B23-Circular Dichroism
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Maryam
Abooali
,
Stephanie
Schlichtner
,
Xi
Lei
,
Nijas
Aliu
,
Sabrina
Ruggiero
,
Sonia
Loges
,
Martin
Ziegler
,
Franziska
Hertel
,
Anna-Lena
Volckmar
,
Albrecht
Stenzinger
,
Petros
Christopoulos
,
Michael
Thomas
,
Elena
Klenova
,
N. Helge
Meyer
,
Stergios
Boussios
,
Nigel
Heaton
,
Yoh
Zen
,
Ane
Zamalloa
,
Shilpa
Chokshi
,
Luca
Urbani
,
Sophie
Richard
,
Kavitha
Kirubendran
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Dietmar
Cholewa
,
Steffen M.
Berger
,
Inna M.
Yasinska
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: V-domain Ig-containing suppressor of T cell activation (VISTA) is a unique immune checkpoint protein, which was reported to display both receptor and ligand activities. However, the mechanisms of regulation of VISTA activity and functions by factors of tumour microenvironment (TME) remain unclear and understanding these processes is required in order to develop successful personalised cancer immunotherapeutic strategies and approaches. Here we report for the very first time that VISTA interacts with another immune checkpoint protein galectin-9 inside the cell most likely facilitating its interaction with TGF-β-activated kinase 1 (TAK1). This process is required for protection of lysosomes, which is crucial for many cell types and tissues. We found that VISTA expression can be differentially controlled by crucial factors present in TME, such as transforming growth factor beta type 1 (TGF-β) and hypoxia as well as other factors activating hypoxic signalling. We confirmed that involvement of these important pathways modulated by TME differentially influences VISTA expression in different cell types. These networks include: TGF-β-Smad3 pathway, TAK1 (TGF-β-activated kinase 1) or apoptosis signal-regulating kinase 1 (ASK1)-induced activation of activating transcription factor 2 (ATF-2) and hypoxic signalling pathway. Based on this work we determined the five critical functions of VISTA and the role of TME factors in controlling (modulating or downregulating) VISTA expression.
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Feb 2025
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B23-Circular Dichroism
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Stephanie
Schlichtner
,
Inna M.
Yasinska
,
Elena
Klenova
,
Maryam
Abooali
,
Gurprit S.
Lall
,
Steffen M.
Berger
,
Sabrina
Ruggiero
,
Dietmar
Cholewa
,
Milan
Milošević
,
Bernhard F.
Gibbs
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-β-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.
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Aug 2023
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: Background: Galectin-9 is a member of the family of lectin proteins and crucially regulates human immune responses, particularly because of its ability to suppress the anticancer activities of T lymphocytes and natural killer cells. Recent evidence demonstrated that galectin-9 is highly expressed in a wide range of human malignancies including the most aggressive tumors, such as high-grade glioblastomas and pancreatic ductal adenocarcinomas, as well as common malignancies such as breast, lung and colorectal cancers. However, solid tumor cells at rest are known to secrete either very low amounts of galectin-9 or, in most of the cases, do not secrete it at all. Our aims were to elucidate whether T cells can induce galectin-9 secretion in human cancer cells derived from solid malignant tumors and whether this soluble form displays higher systemic immunosuppressive activity compared with the cell surface-based protein. Methods: A wide range of human cancer cell lines derived from solid tumours, keratinocytes and primary embryonic cells were employed, together with helper and cytotoxic T cell lines and human as well as mouse primary T cells. Western blot analysis, ELISA, quantitative reverse transcriptase-PCR, on-cell Western and other measurement techniques were used to conduct the study. Results were validated using in vivo mouse model. Results: We discovered that T lymphocytes induce galectin-9 secretion in various types of human cancer cells derived from solid malignant tumors. This was demonstrated to occur via two differential mechanisms: first by translocation of galectin-9 onto the cell surface followed by its proteolytic shedding and second due to autophagy followed by lysosomal secretion. For both mechanisms a protein carrier/trafficker was required, since galectin-9 lacks a secretion sequence. Secreted galectin-9 pre-opsonised T cells and, following interaction with other immune checkpoint proteins, their activity was completely attenuated. As an example, we studied the cooperation of galectin-9 and V-domain Ig-containing suppressor of T cell activation (VISTA) proteins in human cancer cells. Conclusion: Our results underline a crucial role of galectin-9 in anticancer immune evasion. As such, galectin-9 and regulatory pathways controlling its production should be considered as key targets for immunotherapy in a large number of cancers.
