Role of hypoxia inducible factor 1alpha in cobalt nanoparticle induced cytotoxicity of human THP-1 macrophages

Cobalt is one of the main components of metal hip prostheses and cobalt nanoparticles (CoNPs) produced from wear cause inflammation, bone lyses and cytotoxicity at high concentrations. Cobalt ions mimic hypoxia in the presence of normal oxygen levels, and activate hypoxic signalling by stabilising hypoxia inducible transcription factor 1α (HIF1α). This study aimed to assess in vitro the functional role of HIF1α in CoNP induced cellular cytotoxicity. HIF1α, lysosomal pH, tumour necrosis factor α and interleukin 1β expression were analysed in THP-1 macrophages treated with CoNP (0, 10 and 100 μg/mL). HIF1α knock out assays were performed using small interfering RNA to assess the role of HIF1α in CoNP-induced cytotoxicity. Increasing CoNP concentration increased lysosomal activity and acidity in THP-1 macrophages. Higher doses of CoNP significantly reduced cell viability, stimulated caspase 3 activity and apoptosis. Reducing HIF1α activity increased the pro-inflammatory activity of tumour necrosis factor α and interleukin 1β, but had no significant impact on cellular cytotoxicity. This suggests that whilst CoNP promotes cytotoxicity and cellular inflammation, the apoptotic mechanism is not dependent on HIF1α

A FcγRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy

International audienceTrastuzumab is established as treatment of HER2high metastatic breast cancers but many limitations impair its efficacy. Here, we report the design of a Fab-like bispecific antibody (HER2bsFab) that displays a moderate affinity for HER2 and a unique, specific and high affinity for FcγRIII. In vitro characterization showed that ADCC was the major mechanism of action of HER2bsFab as no significant HER2-driven effect was observed. HER2bsFab mediated ADCC at picomolar concentration against HER2high, HER2low as well as trastuzumab-refractive cell lines. In vivo HER2bsFab potently inhibited HER2high tumor growth by recruitment of mouse FcγRIII and IV-positive resident effector cells and more importantly, exhibited a net superiority over trastuzumab at inhibiting HER2low tumor growth. Moreover, FcγRIIIA-engagement by HER2bsFab was independent of V/F158 polymorphism and induced a stronger NK cells activation in response to target cell recognition. Thus, taking advantage of its epitope specificity and affinity for HER2 and FcγRIIIA, HER2bsFab exhibits potent anti-tumor activity against HER2low tumors while evading most of trastuzumab Fc-linked limitations thereby potentially enlarging the number of patients eligible for breast cancer immunotherapy

Posttranslational regulation of Fas ligand function

The TNF superfamily member Fas ligand acts as a prototypic death factor. Due to its ability to induce apoptosis in Fas (APO-1, CD95) expressing cells, Fas ligand participates in essential effector functions of the immune system. It is involved in natural killer cell- and T cell-mediated cytotoxicity, the establishment of immune privilege, and in termination of immune responses by induction of activation-induced cell death. In addition, Fas ligand-positive tumours may evade immune surveillance by killing Fas-positive tumour-infiltrating cells. Given these strong cytotoxic capabilities of Fas ligand, it is obvious that its function has to be strictly regulated to avoid uncontrolled damage. In hematopoietic cells, the death factor is stored in secretory lysosomes and is mobilised to the immunological synapse only upon activation. The selective sorting to and the release from this specific lysosomal compartment requires interactions of the Fas ligand cytosolic moiety, which mediates binding to various adapter proteins involved in trafficking and cytoskeletal reorganisation. In addition, Fas ligand surface expression is further regulated by posttranslational ectodomain shedding and subsequent regulated intramembrane proteolysis, releasing a soluble ectodomain cytokine into the extracellular space and an N-terminal fragment with a potential role in intracellular signalling processes. Moreover, other posttranslational modifications of the cytosolic domain, including phosphorylation and ubiquitylation, have been described to affect various aspects of Fas ligand biology. Since FasL is regarded as a potential target for immunotherapy, the further characterisation of its biological regulation and function will be of great importance for the development and evaluation of future therapeutic strategies

