3.8. NKG2D blockade has no impact on specific HER-2 recognition ................................................ 55 3.8.1. Binding of MICA to NKG2D on NK cells .............................................................................. 55 3.8.2. No down-modulation of NKG2D on NK cells after overnight exposure to soluble recombinant MICA ............................................................................................................................................... 56 3.8.3. Selection of tumor cell lines based on NKG2D ligand expression .......................................... 56 3.8.4. No negative impact of NKG2D blockade on HER-2 specific recognition by CR-engineered NK cells ............................................................................................................................................... 57 3.9. CR-mediated recognition compares favorably with trastuzumab mediated NK cell activation ...... 59 3.10. Tumor challenge model and in vivo imaging ........................................................................ 63 4\. Discussion ............................................................................................................................ 69 4.1. Optimization of a transduction protocol in order to engineer primary human NK cells ................ 70 4.2. Expression and functionality of the CR in vitro........................................................................ 71 4.2.1. HER-2 specific recognition ................................................................................................ 71 4.2.2. Adjusted NK cell activation level based on HER-2 expression level ....................................... 71 4.2.3. IFN-γ and IL-2 are specifically produced by NK cells ........................................................... 72 4.2.4. CR-NK cells exert cytotoxicity towards HER-2 positive targets .............................................. 73 4.3. CR mediated activation overrides inhibition to „self‟ ............................................................... 74 4.4. Freshly isolated tumor cells are similarly targeted by allogenic and autologous NK cells............. 75 4.5. NKG2D blockade does not impair HER-2 recognition .............................................................. 75 4.6. Advantage of the CR-NK approach over trastuzumab-mediated therapy .................................. 77 4.7. Tumor challenge and in vivo imaging ................................................................................... 78 4.8. Improvement of safety and efficacy ..................................................................................... 79 4.9. Prospective studies ............................................................................................................. 81 Summary ................................................................................................................................. 84 Zusammenfassung .................................................................................................................... 85 Abbreviations ............................................................................................................................ 86 References ................................................................................................................................ 90 Publication ............................................................................................................................... 105 Presentations at Conferences .................................................................................................... 105 Curriculum Vitae ...................................................................................................................... 106 Eidesstattliche Erklärung ........................................................................................................... 108NK cells are promising effectors for tumor adoptive immunotherapy, particularly when considering the targeting of MHC class I low or negative tumors. Yet, many tumor cells are resistant to NK killing, which is mainly the case for non-hematopoietic tumors such as carcinomas or melanoma, even when these cells lose MHC class I expression. In this study, primary human NK cells were engineered by gene transfer of an activating chimeric receptor (CR) specific for HER-2, which is frequently overexpressed on carcinomas. To tip the signal balance towards activation, the antigen-binding domain in this CR was fused to the joined CD3ζ and CD28 signaling domains. I found that these targeted NK cells were specifically activated upon recognition of any HER-2 positive tumor cell tested as indicated by high levels of cytokine secretion as well as degranulation and tumor cell lysis. The magnitude of this specific response correlated with the level of HER-2 expression on the tumor cells and was independent of their MHC class I expression level. Remarkably, CR-mediated activation was shown to override inhibition to „self‟ in an autologous setting. Also, freshly isolated HER-2 positive tumor cells from ovarian cancer patients were efficiently targeted by allogeneic and autologous CR-engineered NK cells. Importantly, blocking of NKG2D, which is known to render NK cells less responsive, did not have any impact on this CR-meidated HER-2 specific recognition. Moreover, a side-by-side comparison revealed a superior recognition of the CR over trastuzumab-mediated NK cell activation. Finally, these CR transduced NK cells, but not their mock transduced counterpart, eliminated coinjected tumor cells in NOD/SCID and RAG2 knockout mice as visualized by in vivo imaging. The major advantage of this approach is the direct coupling of the specificity of the chimeric receptor to the function of NK cells that makes it superior to the currently widely used approach based on monoclonal antibodies, which have a short half-life. Moreover, the antibody therapy strongly depends on the patient‟s given FcγR composition. Finally, only 30 % of HER-2 positive tumors are responsive to trastuzumab therapy. Taken together, these results indicate that the expression of this activating chimeric receptor overrides inhibitory signals in primary human NK cells and directs them specifically towards all tested HER-2 expressing tumor cells both in vitro and in vivo.NK-Zellen sind vielversprechende Effektoren im Hinblick auf adoptive Immuntherapie von Tumoren, vor allem wenn man auf Tumore mit wenig oder keiner MHC-Klasse-I-Expression abzielt. Trotzdem sind viele Tumore resistent gegen den Angriff durch NK-Zellen, was besonders für Tumore des nicht- hämatopoietischen Ursprungs wie Karzinome und Melanome zutrifft. In dieser Studie wurden primäre humane NK-Zellen durch Gentransfer mit einem aktivierenden, chimären Rezeptor (CR) spezifisch für HER-2 versehen, welches häufig auf Karzinomen überexprimiert ist. Durch Fusion der Antigen- Bindungsdomäne mit der gekoppelten CD3ζ- und CD28-Signaldomäne wurde die Signal-Balance zugunsten von Aktivierung verschoben. Die CR-modifizierten NK- Zellen wurden aufgrund der Erkennung jeglicher getesteter HER-2 positiver Tumorzellen spezifisch aktiviert, was durch einen hohen Grad von Zytokin- Produktion, Degranulation sowie Tumorzell-Lyse belegt wurde. Das Ausmaß der spezifischen Antwort korrelierte mit dem HER-2-Expressionsniveau und war unabhängig von dem MHC-Klasse-I Expressionsniveau. Die CR-vermittelte Aktivierung hat überdies die Selbstinhibition, welche in einem autologen Milieu das Angreifen eigener Zellen verhindert, aufgehoben, und auch frisch isolierte, HER-2-positive Tumorzellen von Ovarialkarzinom-Patienten wurden effizient durch allogene und autologe CR-modifizierte NK-Zellen erkannt. Des Weiteren hatte die Blockierung von NKG2D keinen Einfluss auf die CR- vermittelte, HER-2-spezifische Erkennung. Im direkten Vergleich war der CR- Ansatz der trastuzumab-vermittelten Erkennung überlegen. Schließlich haben die CR-transduzierten NK-Zellen effizient Tumorzellen in NOD/SCID- sowie RAG2 -knockout-Mäusen beseitigt. Der Vorteil dieser Studie liegt in der Koppelung der Spezifität des CR an die Funktion der NK-Zellen, wodurch dieser Ansatz den zur Zeit eingesetzten monoklonalen Antikörpern überlegen ist, da diese eine kurze Lebensdauer haben. Außerdem hängt die Antikörpertherapie stark von der gegebenen FcγR-Komposition des Patienten ab. Überdies sprechen lediglich 30 % der HER-2-positiven Tumore auf die trastuzumab-Therapie an. Zusammengefasst zeigen diese Ergebnisse, dass die Expression eines aktivierenden, chimären Rezeptors die inhibierenden Signale in primären humanen NK-Zellen aufhebt, und dass diese NK-Zellen gegen alle getesteten HER-2 positiven Tumorzellen sowohl in vitro als auch in vivo spezifisch vorgehen