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Jan 2023
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B23-Circular Dichroism
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Anette Teo Hansen
Selnø
,
Stephanie
Schlichtner
,
Inna M.
Yasinska
,
Svetlana S.
Sakhnevych
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Elena
Klenova
,
Ludmila
Pavlova
,
Bernhard F.
Gibbs
,
Martin
Degen
,
Isabelle
Schnyder
,
Nijas
Aliu
,
Steffen M.
Berger
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear.
Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-β1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-β1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-β1 signalling pathway as a potential highly efficient target for cancer immunotherapy.
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Dec 2020
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B23-Circular Dichroism
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Inna M.
Yasinska
,
N. Helge
Meyer
,
Stephanie
Schlichtner
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Maxwell
Casely-Hayford
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Cloe
Desmet
,
Luigi
Calzolai
,
Luca
Varani
,
Steffen M.
Berger
,
Ulrike
Raap
,
Bernhard F.
Gibbs
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[24509, 20755, 21202]
Open Access
Abstract: Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.
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Nov 2020
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B23-Circular Dichroism
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Inna M.
Yasinska
,
Svetlana S.
Sakhnevych
,
Ludmila
Pavlova
,
Anette
Teo Hansen Selnø
,
Ana Maria
Teuscher Abeleira
,
Ouafa
Benlaouer
,
Isabel
Gonçalves Silva
,
Marianne
Mosimann
,
Luca
Varani
,
Marco
Bardelli
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Dietmar
Cholewa
,
Steffen M.
Berger
,
Bernhard F.
Gibbs
,
Yuri A.
Ushkaryov
,
Elizaveta
Fasler-Kan
,
Elena
Klenova
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[20755]
Open Access
Abstract: Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.
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Jul 2019
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[12578]
Open Access
Abstract: Calcineurin inhibitors potentially prevent pro-allergic mediator release from basophils and mast cells but are rarely used systemically due to ubiquitous expressions of target signaling proteins. However, specific targeting of allergic effector cells with these inhibitors could circumvent unwanted side effects. We recently demonstrated the biocompatibility of gold nanoparticles (AuNPs) as a platform for non-toxic delivery of signaling inhibitors due to unique physicochemical properties of these nanomaterials. Since AuNPs can be conjugated with both anti-allergic drugs and antibodies or other proteins that specifically recognize basophils and mast cells, our aims were to assess specific targeting of allergic effector cell function using AuNPs conjugated with the calcineurin inhibitor ascomycin. Purified human basophils and LAD2 human mast cells were used for investigations with AuNPs conjugated either to CD203c antibodies or containing stem cell factor (SCF), respectively, which were amine-coupled to acidic groups of reduced glutathione (GSH). GSH was also used as a spacer for immobilization of ascomycin on the gold surface. AuNPs conjugated with anti-CD203c and ascomycin strikingly blocked IgE-dependent degranulation of both purified basophils and those present in mixed leukocyte preparations, suggesting specific targeting of these cells. In contrast, LAD2 mast cell responses were not inhibited using anti-CD203c-containing nanoconjugates but were when the conjugates contained SCF. Successful targeting of allergic effector cells using gold nanoconjugates indicates that this technology may have therapeutic potential for the treatment of allergies by specifically delivering highly effective signaling inhibitors with reduced side effects.
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Mar 2019
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B23-Circular Dichroism
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Svetlana S.
Sakhnevych
,
Inna M.
Yasinska
,
Alison M.
Bratt
,
Ouafa
Benlaouer
,
Isabel
Gonçalves Silva
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Bernhard F.