Design and characterization of Squalene-Gusperimus nanoparticles for modulation of innate immunity

Immunosuppressive drugs are widely used for the treatment of autoimmune diseases and to prevent rejection in organ transplantation. Gusperimus is a relatively safe immunosuppressive drug with low cytotoxicity and reversible side effects. It is highly hydrophilic and unstable. Therefore, it requires administration in high doses which increases its side effects. To overcome this, here we encapsulated gusperimus as squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles (NPs) were obtained from nanoassembly of the squalene gusperimus (Sq-Gus) bioconjugate in water, which was synthesized starting from squalene. The size, charge, and dispersity of the Sq-GusNPs were optimized using the response surface methodology (RSM). The colloidal stability of the Sq-GusNPs was tested using an experimental block design at different storage temperatures after preparing them at different pH conditions. Sq-GusNPs showed to be colloidally stable, non-cytotoxic, readily taken up by cells, and with an anti-inflammatory effect sustained over time. We demonstrate that gusperimus was stabilized through its conjugation with squalene and subsequent formation of NPs allowing its controlled release. Overall, the Sq-GusNPs have the potential to be used as an alternative in approaches for the treatment of different pathologies where a controlled release of gusperimus could be required

Bidirectional interference of mesenchymal stromal cells and anti-leukemic effector cells within the bone marrow microenvironment