CXCL12/SDF-1-Dependent Retinal Migration of Endogenous Bone Marrow-Derived Stem Cells Improves Visual Function after Pharmacologically Induced Retinal Degeneration.

Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP(+) cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1(+)) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1(+)) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair

Efficacy of CAR T-cell Therapy in Large Tumors Relies upon Stromal Targeting by IFN gamma

Adoptive T-cell therapy using chimeric antigen receptor-modified T cells (CAR-T therapy) has shown dramatic efficacy in patients with circulating lymphoma. However, eradication of solid tumors with CAR-T therapy has not been reported yet to be efficacious. In solid tumors, stroma destruction, due to MHC-restricted cross-presentation of tumor antigens to T cells, may be essential. However, CAR-Ts recognize antigens in an MHC-independent manner on cancer cells but not stroma cells. In this report, we show how CAR-Ts can be engineered to eradicate large established tumors with provision of a suitable CD28 costimulatory signal. In an HER2-dependent tumor model, tumor rejection by HER2-specific CAR-Ts was associated with sustained influx and proliferation of the adoptively transferred T cells. Interestingly, tumor rejection did not involve natural killer cells but was associated instead with a marked increase in the level of M1 macrophages and a requirement for IFN gamma receptor expression on tumor stroma cells. Our results argue that CAR-T therapy is capable of eradicating solid tumors through a combination of antigen-independent stroma destruction and antigen-specific tumor cell targeting. (C) 2014 AACR