Gibbs
,
Yuri A.
Ushkaryov
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[12578]
Abstract: The progression of acute myeloid leukemia (AML)—the most severe blood/bone marrow cancer—is determined by the ability of malignant cells to escape host immune surveillance. However, the systemic regulatory mechanisms underlying this phenomenon remain largely unknown. In this study, we discovered a fundamental systemic biochemical strategy that allows AML cells to employ physiological systems within the body to survive and escape immune attack. We found that AML cells use a crucial human adrenal cortex hormone (cortisol) to induce the expression of neuronal receptor latrophilin 1 (LPHN1), which facilitates exocytosis. This receptor interacts with the blood plasma protein fibronectin leucine rich transmembrane protein 3 (FLRT3) to cause secretion of the immune suppressor galectin-9, which impairs the anticancer activities of cytotoxic lymphoid cells.
AML is a cancer of the blood and bone marrow that originates from self-renewing malignant immature myeloid cells and rapidly progresses into a systemic, and very often fatal, malignancy.1 AML cells employ physiological systems in the body to produce factors required for proliferation/disease progression.2,3 This includes the hijacking of stem cell factor (SCF), a major hematopoietic growth factor that controls AML progression and thus can become highly oncogenic.2,3 The expression and release of SCF can be triggered by AML cells via cytokines (e.g., interleukin-1β).2 Recent evidence clearly demonstrated that AML cells are also capable of impairing the activities of cytotoxic lymphoid cells (e.g., natural killer (NK) cells and cytotoxic T cells).4 One of the biochemical mechanisms underlying this phenomenon lies in the ability of AML cells to secrete the protein galectin-9. This tandem-type galectin binds the immune receptor Tim-3 and induces a variety of intracellular and cell-to-cell signaling events leading to the inactivation of NK cells, as well as the death of cytotoxic T cells.4,5 We recently reported that the process of galectin-9 secretion in AML cells is stimulated by the unique G protein-coupled receptor LPHN1, which normally functions in neurons to facilitate exocytosis.4,6 LPHN1 is also found in hematopoietic stem cells (HSCs), but its expression disappears at the early stages of their maturation.4,7 However, upon malignant transformation, AML cells preserve their abilities to express LPHN1 and to produce high levels of galectin-9 and Tim-3, in which the latter is involved in trafficking galectin-9 during the secretion process (HSCs express neither galectin-9 nor Tim-34).
It is currently unknown which molecular mechanisms trigger elevated levels of LPHN1 expression in primary human AML cells, and in general, the mechanisms of upregulation of LPHN1 expression at the genomic level remain unclear. It is also unknown whether FLRT3, a natural LPHN1 ligand,4,8 is present in human blood plasma and in other tissues associated with AML. Unraveling these mechanisms is crucial to understanding the pathways that control the ability of AML cells to protect themselves against cytotoxic lymphoid cells and, thus, was the aim of the present study.
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Jun 2018
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B23-Circular Dichroism
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Inna M.
Yasinska
,
Isabel Gonçalves
Silva
,
Svetlana S.
Sakhnevych
,
Laura
Ruegg
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Marco
Bardelli
,
Luca
Varani
,
Ulrike
Raap
,
Steffen
Berger
,
Bernhard F.
Gibbs
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[12578]
Open Access
Abstract: High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.
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Feb 2018
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B23-Circular Dichroism
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Inna M.
Yasinska
,
Giacomo
Ceccone
,
Isaac
Ojea-Jimenez
,
Jessica
Ponti
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Steffen M.
Berger
,
Elizaveta
Fasler-Kan
,
Marco
Bardelli
,
Luca
Varani
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Ulrike
Raap
,
Bernhard F.
Gibbs
,
Luigi
Calzolai
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[12578, 11373]
Open Access
Abstract: In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies. Gold surfaces were also covered with the cytotoxic drug rapamycin which was immobilised using a glutathione linker. These nanoconjugates allowed highly specific and efficient delivery of cytotoxic rapamycin into human malignant blood cells.
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Feb 2018
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