Die Akute Myeloische Leukämie (AML) ist eine hämatologische Krebserkrankung, die durch eine abnormale Expansion myeloischer Vorläuferzellen im Knochenmark ausgeht. Sie wird durch eine kleine Subpopulation ruhender und klonaler leukämischer Stammzellen (LSCs) aufrechterhalten, die nach konventioneller Chemotherapie und Bestrahlung überleben und zu einem Rückfall führen können. Die derzeitigen Überlebensraten bei der Erstdiagnose sind sehr niedrig, was die Notwendigkeit neuer therapeutischer Optionen hervorhebt. Von zytotoxischen T-Lymphozyten (CTL) abgeleitete Therapien bieten einen vielversprechenden alternativen Behandlungsweg, da LSCs eine Reihe von hämatopoetischen spezifischen Antigenen exprimieren, welche als Angriffsziel der Therapien dienen könnten. Die Wirksamkeit einer Immunantwort gegen AML wird jedoch wahrscheinlich durch mehrere Zelltypen der leukämischen Mikroumgebung unterdrückt. Dieser Fakt spiegelt sich in den allgemein enttäuschenden Ergebnissen wieder, die in frühen klinischen Studien erzielt wurden. Von zentraler Bedeutung für die Effizienz immun-basierter Therapien ist es, die „immunologische Schutzzone“ der leukämischen Nische zu verstehen und zu verringern. Ein Schlüsselregulator der CTL-Suppression im Knochenmark sind mesenchymale Stromazellen (MSCs). MSCs sind eine heterogene Gruppe multipotenter Zellen, die die Hämatopoese regulieren und sich in verschiedene funktionelle und strukturelle Vorläuferzellen differenzieren können. Unter inflammatorischen Bedingungen erfahren MSCs eine drastische Veränderung ihres Phänotyps, was zur Expression einer Reihe von entzündungshemmenden und immunsuppressiven Faktoren führt. Frühere in vitro- und in vivo Experimente haben gezeigt, dass MSCs die Expansion von stimulierten CTLs durch kontakt-abhängige und -unabhängige Mechanismen hemmen können. Weniger klar ist die potentielle inhibitorische Rolle von MSCs bezüglich der zytotoxischen Aktivität von CTLs in Bezug auf die AML. In dieser Arbeit haben wir versucht, uns diesem Punkt zu widmen. In verschiedenen in vitro Kokultur-Assays wurde die kontakt-unabhängige Modulation der entzündlichen und proliferativen Kapazitäten von universellen AML-gerichteten chimären Antigenrezeptor (UniCAR) T-Zellen durch MSCs von gesunden Spendern und AML Patienten nachgewiesen. Diese Modulation führt zu einer Hemmung der T-Zell-Expansion, einer CD4+-Anreicherung, sowie zur reduzierten Freisetzung von IFNγ- und IL-2. Darüber hinaus wurde festgestellt, dass MSCs dem entzündlichen Potential der Leukämie- assoziierten WT1- und ROR1-gerichteten CTL-Klonen entgegen wirkten und die Freisetzung von IFNγ, TNFα, IL-2 und IL-4 hemmten. Interessanterweise konnten jedoch T-Zellen, die auf diese Weise gehemmt wurden, ihre zytolytische Aktivität beibehalten. Die Induktion des seneszenten CD28-CD57+-T-Zell-Phänotyps durch parakrine Mechanismen von MSCs konnte unabhängig vom Gedächtnisstadium der T-Zellen, sowie die von der UniCAR-T-Zell-vermittelte Apoptose der MSCs, beobachtet werden. Die umfangreichen Veränderungen von MSCs unter Entzündungsstimulation im spezifischen Kontext der antileukämischen UniCAR-T-Zell-Zytotoxizität wurden abschließend in Transkriptom- und Proteinanalysen beschrieben. Des Weiteren wurde die Expression der Immun-Checkpoint-Moleküle VISTA und HVEM identifiziert, welche bisher im Knochenmark MSCs noch nicht beschrieben wurde. Durch die Analyse des gesamten Transkriptoms wurden zudem genetische Signaturen und molekulare Programme identifiziert, die unter inflammatorischen Bedingungen reguliert wurden. Diese Untersuchungen erbrachten den Nachweis, dass MSCs ihren Phänotyp von multipotenten Modulatoren der extrazellulären Matrix des Knochenmarks, vermittelt durch Wnt-, Hedgehog-, Notch- und BMP-Signalwege, zu Regulatoren der Immunität über die Signalwege Jak-STAT, MAPK und PI3K-Akt-mTOR verlagern können.:Abstract Zusammenfassung Acknowledgements Table of contents Abbreviations List of Tables List of Figures List of Supplemental Figures 1 Introduction 1.1 Acute Myeloid Leukemia 1.1.1 Mutational burden and prognosis 1.1.2 Current therapy 1.1.3 Relapse and the leukemic stem cell theory 1.2 Immunotherapy 1.2.1 Immune engagement in standard AML therapy 1.2.2 Cell surface targets for immunotherapy 1.2.3 Bispecific antibodies 1.2.4 CAR and UniCAR T cells 1.3 Immune evasion and the immunosuppressive AML bone marrow niche 1.3.1 Intrinsic mechanisms of immune escape by AML 1.3.2 Role of the leukemic microenvironment 1.3.3 Mesenchymal stromal cells 1.4 Rationale 2 Materials and Methods 3 Results 3.1 Selection of immunomodulating bone marrow MSCs from healthy donors. 3.2 MSC-mediated interference of anti-leukemic clonal CD8+ T cell activity. 3.3 MSC-mediated bidirectional interference of AML-retargeted UniCAR T cell activity. 3.3.1 MSC-mediated interference of UniCAR T cell activity against AML. 3.3.2 UniCAR T cell-mediated apoptosis of MSCs. 3.3.3 Induction of AML-redirected UniCAR T cell senescence by MSCs. 3.3.4 Licensing of MSCs in the context of UniCAR T cell anti-leukemic activity. 4 Discussion 4.1 Limitations of the study 4.2 MSC interfere with anti-leukemic T cell-mediated inflammation 4.3 MSCs interfere with of UniCAR T cell proliferation. 4.4 UniCAR T cells induce apoptosis of MSCs 4.5 MSCs induce senescence of anti-leukemic T cells 4.6 Anti-leukemic T cell activity activates the immunosuppressive program of MSCs 4.7 Outlook 5 Appendix 6 References 7 DeclarationsAcute myeloid leukemia (AML) is a form of hematological cancer derived from the abnormal expansion of myeloid precursor cells in the bone marrow. It is maintained by a small subpopulation of quiescent and clonal leukemic stem cells (LSCs) that persist after conventional chemotherapy and irradiation, leading to relapse. Current survival rates at first diagnosis are dismal, highlighting the need for novel therapeutic options. Cytotoxic T lymphocyte (CTL)-derived therapies provide a promising alternative avenue for treatment, as LSCs ubiquitously express a range of hematopoietic-specific antigens that could be targeted. However, the effectiveness of an immune response against AML is likely diminished by several constituent cells of the leukemic microenvironment, and this is reflected in the generally disappointing outcomes observed in early clinical trials. Understanding and alleviating the “immunological sanctuary” provided by the leukemic niche is thus central in permitting effective immunity-based therapy. One key regulator of CTL suppression in the bone marrow are mesenchymal stromal cells (MSCs). MSCs are a heterogeneous population of multipotent cells that regulate hematopoiesis and can differentiate into several functional and structural progenitors. Under inflammatory conditions, MSCs undergo a drastic shift in phenotype, leading to the expression of a range of anti-inflammatory and immunosuppressive factors. Previous in vitro and in vivo work has demonstrated that MSCs inhibit the expansion of stimulated CTLs through contact-dependent and independent mechanisms. Less clear is the potential inhibitory role of MSCs concerning the cytotoxic activity of CTLs against their target in the context of AML, which was investigated in this thesis. In prolonged in vitro co-culture assays, a contact-independent modulation of the inflammatory and proliferative capacities of AML-redirected universal chimeric antigen receptor (UniCAR) T cells by healthy donor and patient-derived MSCs was described, inhibiting T cell expansion, CD4+ enrichment, as well as IFNγ and IL-2 release. In addition, MSCs interfered with the inflammatory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, TNFα, IL-2 and IL-4. T cells abrogated in this manner were also shown to retain their cytolytic activity. Induction of the CD28-CD57+ senescent T cell phenotype by MSCs through paracrine mechanisms was demonstrated, a phenomenon that was independent of memory stage, as well as the UniCAR T cell-mediated apoptosis of MSCs. Finally, the expansive changes of MSCs under inflammatory stimulation in the specific context of anti-leukemic UniCAR T cell cytotoxicity were described via transcriptomic and protein analysis, identifying the expression of the immune checkpoint molecules VISTA and HVEM, as-of-yet uncharacterized in bone marrow MSCs. Through whole transcriptome analysis, genetic signatures and molecular programs were identified that were up- or downregulated under inflammation, broadly characterizing MSCs as shifting from multipotent modulators of the bone marrow extracellular matrix mediated by Wnt, Hedgehog, Notch and BMP signaling pathways, to regulators of immunity via the Jak-STAT, MAPK and PI3K-Akt-mTOR pathways. In summary, MSCs were shown to be potent mediators of anti-leukemic immunity, and targeting their modes of action would likely be highly beneficial in a combinatorial approach with AML immunotherapy.:Abstract Zusammenfassung Acknowledgements Table of contents Abbreviations List of Tables List of Figures List of Supplemental Figures 1 Introduction 1.1 Acute Myeloid Leukemia 1.1.1 Mutational burden and prognosis 1.1.2 Current therapy 1.1.3 Relapse and the leukemic stem cell theory 1.2 Immunotherapy 1.2.1 Immune engagement in standard AML therapy 1.2.2 Cell surface targets for immunotherapy 1.2.3 Bispecific antibodies 1.2.4 CAR and UniCAR T cells 1.3 Immune evasion and the immunosuppressive AML bone marrow niche 1.3.1 Intrinsic mechanisms of immune escape by AML 1.3.2 Role of the leukemic microenvironment 1.3.3 Mesenchymal stromal cells 1.4 Rationale 2 Materials and Methods 3 Results 3.1 Selection of immunomodulating bone marrow MSCs from healthy donors. 3.2 MSC-mediated interference of anti-leukemic clonal CD8+ T cell activity. 3.3 MSC-mediated bidirectional interference of AML-retargeted UniCAR T cell activity. 3.3.1 MSC-mediated interference of UniCAR T cell activity against AML. 3.3.2 UniCAR T cell-mediated apoptosis of MSCs. 3.3.3 Induction of AML-redirected UniCAR T cell senescence by MSCs. 3.3.4 Licensing of MSCs in the context of UniCAR T cell anti-leukemic activity. 4 Discussion 4.1 Limitations of the study 4.2 MSC interfere with anti-leukemic T cell-mediated inflammation 4.3 MSCs interfere with of UniCAR T cell proliferation. 4.4 UniCAR T cells induce apoptosis of MSCs 4.5 MSCs induce senescence of anti-leukemic T cells 4.6 Anti-leukemic T cell activity activates the immunosuppressive program of MSCs 4.7 Outlook 5 Appendix 6 References 7 Declaration

Target profiling of PARP inhibitors and necroptosis inhibitors using photoaffinity labelling

Target profiling of a small molecule therapeutic is essential to fully understand how that compound works in the clinic. Photoaffinity labelling (PAL) has become a widely utilised strategy for in-cell target identification campaigns for reversible, small molecule drugs. After an overview of target profiling and PAL, this Thesis discusses the application of PAL to two classes of molecules with incomplete target profiles. The Thesis focusses initially on the generation of the first photo-activatable probe for inhibitors of the PARP family of enzymes, PARPYnD, based on a novel anti-cancer PARP1/2/6 inhibitor AZ0108 with unexplained off-target toxicity. The design, synthesis and validation of the probe is discussed, along with the application of PARPYnD to PAL studies. Herein, simultaneous live-cell target engagement of PARP1/2 is shown for the first time by a photo-activatable probe, and this labelling is used to quantify live-cell engagement of these PARPs by known PARP inhibitors in competitive PAL experiments. For AZ0108 and clinical PARP inhibitor olaparib, novel off-targets are identified, demonstrating the power of PAL to capture weaker, secondary binders. Finally, PARPYnD fails to label PARP6 in live cells, but is able to label recombinant PARP6, highlighting a biomolecular disparity that raises questions about the proposed mechanism of action of AZ0108. PAL is then applied to a novel series of inhibitors of necroptosis, an inflammatory form of cell death, with an unknown mechanism of action. Design and synthesis of cell-active photo-activatable probe 7PQYnD1 is presented, along with the development of a bespoke live-cell necroptosis assay to evaluate necroptosis inhibitors in-house. 7PQYnD1 is then applied to the PAL workflow and five bona fide target proteins are identified through proteomics. Preliminary functional analysis of these hits is then undertaken to begin to identify the target interaction(s) responsible for the anti-necroptosis phenotype of these compounds.Open Acces

A brighter side to thalidomide: its potential use in immunological disorders

Thalidomide and its derivatives are immunomodulatory drugs (IMiDs) known for their sedative, teratogenic, anti-angiogenic, and anti-inflammatory properties. Commonly used in the treatment of cancers such as Multiple Myeloma and Myelodysplastic Syndrome, IMiDs have also been used in the treatment of an inflammatory skin pathology associated with Hansen’s disease/Leprosy and show promise in the treatment of autoimmune disorders including Systemic Lupus Erythmatosus and Inflammatory Bowel Disease. Recent structural and experimental observations have revolutionized our understanding of these properties by revealing the fundamental molecular events underpinning IMiD activity. Here, we review these findings, their relevance to IMiD therapy in immunological disorders, and discuss how further research might unlock the vast clinical potential of these compounds

Regulation of cell fate by lymphotoxin (LT) receptor signalling: Functional differences and similarities of the LT system to other TNF superfamily (TNFSF) members

The role of TNFR family members in regulating cell fate both in the immune system and in non-lymphoid tissues has been under extensive research for decades. Moreover, the ability of several family members (death receptors) to induce death (mainly via apoptosis) represents a promising target for cancer therapy. Many studies have focused mostly on death receptors such as TNFRI, Fas and TRAIL-R due to their strong pro-apoptotic potential. Yet, cell death can be triggered via non-classical death receptors, and the Lymphotoxin (LT) system represents a very good example of such a TNFR subfamily. Here we provide a comprehensive review of intracellular signalling pathways and cellular responses to LTspecific signalling, and compare for the first time the LT system to other TNFRs, such as CD40. Our aim is to highlight that non-classical TNFR-TNFL dyads such as the LT system demonstrate more complex, cell-type and context-specific capabilities. Understanding these complexities will permit a better understanding of the biological mechanisms via which nondeath domain-containing TNFRs induce cell death, but may also allow the design of better therapeutic strategies